Article

A Direct Demonstration of Closed-State Inactivation of K Channels at Low pH

Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, BC, Canada.
The Journal of General Physiology (Impact Factor: 4.79). 06/2007; 129(5):437-55. DOI: 10.1085/jgp.200709774
Source: PubMed

ABSTRACT

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker alpha-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K(+) or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at -80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.

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Volume 129 Number 5 May 2007 437–455
http://www.jgp.org/cgi/doi/10.1085/jgp.200709774
437
ARTICLE
A Direct Demonstration of Closed-State Inactivation of K
Channels
at Low pH
Thomas W. Claydon,
1
Moni Vaid,
2
Saman Rezazadeh,
2
Daniel C.H. Kwan,
2
Steven J. Kehl,
2
and David Fedida
1,2
1
Department of Anesthesiology, Pharmacology, and Therapeutics and
2
Department of Cellular and Physiological Sciences,
University of British Columbia, Vancouver, V6T 1Z3 Canada
Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-
clamp  uorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring  uorescence emis-
sion from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor
domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker -helix (A359C and
R362C) reported a pH-induced acceleration of the slow phase of  uorescence quenching that represents P/C-type
inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C  uorescence
demonstrated slow unquenching that also re ects channel inactivation and this too was accelerated at low pH. In
addition, however, acidic pH caused a reversible loss of the  uorescence signal (pKa 5.1) that paralleled the re-
duction of peak current amplitude (pKa 5.2). Protons decreased single channel open probability, suggesting
that the loss of  uorescence at low pH re ects a decreased channel availability that is responsible for the reduced
macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K
or the mutation
T449V) prevented  uorescence loss at low pH, and the  uorescence report from closed Shaker ILT S424C channels
implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the  uorescence
amplitude (pKa 5.9) in channels held continuously at 80 mV. This suggests that low pH stabilizes closed-inactivated
states. Thus,  uorescence experiments suggest the major mechanism of pH-induced peak current reduction is
inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to
acceleration of P/C-type inactivation from the open state.
INTRODUCTION
The kinetic properties of many voltage gated potassium
(Kv) channels are affected by external pH. Low extra-
cellular pH reduces the peak current in Shaker channels
as well as in Kv1.3, Kv1.4, and Kv1.5 from the related Kv1
family of mammalian channels (Deutsch and Lee, 1989;
Lopez-Barneo et al., 1993; Perez-Cornejo, 1999; Steidl
and Yool, 1999; Claydon et al., 2000; Jäger and Grissmer,
2001; Kehl et al., 2002; Starkus et al., 2003; Somodi et al.,
2004) in a manner that is independent of the depolarizing
shift of the voltage dependence of channel opening that
is caused by a proton-induced charge screening effect
(Deutsch and Lee, 1989; Kehl et al., 2002; Trapani and
Korn, 2003). In pH-sensitive mammalian Kv1 channels,
a histidine residue in the outer pore is responsible for
the inhibition of current and consistent with this, the
pK
a
(with physiological external K
concentrations) is
pH 6.1–6.2 (Steidl and Yool, 1999; Claydon et al., 2000;
Kehl et al., 2002). In the case of the Shaker channel,
which does not possess a histidine residue within the
outer pore, the pK
a
is pH 4.7–5.0 suggesting that the pH
sensor may be an aspartate or glutamate residue (Perez-
Cornejo, 1999; Starkus et al., 2003).
In the absence of N-type inactivation, acidic pH not
only reduces peak current amplitude, but also enhances
the decay of current in Shaker, Kv1.4, and Kv1.5 chan-
nels during the depolarizing pulse (Perez-Cornejo,
1999; Claydon et al., 2002; Starkus et al., 2003; Zhang
et al., 2003). It has been suggested that the decrease in
peak current observed with extracellular acidi cation
occurs as a direct consequence of the pH-induced en-
hancement of open-state inactivation, which results in
an accumulation of channels in the P/C-type inacti-
vated state (Steidl and Yool, 1999; Starkus et al., 2003;
Zhang et al., 2003). In support of this, the acceleration
of current decay and the decrease of peak current am-
plitude at low pH is abolished in Shaker, Kv1.4, and
Kv1.5 mutant channels in which inactivation is compro-
mised by mutation of the outer pore residue equivalent
to T449 in Shaker (K532 and R487 in Kv1.4 and Kv1.5,
respectively), to a valine or tyrosine residue (Claydon
et al., 2002; Starkus et al., 2003; Zhang et al., 2003;
Kurata and Fedida, 2006).
However, an additional mechanism to explain the de-
creased peak Kv channel current at low pH has also
Correspondence to David Fedida: fedida@interchange.ubc.ca
Abbreviations used in this paper: PMT, photomultiplier tube; TMRM,
tetramethylrhodamine-5-maleimide.
Page 1
438 pH and Closed-State Inactivation
been proposed from our previous work. During extra-
cellular acidi cation (to pH 5.9), Kv1.5 current inhibi-
tion did not demonstrate use dependence as would be
expected if the current was being reduced by an accu-
mulation of open-state inactivation (Kehl et al., 2002).
In addition, a 2-min rest interval with pH 5.9 bath solu-
tion, which would allow enough time for recovery from
pH-induced open-state inactivation, did not relieve the
current inhibition, which suggests that inactivated chan-
nels cannot recover easily at low pH. This idea of stabili-
zation of closed-state inactivated channels at low pH is
supported by a recent study investigating the effect of
acidic pH on single channel Kv1.5 current. Acidic pH
did not alter the Kv1.5 single channel conductance, but
increased the number of null sweeps and induced gat-
ing behavior in which the channel switched between
available and unavailable modes (Kwan et al., 2006).
This suggests that extracellular acidi cation reduces
channel availability. Since low pH apparently also accel-
erated open-state inactivation, as the mean burst dura-
tion was decreased and the interburst interval was
increased, these data suggest that extracellular acidi ca-
tion enhances both open- and closed-state inactivation,
and that the two processes can coexist in Kv channels.
Voltage-clamp fluorimetry allows real-time observa-
tion of the conformational changes associated with
channel gating, including those that do not result in
ionic current  ow (Mannuzzu et al., 1996; Cha and
Bezanilla, 1997). Changes in the emission observed from
a  uorophore attached at speci c residues are due to
changes of its microenvironment as channels activate
and inactivate (Cha and Bezanilla, 1997; Loots and
Isacoff, 1998; Bezanilla, 2002). Here, we have used this
technique to investigate the fate of channels at low pH
by directly monitoring the effect of acidi cation on the
channel rearrangements associated with P-type, C-type,
and closed-state inactivation of Shaker channels. We
show that low extracellular pH not only enhances P-type
inactivation of channels, but also their progression to
the C-type inactivated state, and in addition that pro-
tons stabilize an unavailable state in which channels are
inactivated at rest. We demonstrate that stabilization of
closed-inactivated states at low pH is largely responsible
for the decrease in peak current amplitude, while stabi-
lization of open-inactivated states is responsible for the
enhanced current decay. In parallel, we have obtained
single channel and gating current data that are in ac-
cord with this hypothesis. These data provide a direct
demonstration that protons alter both open- and closed-
state inactivation of Kv channels.
MATERIALS AND METHODS
Solutions
For Xenopus oocytes, Barth’s medium contained (in mM) 88 NaCl,
1 KCl, 2.4 NaHCO
3
, 0.82 MgSO
4
, 0.33 Ca(NO
3
)
2
, 0.41 CaCl
2
,
20 HEPES, titrated to pH 7.5 using NaOH. ND96 bath solution
contained (in mM) 96 NaCl, 3 KCl, 1 MgCl
2
, 0.3 CaCl
2
, titrated
to pH 7.5, 6.0, 5.0, or 4.0 using NaOH or HCl. HEPES (5 mM)
was used as the buffer for pH 7.5 and pH 6.0 solutions, while
5 mM MES was used to buffer pH 5.0 and pH 4.0 solutions. Tetra-
methylrhodamine-5-maleimide (TMRM; Invitrogen) labeling of
oocytes was performed in a depolarizing solution consisting of (in
mM) 99 KCl, 1 MgCl
2
, 2 CaCl
2
, and 5 HEPES, titrated to pH 7.5
using KOH and which contained 50 M TMRM. To record Shaker
channel gating currents from tsa201 cells, the pipette contained
(in mM) 140 NMG, 1 MgCl
2
, 10 EGTA, 10 HEPES, titrated to pH
7.5 using HCl, and the bath solution contained (in mM) 140
NMG, 1 MgCl
2
, 10 HEPES, titrated to pH 7.5 or pH 4.0 using HCl.
To record Shaker single channel activity from ltk cells, the pi-
pette contained (in mM) 130 KCl, 10 EGTA, 1.38 MgCl
2
(free
concentration, 1 mM), 4.75 CaCl
2
(free concentration, 50 nM),
10 HEPES, titrated to pH 7.4 using KOH, and the bath solution
contained (in mM) 3.5 KCl, 140 NaCl, 2 CaCl
2
, 1 MgCl
2
, 10 HEPES
or MES, titrated to pH 7.4 or pH 4.0 using NaOH or HCl. Unless
stated, all chemicals were purchased from Sigma-Aldrich.
Molecular Biology and RNA Preparation
The N-terminal deletion mutant Shaker 6–46 (Shaker-IR) that is
fast inactivation removed (Hoshi et al., 1991) was expressed in
Xenopus oocytes using a modi ed pBluescript SKII expression vec-
tor (pEXO) (a gift from A. Sivaprasadarao, University of Leeds,
Leeds, UK) and in ltk and tsa201 mammalian cells using the
GW1 vector (a gift from G. Panyi, University of Debrecen, Debre-
cen, Hungary). For oocyte  uorescence experiments, cysteine res-
idues were introduced at speci c sites (M356-R362) in the S3–S4
linker and S4 voltage sensor, and S424 in the S5-P turret for TMRM
labeling and the only externally accessible cysteine residue, found
in the S1–S2 linker, was replaced with a valine residue (C245V) to
prevent nonspeci c dye labeling. For clarity, we use the terms
Shaker A359C and Shaker S424C to describe the Shaker 6–46
C245V A359C and Shaker 6–46 C245V S424C mutant channels,
respectively. The T449V mutation in Shaker was used to inhibit
slow inactivation (Lopez-Barneo et al., 1993) and the W434F mu-
tation was used to permanently P-type inactivate channels (Perozo
et al., 1993; Yang et al., 1997). The ILT mutant channel was a gift
from E. Isacoff (University of California, Berkeley, CA) and was
used to isolate the independent voltage sensor transitions from
the concerted opening transition (Smith-Maxwell et al., 1998a,b).
Point mutations were generated using the Stratagene Quikchange
kit (Stratagene). All primers were synthesized by Sigma Genosys.
All constructs were sequenced at the University of British Colum-
bia core facility. cDNA was linearized using BstEII or HindIII (for
ILT channels) and cRNA was synthesized using the mMessage
mMachine T7 Ultra cRNA transcription kit (Ambion).
Oocyte Preparation and Injection
Xenopus laevis oocytes were prepared and isolated as described
previously (Claydon et al., 2000). In brief, gravid frogs were termi-
nally anesthetized, and stage V–VI oocytes were isolated and de-
folliculated using a combination of collagenase treatment (1 h in
1 mg/ml collagenase type 1; Sigma-Aldrich) and manual defolli-
culation. Oocytes were injected with 50 nl (5–10 ng) cRNA using
a Drummond digital microdispenser (Fisher Scienti c) and then
incubated in Barth’s medium at 19C. Currents were recorded
1–7 d after injection.
Voltage-Clamp Fluorimetry
Voltage-clamp  uorimetry was performed as described previously
(Claydon et al., 2006). In brief, introduced cysteine residues were
labeled with 50 M TMRM in oocyte depolarizing solution. Fluo-
rimetry was performed using a Nikon TE300 inverted microscope
with epi uorescence attachment and a 9124b Electron Tubes
Page 2
Claydon et al. 439
photomultiplier tube (PMT) module (Cairn Research). TMRM
was excited by light  ltered with a 525-nm band pass excitation
lter and the  uorescence emission was collected using a 20 ob-
jective and  ltered through a 565-nm long pass emission  lter be-
fore being passed to the PMT recording module. Voltage signals
from the PMT were then digitized using an Axon Digidata 1322
A/D converter and passed to a computer running pClamp9 soft-
ware (Axon Instruments) to record the  uorescence emission in-
tensity. To minimize  uorophore bleaching, a computer-controlled
shutter (Uniblitz; Vincent Associates) was opened shortly before
voltage-clamp pulses were applied. Fluorescence signals were  l-
tered at 1 kHz with a sampling frequency of 20 kHz (when the
pulse duration was 100 ms), 10 kHz (when the pulse duration was
7 s), or 2 kHz (when the pulse duration was 42 s). Traces were not
averaged, except for those recorded during 100-ms pulses, which
represent the average of  ve sweeps. To account for the bleaching
of the  uorescence signal during 7- or 42-s pulses,  uorescence
recorded for 7 or 42 s at the holding potential of 80 mV was
subtracted. Simultaneous voltage clamp of the oocyte and acquisi-
tion of the current and voltage signals was achieved using the
two-microelectrode voltage-clamp technique with a Warner In-
struments OC-725C ampli er, Axon Digidata 1322, and pClamp9
software. Microelectrodes were  lled with 3 M KCl and had a re-
sistance of 0.2–0.5 M.
Unlabeled wild-type Shaker channels inactivate with a time
constant of 5.7 0.7 s, and unlabeled Shaker A359C and S424C
mutant channels inactivate with time constants of 5.6 0.5 and
1.5 0.2 s, respectively. This suggests that the introduction of a
cysteine residue at S424, but not A359, speeds inactivation signif-
icantly. Despite this change, we believe that both the inactivation
process and the nature of the  uorescence changes are qualita-
tively similar in their fast and slow components. First, we have
shown that low pH accelerates inactivation of wild-type, A359C,
and S424C channels similarly (9.4-, 6.9-, and 5.7-fold, respec-
tively; the time constants of inactivation were reduced to 0.61
0.11 s, 0.81 0.18 s, and 0.25 0.05 s, respectively). As well,
other interventions that are known to modify inactivation such
as raised external K
, the mutation C462A in S6, and also low pH
(Loots and Isacoff, 1998) result in consistent changes in ionic
current and  uorescence measurements. This suggests that the
underlying processes being examined are not seriously per-
turbed in these two cysteine-substituted channels. TMRM-labeled
Shaker A359C and S424C channels inactivate with time constants
of 2.7 0.2 and 2.0 0.3 s, respectively. Loots and Isacoff (1998)
reported an inactivation time constant for TMRM-labeled S424C
of 2.5 s, but a value for inactivation of ionic current in A359C
mutant channels has not been reported previously. Our data
suggest that TMRM labeling of A359C, but not S424C, speeds in-
activation twofold (Table I). However, as suggested above, inacti-
vation in TMRM-labeled A359C channels remains modi able by
low pH (the time constant of inactivation is reduced 4.6-fold to
0.58 0.08 s at pH 4.0) and the overall effect is relatively un-
changed. This also holds true for the pH sensitivity of TMRM-
labeled S424C channels, which was somewhat greater than in
unlabeled channels.
Single Channel and Gating Current Recordings
Single channel and gating currents were recorded using an Axo-
patch 200A ampli er (Axon Instruments) with computer-driven
protocols (pClamp9 software and Digidata 1200B interface; Axon
Instruments) or with an EPC-7 patch-clamp ampli er and Pulse
PulseFit software (HEKA Electronik). Single channel recordings
were obtained from outside-out patches excised from ltk cells
expressing Shaker 6–46 channels. Currents were recorded dur-
ing 500-ms voltage-clamp pulses to 100 mV from a holding po-
tential of 80 mV (the pulse interval was 15 s). Data were sampled
at 10 kHz and  ltered at 1 kHz. Thin-walled borosilicate micro-
electrodes were coated with Sylgard (Dow Corning) and typically
had a resistance of 8–12 M. Gating current records were ob-
tained in the whole-cell patch clamp con guration from tsa201
cells expressing Shaker 6–46 channels. Currents were recorded
during 20-ms voltage-clamp pulses from 80 to 80 mV in 10-mV
increments from a holding potential of 80 mV. Leak subtraction
was performed using a P/4 protocol from a holding potential of
80 mV. Data were sampled at 100 kHz and  ltered at 10 kHz.
Microelectrodes had a resistance of 1.5–2.0 M. Experiments
were performed at 20–25C.
Data Analysis
G-V curves were derived using the normalized chord conduc-
tance, which was calculated by dividing the maximum current
during a depolarizing step by the driving force derived from the
K
equilibrium potential (the internal K
concentration was as-
sumed to be 99 mM). G-V, F-V, and Q-V curves were  tted with a
single Boltzmann function:
=+ -
1/2
1/(1 exp(( )/ )),yVVk
(1)
where y is the conductance normalized with respect to the maxi-
mal conductance, V
1/2
is the half-activation potential, V is the test
voltage, and k is the slope factor. Data throughout the text and
gures are shown as mean SEM.
TABLE I
Time Constants of Ionic Inactivation and Fluorescence Decay
TMRM TMRM
(s) at pH 7.5 (s) at pH 4.0 (s) at pH 7.5 (s) at pH 4.0
Ionic current
Wild-type 5.7 0.7 (8) 0.61 0.11 (8) 3.9 0.5 (4) 0.49 0.07 (3)
A359C 5.6 0.5 (10) 0.81 0.18 (9) *2.7 0.2 (14) 0.58 0.08 (11)
S424C *1.5 0.2 (7) 0.25 0.05 (7) *2.0 0.3 (9) 0.26 0.03 (5) (pH 5.0)
Fluorescence decay
A359C 2.4 0.3 (14) 1.2 0.1 (14)
S424C
1
2.6 0.8 (9)
1
0.33 0.08 (5) (pH 5.0)
2
43.7 22.2 (9)
2
61.2 52.4 (5) (pH 5.0)
Introduction of a cysteine residue at A359 had no effect on the inactivation of ionic current, but TMRM labeling of A359C accelerated inactivation to a
certain degree. Introduction of a cysteine residue at S424C accelerated the inactivation of ionic current, but TMRM labeling had no additional effect.
During prolonged depolarizing pulses (42 s), S424C  uorescence decay was best described by a biexponential function, which represents P- and C-type
inactivation processes. Numbers in parentheses represent the number of oocytes measured. * represents inactivation time constants that were signi cantly
different (P < 0.05) from the time constant of wild-type channel ionic current inactivation at pH 7.5 in the absence of TMRM (TMRM, pH 7.5).
Page 3
440 pH and Closed-State Inactivation
RESULTS
Acidic pH Reduces the Conductance of Shaker
6–46 Channels
Shaker 6–46 currents are inhibited by extracellular
protons (pK
a
4.7–5.0) and this has been attributed to
enhanced P/C-type open-channel inactivation at low
pH (Perez-Cornejo, 1999; Starkus et al., 2003) that ac-
cumulates during repetitive stimulation (Starkus et al.,
2003). However, we found that acidic extracellular pH
reduced current amplitude even when the acceleration
of inactivation during the depolarization was minimal,
suggesting that protons also reduce the macroscopic
conductance. This is shown in Fig. 1 A in the compari-
son of conductance–voltage relations at different exter-
nal pH, which were constructed from currents recorded
in response to short voltage-clamp pulses (100 ms) and
with long interpulse intervals (10 s) to avoid cumulative
inactivation. In addition to a rightward shift of the volt-
age dependence of channel opening that is attributed
to a surface charge-screening effect (Deutsch et al., 1989;
Hille, 2001; Kehl et al., 2002; Trapani and Korn, 2003),
the maximal conductance was reduced at pH 5.0 by
21 7% and at pH 4.0 by 47 5% (n 3).
Acidic pH Alters the Fluorescence Report from Shaker
A359C Channels
Using voltage-clamp  uorimetry, it is possible to track
the protein conformational rearrangements that are as-
sociated with ion channel gating (Mannuzzu et al., 1996;
Cha and Bezanilla, 1997). Of particular importance to
this study, this technique allows observation of electri-
cally silent rearrangements that do not result in channel
opening. We therefore used voltage-clamp  uorimetry
to understand the fate of Shaker channels at low pH.
Data in Fig. 1 (B–E) show ionic current traces and
uorescence signals recorded from Shaker A359C chan-
nels with an extracellular pH of either 7.5 or 4.0. TMRM
Figure 1. Low pH reduces the
conductance of Shaker chan-
nels. (A) G-V relations of Shaker
6–46 channels recorded with
the indicated external pH
(n 3). Conductance was cal-
culated from currents recorded
during 100-ms voltage pulses
applied from 80 to 100 mV
at 10-s intervals in 10-mV in-
crements (holding potential
80 mV). (B–E) Typical ionic
currents (B and C) and  uo-
rescence signals (D and E) re-
corded from Shaker A359C
channels in response to 100-ms
voltage pulses applied from
80 to 100 mV at 10-s in-
tervals (holding potential
80 mV) at pH 7.5 and pH 4.0.
Although pulses were applied
in 10-mV increments, only
selected pulses are shown for
clarity. Current and  uores-
cence traces at pH 4.0 (C and
E) are shown at more depolar-
ized potentials than in B and
D to account for the charge
screening effect of protons.
Contributions of the fast and
slow phases to the overall  uo-
rescence signal were measured
from biexponential  ts of the
uorescence traces at 60 mV
for pH 7.5 and at 100 mV for
pH 4.0. Arrows and percent-
ages in D and E show the con-
tribution of the slow phase at
pH 7.5 and pH 4.0. The mean
slow phase contribution was 14
2% at pH 7.5 and 71 3%
at pH 4.0 (n 8).
Page 4
Claydon et al. 441
labeling of A359C has previously been shown to faith-
fully report local environmental changes associated
with voltage sensor movement in response to mem-
brane depolarization (Mannuzzu et al., 1996; Cha and
Bezanilla, 1997). At pH 7.5, there was no obvious channel
opening at the holding potential of 80 mV in Fig. 1 B
and no voltage sensor movement in Fig. 1 D. On depolar-
ization to 60 mV, despite the low open probability, there
was a signi cant uorescence report consistent with volt-
age sensor movement (Fig. 1 D). Further depolarization
to 60 mV induced robust ionic currents that activated
rapidly and showed only minimal decay (Fig. 1 B). This
was re ected in the  uorescence signals (Fig. 1 D),
which show a large  uorescence de ection from Shaker
A359C channels with a fast phase of  uorescence quench-
ing that accounted for 86 2% (n 8) of the total
uorescence de ection and occurred with a time course
similar to that for channel activation. Biexponential  ts
to the  uorescence relaxation showed that this rapid
component was followed by a slow phase that accounted
for 14 2% of the de ection and was associated with
channel inactivation (Claydon et al., 2006).
At pH 4.0, peak ionic current was reduced by 57
4% (Fig. 1 C; n 21) even when charge screening was
accounted for by the application of a depolarizing pulse
to 100 mV, at which open probability was maximal
(Fig. 1 A). However, the corresponding  uorescence
signals in Fig. 1 E show that the  uorophore continues
to report the same extent of environmental change (al-
though with altered kinetics; see below), which suggests
that although a proportion of the channels does not
conduct at acidic pH, voltage sensor movement is pre-
served. The mean peak amplitude of the  uorescence
deflection at pH 4.0 was 116 11% of that at pH 7.5
(n 7; t test, not signi cantly different).
Another measure of voltage sensor movement can
be obtained from gating current measurements. We re-
corded gating currents from wild-type Shaker channels at
pH 7.5 and pH 4.0 (Fig. 2) and a typical family of rec-
ords obtained at pH 7.5 during 20-ms voltage-clamp
pulses ranging from 80 to 60 mV (the holding po-
tential was 80 mV) is shown in Fig. 2 A (current during
the  rst 6 ms of the pulse is shown to highlight on-gating
events). In Fig. 2 B, the integrals of the on-gating cur-
rents recorded at pH 7.5 and pH 4.0 in the same cell are
plotted. These show that acidic pH shifted the voltage
dependence of charge movement to more depolarized
potentials, but did not signi cantly alter the total gating
charge movement. This is shown more clearly by the
mean Q-V relations in Fig. 2 C. Acidic pH shifted the V
1/2
of the Q-V relation by 35 mV from 27.9 0.4 mV
(k was 9.5 0.4 mV) at pH 7.5 to 5.9 1.2 mV (k was
12.4 1.0 mV) at pH 4.0, without altering the total gat-
ing charge, which suggests that the number of activat-
able channels (i.e., the number of channels in which the
voltage sensors are available to move) is not changed.
It is clear from the  uorescence signals recorded in
Fig. 1 E that acidic pH did alter the kinetics of the voltage
sensor report. The contribution of the fast phase of the
uorescence signal was reduced to 29 3% of the total
signal, because the slow phase was dramatically increased
to account for 71 3% of the signal (n 8; P < 0.001,
Figure 2. Acidic pH does not alter total gating charge movement
in Shaker channels. (A) Typical wild-type Shaker 6–46 (Shaker
A359) gating current records obtained from tsa201 cells in the
whole-cell patch clamp con guration during 20-ms voltage-clamp
pulses from 80 to 60 mV (in 10-mV increments) from a hold-
ing potential of 80 mV. Currents during every other pulse are
shown for clarity and only the  rst 6 ms of the depolarizing pulses
are shown to highlight the on-gating current. (B) Typical inte-
grals of on-gating currents, such as those in A, recorded in the
same cell under control conditions (pH 7.5) and at pH 4.0. Acidic
pH did not alter the total gating charge movement in these chan-
nels. (C) Mean Q-V relationships recorded at pH 7.5 and pH 4.0
(n 2–4). At pH 4.0, the Q-V relation was shifted by 35 mV
from 27.9 0.4 mV at pH 7.5 to 5.9 1.2 mV.
Page 5
442 pH and Closed-State Inactivation
paired t test, when compared with pH 7.5). Since it has
previously been shown that a  uorophore labeling A359C
in the S3–S4 linker reports on conformational changes
associated with inactivation as well as those associated
with voltage sensor movement (Loots and Isacoff, 1998;
Claydon et al., 2006), we hypothesized that the enhance-
ment of the slow phase of the  uorescence de ection
represented the accelerated onset of inactivation previ-
ously reported to occur in Shaker channels at low pH
(Perez-Cornejo, 1999; Starkus et al., 2003). To test this,
we investigated the effect of low pH on the  uorescence
report from Shaker A359C channels during long depolar-
izing pulses that induced much more inactivation.
Acidic pH Accelerates the Inactivation Report from Shaker
A359C Channels
The effect of acidic pH on Shaker A359C channel inacti-
vation during 7-s depolarizations to 60 mV is shown in
Fig. 3 (see also Table I). The  uorescence signals in Fig.
3 B show a clear slow phase that corresponds to the inac-
tivation of ionic current in panel A. At pH 7.5, ionic cur-
rent decayed with a
of 2.7 0.2 s (Fig. 3 A; n 14) and
the slow phase of  uorescence decayed with a similar
of 2.4 0.3 s, and contributed 53 4% to the total sig-
nal amplitude (Fig. 3 B; n 14). Reducing the extracel-
lular pH to 5.0 had only a minor effect on ionic current
and the  uorescence signal from Shaker A359C channels
(Figs. 3, A and B). However, pH 4.0 both reduced the
peak current amplitude and enhanced the decay of the
remaining current (Fig. 3 A;
was 579 84 ms, n 11).
The  uorescence signals in Fig. 3 B, which were recorded
simultaneously, show that at pH 4.0 both the contribu-
tion and the decay of the slow de ection were enhanced;
the contribution of the slow phase was increased to 76
3% and the
of the slow decay was reduced to 1.2 0.1 s
(n 14). These data con rm the previous reports that
low pH enhances inactivation of Shaker channels (Perez-
Cornejo, 1999; Starkus et al., 2003) and suggest that
acidic pH enhances the conformational rearrangements
associated with inactivation, which are reported by a  uoro-
phore at the outer end of the voltage sensor.
The  uorescence signal recorded from Shaker A359C
channels returns to baseline upon repolarization and
also shows a fast and a slow phase, which represent, re-
spectively, the return of the voltage sensor to its resting
conformation during deactivation and the recovery from
inactivation (Loots and Isacoff, 1998; Claydon et al.,
2006). Both phases were altered by acidic pH (Fig. 3 B);
the
of the fast phase was reduced from 63 16 ms at
pH 7.5 to 21 1 ms at pH 4.0 (n 17; P < 0.05, paired
t test), which is consistent with the previous observation
that acidic pH accelerates deactivation (Starkus et al.,
2003) likely as a result of charge screening, and the of
the slow phase was reduced from 699 58 ms at pH 7.5
to 202 20 ms at pH 4.0 (n 17; P < 0.001, paired t
test). Since the slow phase of Shaker A359C  uorescence
return upon repolarization re ects immobilization of
the voltage sensor associated with inactivation, it is possi-
ble that further slow changes in recovery occur that are
beyond the recording period of the experiment.
Figure 3. Acidic pH enhances the inactivation report from Shaker
A359C. Typical ionic currents (A) and  uorescence signals (B)
recorded from Shaker A359C channels during 7-s voltage-clamp
pulses to 60 mV at the indicated external pH (holding potential
of 80 mV). (C) Typical ionic currents and  uorescence signals
recorded at pH 7.5 and pH 4.0 from TMRM-labeled channels in
which a cysteine residue was not engineered at position A359
(Shaker A359). These channels lack an externally labelable cyste-
ine residue (the only external cysteine, in the S1–S2 linker [C245],
has been removed) and act as a control demonstrating the lack of
a voltage-dependent  uorescence de ection in the absence of an
introduced cysteine and also the pH independence of emission of
the TMRM dye.
Page 6
Claydon et al. 443
To demonstrate the speci city of the response of the
uorophore attached at position A359 to changes in ex-
ternal pH, we compared the effect of changing the pH on
TMRM-labeled Shaker channels lacking an externally labe-
lable cysteine residue (Shaker A359; Fig. 3 C and Table I).
The decay of ionic current was accelerated from 3.9
0.5 s at pH 7.5 to 0.49 0.07 s at pH 4.0 (n 3–4; Table I;
these values are not signi cantly different from wild
type in the absence of TMRM). The  uorescence sig-
nals from Shaker A359 demonstrate that there are no
voltage-dependent conformational changes reported
by TMRM that labels nonspeci c sites in endogenous
membrane proteins. Furthermore, the  uorescence sig-
nal is not altered by changes of the extracellular pH.
These results emphasize the ability of site-speci c attach-
ment of TMRM to detect gating-induced rearrangements
of channel conformation, and in addition, con rm that
the emission from TMRM is pH insensitive (Cha and
Bezanilla, 1998; see also Molecular Probes website at
http://probes.invitrogen.com).
Detection of the pH-induced Acceleration of Inactivation
by the Voltage Sensor Is Site Specifi c
To understand which structural domains are involved in
the pH-induced changes in the  uorescence signal from
TMRM attached at A359C and the local rearrangements
that they represent, we investigated the effect of acidic
pH on the  uorescence signal from TMRM attached at
each position from M356C in the S3–S4 linker to the
outermost arginine, R362C, in the S4 domain. Fluores-
cence signals recorded during 7-s depolarizing pulses to
60 mV from each of these mutant channels at either
pH 7.5 or pH 4.0 extracellular solution are shown in
Fig. 4. Each site reported robust  uorescence signals
on depolarization with the exception of I360C and
L361C, from which only baseline signals could be de-
tected (unpublished data). This lack of signal was unex-
pected given the  uorescence report from these residues
described previously (Loots and Isacoff, 2000). However,
we were unable to detect voltage-dependent signals in
any of 17 oocytes, which displayed large ionic currents.
Close examination of the effect of acidic pH on the
uorescence signal from each mutant channel reveals
that only A359C and R362C reported pH-dependent
changes. With TMRM attached at R362C, the
of the
slow phase was reduced from 2.6 0.4 s at pH 7.5 to 1.4
0.2 s at pH 4.0 (n 3; P < 0.05, paired t test). Since
A359C and R362C are likely positioned on the same
side of the S4 helix, this suggests that this face senses
the rearrangements associated with the pH modulation
of inactivation.
Fluorescence Signals from the Pore Report Two Effects
of Acidic pH
Although the  uorescence records from Shaker A359C
report the rearrangements underlying the acceleration
of inactivation at low pH, they do not provide much
insight into the basis for the decline of peak current
amplitude following extracellular acidi cation. It has
previously been reported that a TMRM  uorophore at-
tached at S424C within the outer pore is a very good re-
porter of the rearrangements of the Shaker channel
pore that are associated with inactivation (Loots and
Isacoff, 1998). Ionic currents and  uorescence signals
recorded from Shaker S424C channels in response to 42-s
depolarizing pulses to 60 mV at pH 7.5, pH 6.0, and
pH 5.0 are shown in Fig. 5. Ionic current at pH 7.5 inac-
tivated with a time constant of 2.0 0.3 s (n 9; Table I).
As in Shaker A359C, acidic pH reduced the peak current
amplitude and enhanced the decay of the remaining
current in Shaker S424C channels (Fig. 5 A and Table I)
although this mutant channel was more sensitive to
acidic pH than Shaker A359C as current amplitude was
signi cantly reduced at pH 5.0 (see Fig. 7 A) and ionic
current decayed more quickly with a time constant of
264 32 ms (n 7; Table I). The  uorescence traces in
Figure 4. Voltage sensor detection of pH-induced enhancement of inactivation is site speci c. Typical  uorescence signals from TMRM
attached at different sites in the S4 voltage sensor and S3–S4 linker from M356C to R362C recorded during a 7-s pulse to 60 mV at pH
7.5 and pH 4.0 (holding potential 80 mV). Robust  uorescence de ections were recorded from each site in the scan with the excep-
tion of I360C and L361C, from which we were unable to record voltage-dependent  uorescence signals. Only A359C and R362C report
the pH-induced enhancement of inactivation.
Page 7
444 pH and Closed-State Inactivation
Fig. 5 B show that TMRM attached at S424C reports the pH-
induced inactivation gating modi cation well. At pH 7.5,
the  uorescence signal from Shaker S424C shows fast
and slow phases on depolarization as reported previ-
ously (Loots and Isacoff, 1998; Claydon et al., 2006). The
fast phase, re ecting movement of the voltage sensor,
was small and contributed only 20 5% of the total
signal, whereas the slow phase, representing the confor-
mational rearrangements of the pore during inactiva-
tion, contributed 80 5%. The slow phase was best  t
with a biexponential function with time constants of 2.6
0.8 and 43.7 22.2 s (n 9). The time constant of the
faster component is not signi cantly different from that
of the ionic current decay during P-type inactivation
(2.0 0.3 s), while the slower component most likely
re ects the slower C-type inactivation process that oc-
curs after ionic current has decayed to baseline. A sim-
ilar report of C-type inactivation during prolonged
depolarization was previously described from the very
slow component of  uorescence de ection recorded
from A359C (Loots and Isacoff, 1998).
Unlike the report from Shaker A359C, the  uorescence
signals from Shaker S424C at acidic pH demonstrate two
effects of protons on channel gating. The  rst is an ac-
celeration of the slow phase of  uorescence that is con-
sistent with the faster inactivation of ionic current (Fig.
5, A and B). The acceleration of the slow  uorescence
report is shown more clearly in Fig. 5 C, which plots the
same  uorescence de ections as those in panel B nor-
malized to their peak value. The
of the faster compo-
nent of the slow phase was reduced (n 5; P < 0.05,
paired t test) from 2.6 0.8 s at pH 7.5 to 331 79 ms
at pH 5.0 (Table I). The second effect of acidic pH was a
decrease of the peak amplitude of the  uorescence sig-
nal (Fig. 5 B). Following a transient increase in the  uo-
rescence signal that lasts 10 s at pH 6.0 (that is due to
the acceleration of inactivation upon depolarization and
has been described previously, Loots and Isacoff, 1998,
for depolarizations of approximately this duration), the
uorescence signal from Shaker S424C was reduced by
53 6% at pH 5.0 (n 5) and this was completely re-
stored after returning to pH 7.5 (see Fig. 10 C).
To understand why the macroscopic conductance and
the  uorescence report from TMRM at S424C was re-
duced with acidic pH (Fig. 5), while the report from
A359C was not (Figs. 1 and 3), we measured Shaker-IR
single channel currents at pH 7.4 and pH 4.0 (Fig. 6),
since the reduced report from S424C may re ect an ef-
fect of protons on channel open probability or the sin-
gle channel current amplitude. Channel activity was
assessed during 500-ms voltage-clamp pulses to 100 mV
from outside-out patches (the pulse interval was 15 s).
At pH 7.4, the single channel current amplitude (at
100 mV) was 2.7 0.3 pA and the representative
traces show high channel availability. We calculated the
availability by determining the proportion of sweeps
showing channel activity during the  rst 50 ms of the
pulse, and the average availability from 11 patches was
0.87 at pH 7.4. Application of pH 4.0 to the same patch
(Fig. 6) markedly reduced availability, resulting in a
number of null sweeps (the average availability from  ve
patches was 0.14), but did not alter the single channel
current amplitude (2.7 0.3 pA). These data suggest
Figure 5. Two effects of acidic pH are detected from TMRM at-
tached within the pore. Typical ionic currents (A) and  uores-
cence signals (B) recorded from Shaker S424C channels during
42-s voltage-clamp pulses to 60 mV at the indicated external pH
(holding potential 80 mV). Acidic pH decreased peak and ac-
celerated inactivation of the ionic current, and this was detected
in the  uorescence signals as a decrease in the peak  uorescence
amplitude and an enhancement of the slow phase of the remain-
ing  uorescence de ection. (C) Shaker S424C channel  uores-
cence de ections from B scaled to the maximum signal amplitude
to highlight the pH-induced enhancement of the slow phase of
uorescence de ection.
Page 8
Claydon et al. 445
that the loss of  uorescence observed in Fig. 5 is not
due to reduced single channel conductance, but rather
reduced channel availability.
Further analysis of the  uorescence data from Shaker
S424C allowed us to measure the extent of the loss of
the  uorescence signal at low pH and show that it was
quantitatively correlated to the reduction of peak cur-
rent amplitude (Fig. 7). Fig. 7 A shows conductance–
voltage relationships constructed from Shaker S424C
currents recorded during 100-ms voltage pulses from
80 to 100 mV at either pH 7.5 or pH 5.0 (n 3). The
voltage dependence of the conductance decrease (Fig.
7 A) was compared with the voltage dependence of the
pH-induced loss of  uorescence during 42-s voltage
pulses (Fig. 7 B) from the same group of oocytes as those
in panel A. Long pulses were required to make this com-
parison, because it was necessary to measure the effect
of low pH on the  uorescence amplitude once depolar-
ization-induced inactivation had proceeded to near
steady state. In this way the reduction in conductance
could be directly compared with the  uorescence report
of the proportion of channels that were no longer avail-
able to make the transition to the inactivated state.
It is clear that the extent of the loss of  uorescence at
pH 5.0 (Fig. 7 B) matches the decrease in conductance
(Fig. 7 A) at all potentials. At 60 mV there was a
53 6% loss of the  uorescence signal, which was not
signi cantly different from the 51 3% reduction in
conductance (n 3; t test). In Fig. 7 C, the relative  uo-
rescence amplitude at 60 mV is plotted alongside the
relative conductance at 60 mV with different external
pH. The loss of  uorescence and reduction of conduc-
tance showed the same pH dependency with pKa values
of 5.1 and 5.2, respectively.
The data in Figs. 5–7 demonstrate that acidic pH has
two effects: an acceleration of the slow phase of  uores-
cence from Shaker S424C that represents the enhance-
ment of inactivation upon depolarization, and a loss of
the  uorescence report from S424C that, in the light of
the observed reduction in single channel open proba-
bility, suggests a reduction of channel availability and
consequently the peak current amplitude. The pH-
induced loss of  uorescence signal is of particular inter-
est because it contrasts with the lack of effect of acidic
pH on the  uorescence amplitude recorded from Shaker
A359C channels (Fig. 1 F and Fig. 3 B) and allows a
quantitative correlation to be made between the  uo-
rescence loss and conductance decrease at low pH.
Acidic pH Enhances Rearrangements Associated
with P- and C-type Inactivation
P/C-type inactivation involves two sequential processes,
with a concerted closure of the pore resulting in the
collapse of conductance (P-type inactivation) followed
Figure 6. Acidic pH decreases open probabil-
ity without altering single channel current am-
plitude. Typical recordings of single channel
events obtained from outside-out patches of
ltk cells expressing Shaker 6–46 channels
during consecutive 500-ms voltage-clamp pulses
to 100 mV (pulse interval 15 s) from a hold-
ing potential of 80 mV (the voltage protocol
is shown). In this example the patch contains a
single channel. Channel activity from the same
patch was recorded during control conditions
(pH 7.4) and at pH 4.0. Dashed lines mark the
zero current level. Acidic pH markedly reduced
channel availability (calculated by determining
the proportion of sweeps showing channel ac-
tivity during the  rst 50 ms of the pulse) from
0.87 at pH 7.4 to 0.14 at pH 4.0 without effect
on the single channel current amplitude. At
100 mV, the single channel current ampli-
tude was 2.7 0.3 pA at pH 7.4 and 2.7 0.3
pA at pH 4.0 (data were collected from 11
patches at pH 7.4 and 5 patches at pH 4.0). The
84% reduction in channel availability (from
0.87 to 0.14) was greater than the reduction
of macroscopic conductance measured from
Shaker channels expressed in Xenopus oocytes
(Fig. 1), but similar to the decrease in peak
macroscopic current measured from Shaker
channels expressed in mammalian cells at pH
4.0 (91 8%; n 5). This difference between
expression systems is most likely due to incom-
plete exchange of solution over the entirety of
the large invaginated oocyte membrane.
Page 9
446 pH and Closed-State Inactivation
by a stabilization of the voltage sensor in its activated
conformation (C-type inactivation), which reports as
an immobilization of the return of gating charge on re-
polarization (De Biasi et al., 1993; Olcese et al., 1997;
Yang et al., 1997; Loots and Isacoff, 1998). Channels
with the mutation W434F are thought to be perma-
nently P-type inactivated (Perozo et al., 1993; Yang et al.,
1997), and the  uorescence signal from TMRM attached
at S424C in these channels reports well on activation, as
if P-type inactivation moves the  uorophore into closer
proximity with the voltage sensor and allows it to track
the transition of channels to a W434F-like inactivated
state (Loots and Isacoff, 1998). This idea is consistent
with the close proximity of the S5-P linker with S4 (of
an adjacent subunit) in the Kv1.2 channel crystal struc-
ture (Long et al., 2005). Since, in the present study,
acidic pH accelerates the slow  uorescence report from
S424C, this suggests that protons enhance P-type inacti-
vation and we therefore used a protocol designed to
monitor the effect of pH on the ability of TMRM at-
tached at S424C to detect voltage sensor movement.
Fig. 8 A shows ionic current and  uorescence signals
recorded from Shaker S424C TMRM-labeled channels
during a 2-s test pulse to 60 mV applied either 200 ms,
12 s, or 42 s after a 5-s conditioning pulse to 60 mV.
When the interval between pulses was short (200 ms),
only a small percentage of channels recovered from in-
activation and so the  uorescence signal during the
test pulse reports mostly from inactivated channels. In
this case, the amplitude of the fast phase of the  uores-
cence signal was increased from 26 3% in the condi-
tioning pulse to 60 3% during the test pulse (n 6;
P < 0.01, paired t test), suggesting that many channels
became P-type inactivated during the conditioning
pulse as the  uorophore now reports activation more
clearly. With a 42-s interval that allowed for more com-
plete recovery from inactivation, the  uorescence sig-
nal during the test pulse recovered its slow phase and
the fast phase diminished back to 17 7%. Fig. 8 B
shows the effect of acidic pH on the progression of
channels to the P-type inactivated state. The double
pulse protocol shows that the fast phase dominated
during the test pulse at pH 5.0, suggesting that the ma-
jority of the channels sense activation well and are
therefore P-type inactivated. The contribution of the
fast phase in the test pulse after a 200-ms interval at pH
5.0 was 82 4%, which is signi cantly greater (n 5;
P < 0.01, paired t test) than the 60 3% seen at pH 7.5
(Fig. 8 A). These data demonstrate that the enhance-
ment of current decay at acidic pH is a result of en-
hanced P-type inactivation.
We next asked whether the progression of channels
to the C-type inactivated state could also be altered by
changes of pH. To test this, we studied the  uorescence
report from TMRM attached at S424C in the presence
of the W434F mutation, which permanently P-type
inactivates channels (Yang et al., 1997) and allows the
rearrangements coupled to C-type inactivation to be ob-
served in isolation. The  uorescence signal shown in
Fig. 9 A was recorded during a 7-s pulse to 60 mV at
pH 7.5. All signals recorded from this mutant channel
were relatively small because the report of the transition
to the P-type inactivated state that was evident in Fig.
5 B is absent since W434F mutant channels are P-type
Figure 7. The loss of  uorescence at low pH is
similar to the reduction of peak current ampli-
tude. (A) G-V relations of Shaker S424C channels
recorded with the indicated external pH (n 3).
Using Eq. 1 (the intracellular K
concentration
was assumed to be 99 mM), conductance was cal-
culated from currents recorded during 100-ms
voltage pulses applied from 80 to 100 mV at
10-s intervals in 10-mV increments (holding po-
tential 80 mV). (B) F-V relations of the Shaker
S424C channels recorded at the indicated pH
during 42-s pulses from the same group of oo-
cytes as A (n 3). All  uorescence amplitudes
were normalized to the  uorescence amplitude at
100 mV with pH 7.5. Lines represent no mathe-
matical signi cance and are simply to guide the
eye. The loss of  uorescence at low pH was simi-
lar to the decrease in con ductance at all poten-
tials. (C) Plot of the dependence on the external
pH of Shaker S424C channel  uorescence am-
plitude at 60 mV or conductance at 60 mV
(n 2–7). Fluorescence and conductance values
were normalized to those at pH 7.5. Conductance
values were calculated from peak currents re-
corded at the same time as  uorescence de ections during 42-s pulses so as to directly compare the decrease in conductance with the
loss of  uorescence from simultaneous measurements from the same oocyte. Data were  tted with a standard Hill equation (assuming
a Hill coef cient, n, of 1). pKa values were 5.1 and 5.2 for the loss of  uorescence and reduction of conductance, respectively.
Page 10
Claydon et al. 447
inactivated at rest. There was a prominent fast phase
because the P-type inactivated channels reported well
on the fast transitions associated with channel activa-
tion. Examination of the  uorescence trace in Fig. 9 A
shows an additional decaying component of  uores-
cence, which suggests that a transient reorganization
of the pore, sensed by TMRM at S424C, occurs in P-type
inactivated channels on depolarization. The amplitude
of the decaying component of  uorescence was in-
creased 2.1 0.4-fold at pH 5.0 (n 3; P < 0.001,
paired t test, when compared with pH 7.5). To con rm
that this signal represents the progression of channels
from the permanent P-type inactivated state to the C-
type inactivated state, the voltage dependence of the
transition was compared (Fig. 9 B) with that of voltage
sensor movement (Shaker A359C W434F F-V) and the
open probability (Shaker A359C G-V). The C-type inac-
tivation  uorescence–voltage relationship at pH 7.5
() and pH 5.0 () had V
1/2
and slope factor, k, values
of 19.2 2.2 mV and 15.5 1.9 mV, and 21.6
2.1 mV and 19.4 1.8 mV, respectively. For compari-
son, the peak  uorescence–voltage relation (F-V) of
Shaker A359C W434F channels (), which reports the
voltage dependence of voltage sensor movement, and
the conductance–voltage relation (G-V) of Shaker
A359C channels (), which reports the voltage depen-
dence of channel opening, are also plotted in Fig. 9 B.
It is clear that the C-type inactivation transition
reported by Shaker S424C W434F lies to the right of
the Shaker A359C W434F F-V curve and more closely
follows the G-V relation. This suggests that although
the pore is P-type inactivated, the transition to C-type
inactivation is dependent on opening of the intracellu-
lar gate and not on early independent voltage sensor
movements. The data in Fig. 9 B show that this transi-
tion is enhanced by acidic pH and, taken together with
the data in Figs. 5 and 7, suggest that acidic pH stabi-
lizes both P-type inactivated and C-type inactivated
channel states.
The Loss of Fluorescence with Acidic pH Is Determined
by Channel Inactivation
The rate of Shaker channel inactivation is dependent on
the external K
concentration. Raising external K
in-
creases ion occupancy of the selectivity  lter and pre-
vents constriction of the outer pore (Lopez-Barneo
et al., 1993). We reasoned that if the mechanistic basis
for the loss of  uorescence from S424C with acidic pH
was an inactivation process, then raising external K
should prevent the changes of  uorescence caused by
changing pH. The effect of raising the external K
con-
centration on the  uorescence report from Shaker S424C
is shown in Fig. 10. In the  rst set of tracings (Fig. 10 A),
the  uorescence emission from Shaker S424C channels
was recorded during control conditions (pH 7.5, 3 mM
external K
). Application of solution with 3 mM K
at
pH 5.0 (Fig. 10 B) resulted in a loss of  uorescence as
described in Fig. 5, and this was completely reversed
on wash with control solution (Fig. 10 C). Solution
containing high (99 mM) K
was then applied to the
same cell (Fig. 10 D). Fig. 10 E shows the effect of acidic
pH on the  uorescence in the presence of high external
K
. With 99 mM K
, pH 5.0 no longer resulted in a loss of
the  uorescence signal, suggesting that the loss observed
Figure 8. Low pH enhances
P-type inactivation. Typical
ionic currents and  uores-
cence signals recorded at pH
7.5 (A) and pH 5.0 (B) from
Shaker S424C channels during
a 2-s test pulse to 60 mV ap-
plied either 200 ms, 12 s, or
42 s after a 5-s conditioning
pulse to 60 mV (holding
potential 80 mV). Similar
records were obtained from
ve other oocytes.
Page 11
448 pH and Closed-State Inactivation
with 3 mM K
is an inactivation-dependent phenome-
non. The increased external K
also slowed the decay
and largely prevented the reduction of peak ionic cur-
rent at pH 5.0; the of ionic decay was increased from
285 89 ms with 3 mM K
at pH 5.0 to 2.9 1.3 s with
99 mM K
at pH 5.0, and the peak ionic current was re-
duced by 51 12% with 3 mM K
at pH 5.0, but only by
9 15% with 99 mM K
at pH 5.0 (n 4). Responses
obtained following wash with pH 7.5 and 3 mM external
K
are shown in Fig. 10 F.
Inactivation of Shaker channels is also largely pre-
vented by the mutation T449V in the outer pore (Lopez-
Barneo et al., 1993). Fluorescence signals recorded
from Shaker S424C T449V with the bath solution ad-
justed to either pH 7.5 or pH 5.0 are shown in Fig. 11.
Neither the amplitude nor rate of decay of the  uores-
cence emission (Fig. 11) or ionic current (not depicted)
on depolarization was altered by application of acidic
pH when inactivation was inhibited by the T449V muta-
tion. These data, along with those from the experiments
presented in Fig. 10, show that the loss of the  uores-
cence signal during acidic pH can be prevented in chan-
nels when inactivation is compromised.
Acidic pH Induces Conformational Changes
in Closed Channels
The  uorescence data presented so far have demon-
strated that the environmental changes around TMRM
that are associated with both P-type and C-type inactiva-
tion are accelerated at low pH (Figs. 3, 5, and 7–9), and
the diminished report of TMRM from the pore along
with the decreased single channel open probability sug-
gests that protons cause a decrease of channel availabil-
ity upon depolarization (Figs. 5–7). Further, we have
shown that reduction of inactivation prevents this loss of
uorescence at low pH (Figs. 9 and 10). We therefore
hypothesized that the loss of  uorescence at acidic pH,
and consequently the reduction of channel availability
and peak current amplitude, was due to a stabilization of
channels in closed-inactivated states. To test this hypoth-
esis, we used the ILT mutant Shaker channel (which con-
tains three mutations in the S4 voltage sensor: V369I,
I372L, and S376T), as it isolates the early independent
voltage sensor movements from the cooperative open-
ing transition of channels (Smith-Maxwell et al., 1998a,b).
By applying an appropriate depolarizing pulse amplitude,
the effect of acidic pH can be tested on channels in
Figure 9. Low pH enhances C-type inactivation. (A) Typical  uorescence signals recorded from the same cell expressing Shaker S424C
W434F channels during 7-s depolarizing pulses to 60 mV (applied after at least 3 min at the holding potential of 80 mV) at pH 7.5,
pH 5.0, and on return to pH 7.5. Oocytes were held at 80 mV for 3 min between measurements. The W434F mutation permanently
P-type inactivates channels enabling observation of C-type inactivation rearrangements during depolarizing pulses. The amplitude of the
decaying component of  uorescence was approximately twofold larger at pH 5.0 than at pH 7.5. The dotted lines highlight the extent
of the decay. The decay of  uorescence was biexponential with values of 21 1 ms and 1.8 0.1 s at pH 7.5, and 21 1 ms and 0.36
0.03 s at pH 5.0 (n 3; P < 0.001, paired t test, when compared with pH 7.5). (B) Plot of the voltage dependence of the amplitude of
the decaying component of  uorescence that re ects C-type inactivation (n 3). The amplitude of the decaying component was mea-
sured at each potential and normalized to that at 100 mV (F decay). Also plotted is the G-V relationship of Shaker A359C channels
and F-V relationship of Shaker A359C W434F channels for comparison of the voltage dependence of pore opening and voltage sensor
movement, respectively (n 5–21).
Page 12
Claydon et al. 449
which the majority of the gating charge has moved, but
that still remain closed. Fig. 12 A shows  uorescence
records from ILT mutant channels with TMRM attached
at S424C during 200-ms depolarizations from 80 to
0 mV. At pH 7.5, the report was small, likely because the
uorophore at S424C does not report well on activation
(compare with Shaker S424C, Fig. 5), and because the
uorophore does not report the large  uorescence de-
ection that is associated with inactivation since these
channels do not open (at the potentials used in this
experiment) and therefore do not make signi cant
transition to the inactivated state. The  uorescence re-
port is reminiscent of gating current recordings from
Shaker and Kv channels with a fast transient upward  uo-
rescence de ection followed upon repolarization by
a slower transient downward fluorescence deflection
(Bezanilla et al., 1994; Fedida et al., 1996). At pH 5.0, the
uorescence report from Shaker ILT S424C channels
was markedly different (Fig. 12 B). In contrast to the
weak report at pH 7.5, there was a robust  uorescence
de ection with acidic pH that occurred at potentials at
which the channels remained closed. Fluorescence de-
ections in response to voltage pulses from 80 to 80
mV are shown in Fig. 12 B; a stronger depolarization was
required because surface charge screening at pH 5.0
shifted activation to more depolarized potentials. In Fig.
12 C, the mean amplitude of the  uorescence de ec-
tion from four oocytes at pH 7.5 and pH 5.0 is plotted.
It is clear that low pH induced rapid conformational
changes that were sensed by the  uorophore in the
pore. Furthermore, the pH-induced  uorescence shows
a clear voltage dependence that is shifted 47 mV to
the right of the  uorescence report of voltage sensor
movement from Shaker ILT A359C W434F channels due
to the charge screening effect of protons (the pH-
induced shift is similar to that observed in Shaker A359C
channels in Fig. 1, D and E). In addition, the time constant
of the fast phase of  uorescence induced at pH 5.0 in
Shaker ILT S424C channels displayed a clear voltage depen-
dence that was similar to that of the fast phase in Shaker
ILT A359C W434F channels at pH 7.5 (Fig. 12 D). These
data show that low pH enhances the ability of TMRM
Figure 10. Inhibition of inactivation by raising external K
rescues the loss of  uorescence at low pH. (A–F) Typical  uorescence signals
recorded from the same oocyte expressing Shaker S424C channels during 42-s depolarizing pulses to 60 mV during the indicated ma-
nipulations of the external K
and proton concentration. After control  uorescence recordings were obtained with 3 mM K
and pH
7.5 (A), the pH was reduced to pH 5.0 (with 3 mM K
) to demonstrate the decrease of  uorescence (B). Fluorescence signals were then
recorded on return to pH 7.5 with 3 mM K
(C) before application of solution with 99 mM K
at pH 7.5 (D). The  uorescence was then
measured with 99 mM K
and pH 5.0 (E), before the oocyte was washed with control solution (3 mM K
and pH 7.5) again (F). Similar
records were obtained from three other oocytes.
Figure 11. Inhibition of inactivation by the mutation T449V res-
cues the loss of  uorescence at low pH. Typical  uorescence sig-
nals recorded from Shaker S424C T449V channels during 7-s
depolarizing pulses to 60 mV at pH 7.5 and pH 5.0 (holding po-
tential 80 mV). Inhibition of inactivation prevented the loss of
uorescence at low pH.
Page 13
450 pH and Closed-State Inactivation
attached at S424C to detect the rapid activation move-
ments of the voltage sensor on depolarization in channels
that remain closed, and therefore suggest that acidic pH
induces a W434F-like inactivated conformation of the
pore in closed channels.
Another direct demonstration of closed-state rear-
rangements at low pH is shown in Fig. 13. The diary plot
in Fig. 13 A shows  uorescence emission from Shaker
ILT S424C channels sampled at 5-s intervals while hold-
ing channels at 80 mV and changing the pH of the so-
lution. The  uorescence emission from closed channels
was clearly altered by the pH. Fig. 13 B shows a plot of
the mean relative  uorescence change from that at pH
7.5 at each pH in three oocytes. The  uorescence de-
ection was 112 2, 111 2, 105 2, and 100 3%
that of the de ection at pH 7.5 during exposure to pH
4.0, 5.0, 6.0, and 8.0, respectively (n 3). The pH de-
pendence of the closed-state rearrangements had a pKa
of 5.9, which is similar to that reported above for the pH
dependence of the loss of the  uorescence and the con-
ductance decrease (pH 5.1 and 5.2, respectively). Similar
changes of pH had no effect on the  uorescence emis-
sion of TMRM-treated Shaker channels that lack an ex-
ternal cysteine (Shaker A359, Fig. 13 C). These data
demonstrate that low pH induces conformational rear-
rangements in closed channels that are detected by
S424C and taken together with the data presented above
suggest that acidic pH induces inactivation-dependent
conformational rearrangements of channels while they
reside in closed states.
DISCUSSION
Loss of Conductance at Low pH
The  uorescence results presented here support the idea
that acidic pH stabilizes both open- and closed-inactivated
channel states. Upon depolarization, low pH accelerates
inactivation from the open state (depolarization-induced
inactivation), which is manifest as an enhancement of
the rate of decay of ionic current and of the  uorescence
report of P- and C-type inactivation in Shaker A359C
and Shaker S424C channels (Figs. 1, 3–5, and 8–11), as
discussed below. However, this is not the major mecha-
nism by which peak current is reduced. Instead, our
uorescence studies demonstrate that low pH also in-
duces inactivation of channels from resting closed
states, which is manifest as the loss of the  uorescence
signal in Shaker S424C channels and the decrease in
peak current amplitude (Figs. 5 and 7–13). We propose
that the loss of  uorescence reports the decrease of
conductance seen in Fig. 1 A and Fig. 7 A as it is clear
from the report from A359C (Fig. 1, D and E) and the
gating current measurements (Fig. 2 C) that the number
Figure 12. Acidic pH induces
conformational changes in
closed channels that are
associated with inactivation.
Typical  uorescence signals
recorded from Shaker ILT
S424C channels during 100-ms
voltage pulses from 80 to
0 mV at pH 7.5 (A) and from
80 to 80 mV at pH 5.0 (B).
Although pulses were applied
in 10-mV increments, only ev-
ery other pulse is shown for
clarity. A voltage pulse to 0 mV
at pH 7.5 or to 80 mV at
pH 5.0 (shift in voltage depen-
dence is due to surface charge
screening by protons) evokes
maximum voltage sensor
movement without any chan-
nel opening. Reduction of the
extracellular pH induced con-
formational changes in closed
channels that are consistent
with inactivation. (C) Mean F-V
relationships of the  uores-
cence recorded from closed
channels at pH 7.5 and pH 5.0
from Shaker ILT S424C channels (n 4), and from Shaker ILT A359C W434F channels (n 4). The V
1/2
and k values were 37.1 0.8 mV
and 16.1 0.7 mV for Shaker ILT S424C channels at pH 5.0, and 83.0 0.4 mV and 15.7 0.4 mV for Shaker ILT A359C W434F channels
at pH 7.5, respectively. (D) Mean
-V relationships for the fast phase ( tted with a single exponential) of the  uorescence de ection from
Shaker ILT S424C channels at pH 5.0, and from Shaker ILT A359C W434F channels (n 4). The voltage dependence and kinetics of the  uo-
rescence report from Shaker ILT S424C channels was similar to that of voltage sensor movement reported by Shaker A359C W434F.
Page 14
Claydon et al. 451
of activatable channels was not altered by acidic pH
(i.e., total gating charge movement was not reduced by
protons) and there is no evidence from the single chan-
nel recordings that external protons reduce single
Shaker channel current amplitude (Fig. 6). It is possible
that the  uorophores in a proportion of the channels
no longer report during exposure to acidic pH because
they have moved into a position that no longer senses
the collapse of the pore during depolarization-induced
inactivation. However, the loss of  uorescence was pre-
vented by interventions that inhibited inactivation,
such as raising the external K
concentration (Fig. 10)
and the mutation T449V (Fig. 11), which indicates that
this population of channels no longer report simply be-
cause the channels are already inactivated. In agree-
ment with this, use of the ILT mutant channel to isolate
voltage sensor movement from pore opening (Smith-
Maxwell et al., 1998a,b) demonstrates that, at poten-
tials at which channels do not open, low pH induces
uorophore movement into a position from which it
better detects voltage sensor movement (Fig. 12). The
improved report of activation on going from pH 7.5 to
pH 5.0 suggests that, even though these channels do
not open (at the test potential used), acidic pH stabi-
lizes an inactivated W434F-like conformation and this is
consistent with the decreased single channel availabil-
ity observed in Fig. 6. In support of this, we were able to
directly observe conformational rearrangements in chan-
nels held at 80 mV when the pH of the solution was
lowered (Fig. 13).
It is interesting that the large  uorescence de ection
observed with Shaker ILT S424C (Fig. 12) was not ob-
served in Shaker S424C channels at low pH (Fig. 5).
Those channels that are closed inactivated should re-
port a rapid  uorescence de ection on depolarization,
but this is not evident. This may be because this report
is masked by the robust signal from open and inactivat-
ing Shaker S424C channels at low pH. Indeed, Fig. 9 A
shows that permanently inactivated Shaker S424C chan-
nels produce only a small  uorescence de ection (see
also Fig. 4 C in Loots and Isacoff, 1998). It appears that
the ILT mutation can enhance the report of activation
in channels that are closed-state inactivated in some way
that is not fully understood at the present time.
A positive correlation between the rate of depolariza-
tion-induced P/C-type inactivation and the prevalence
of closed-state inactivation has been reported previously
(Lopez-Barneo et al., 1993). These authors showed that
reducing external K
reduced channel availability (i.e.,
decreased the proportion of channels able to conduct
ions on depolarization) more signi cantly in mutant
Shaker channels that displayed accelerated depolarization-
induced inactivation. This conclusion is also supported
by the demonstration that the loss of  uorescence and
the decrease of macroscopic conductance share a very
similar pH dependence with pKa values of 5.1 and
5.2, respectively (Fig. 7), and is consistent with the ob-
servation that low pH markedly reduced the open prob-
ability of Shaker single channels (Fig. 6). Similar  ndings
are reported for the effect of acidic pH on Kv1.5 chan-
nels in which single channels switch between available
and unavailable states (Kwan et al., 2006). This predicts
the loss of the macroscopic  uorescence signal and maxi-
mal conductance that we observe since these measure-
ments represent the proportion of channels that are in
Figure 13. Demonstration of pH-induced closed-state rearrange-
ments. Typical diary plots of the effect of changing the pH on the
uorescence emission from Shaker ILT S424C (A) or Shaker A359
(C, as a control) channels held continuously at 80 mV. To mini-
mize bleaching of the  uorophore,  uorescence was sampled ev-
ery second (although only every  fth recording is shown for
clarity) by opening the shutter for 100 ms. Low pH induced rear-
rangements at 80 mV that were detected by TMRM at S424C.
(B) Plot of the dependence of the closed-state  uorescence
changes on the external pH (n 3). The relative  uorescence
change from that at pH 7.5 is plotted. Data were  tted with a stan-
dard Hill equation (assuming a Hill coef cient, n, of 1). The pKa for
the closed-state  uorescence change was pH 5.9. The value at pH
7.5 represents the mean of all pre- and post-treatment conditions
since the order of pH changes was not necessarily consistent.
Page 15
452 pH and Closed-State Inactivation
the unavailable state. Our  uorescence data from Shaker
channels demonstrate that the pKa for the transition to
the unavailable closed-inactivated state is pH 5.1–5.9
(Fig. 7 C and Fig. 13 B). The pKa values for the stabiliza-
tion of closed-state inactivation and loss of conductance
(pH 5.2, Fig. 7 C) are similar to previously reported pKa
values, pH 4.7–5.0, for the effect of protons on P/C-type
inactivation from the open state (Perez-Cornejo, 1999;
Starkus et al., 2003). In Kv1.5 channels, the pKa values
for the inhibition of maximal conductance and the ac-
celeration of inactivation by external acidi cation only
differ by 0.3 pH units (pKa pH 6.2 and pH 6.5, respec-
tively; unpublished data). This suggests that the effects
of acidic pH on closed- and open-state inactivation may
involve the same mechanism.
An argument against this idea is the differential sensi-
tivity of pH-induced closed- and open-state inactivation
to external K
. For example, raising external K
from 3
to 99 mM completely reversed the loss of the  uores-
cence signal caused by acidic pH, suggesting that 99 mM
K
prevents closed-state inactivation entirely (Fig. 10).
However, the report of P/C-type inactivation was only
slowed approximately twofold (from 2.0 0.6 to 3.6
0.4 s; n 3) and not completely prevented (Fig. 10).
Differential sensitivities of P/C-type inactivation and the
inhibition of maximal conductance by acidic pH to ex-
ternal K
are also evident in Kv1.5 channels (Fedida
et al., 1999; Zhang et al., 2005). However, this difference is
probably due to the fact that the K
-sensitive site in the
pore that mediates inactivation is  ooded by ef ux of
internal K
in open channels such that it is relatively in-
sensitive to changes in external K
, whereas the absence
of K
ef ux in closed channels reveals the sensitivity of
inactivation to the external K
concentration, as is the
case with depolarization-induced inactivation in the
presence of an internal pore blocker or with the removal
of internal K
(Baukrowitz and Yellen, 1995).
Enhancement of P-type Inactivation at Low pH
It is well established that acidic extracellular pH en-
hances the rate of inactivation upon depolarization in a
number of Kv channels, such as Shaker, Kv1.4, and Kv1.5,
although their sensitivity to protons differs (Steidl and
Yool, 1999; Claydon et al., 2000; Kehl et al., 2002; Starkus
et al., 2003). Our  uorescence studies enable direct ob-
servation of the rearrangements associated with the col-
lapse of the pore during P-type inactivation from the
report of TMRM attached either within the pore at
S424C or at the top of the voltage sensor at A359C.
Acidic pH enhanced the slow phase of the  uorescence
report from TMRM attached at A359C and at S424C
that reports the dynamic structural changes associated
with P-type inactivation (Fig. 3 B and Fig. 5 B). In addi-
tion, the experiments in Fig. 6 suggest that a greater
proportion of channels make the transition at rest to
the W434F-like P-type inactivated state at pH 5.0 than at
pH 7.5. These  ndings are consistent with the enhanced
decay of current seen in Fig. 3 A and Fig. 5 A and also
with data from previous studies during exposure to
acidic pH (Claydon et al., 2002; Kehl et al., 2002; Starkus
et al., 2003; Zhang et al., 2003), as well as the enhanced
decay of  uorescence that was previously observed from
TMRM attached at S424C (Loots and Isacoff, 1998).
Enhancement of C-type Inactivation at Low pH
W434F mutant channels are thought to be permanently
P-type inactivated (Perozo et al., 1993; Yang et al., 1997),
and therefore transitions to the C-type inactivated state
should be observable on depolarization from a  uoro-
phore placed in the outer pore. Our  uorescence re-
cords from TMRM attached at S424C in W434F mutant
channels (Fig. 9 A) showed a large fast de ection con-
sistent with previous  uorescence observations in this
channel (Loots and Isacoff, 1998) and also the notion
that the  uorophore in P-type inactivated channels oc-
cupies a position that senses the movement of the volt-
age sensors very well (Loots and Isacoff, 1998). However,
in addition to the rapid  uorescence de ection, we ob-
served a secondary  uorescence de ection that most
likely represents the progression of channels to the
C-type inactivated state, and this was enhanced during
acidic pH (Fig. 9, A and B). The C-type inactivation  u-
orescence transition was best  t with a double exponen-
tial function, and although the mechanistic basis for
this is uncertain, only the slower time constant was
reduced by acidic pH. The voltage dependence of the
total amplitude of the  uorescence de ection followed
that expected for channel opening (Fig. 9 C). This sug-
gests that, although these channels are permanently
P-type inactivated, activation of the voltage sensors is
not suf cient to cause C-type inactivation, and that tran-
sition to the C-type inactivated state can only occur once
the intracellular gate opens. Furthermore, this suggests
that, unlike P-type inactivation, C-type inactivation can-
not occur from resting closed states.
Although we have demonstrated a clear secondary
uorescence de ection that probably re ects C-type in-
activation, it was not evident in the previous report of
uorescence from S424C in W434F mutant channels
(Loots and Isacoff, 1998). This could be due to an addi-
tional mutation (C462A) made in those studies, which
enhanced the rate of inactivation 20-fold (Loots
and Isacoff, 1998) and perhaps made the transition to
C-type inactivation too rapid to resolve.
A Discrete Site in S4 Reports the pH-induced Alteration
of Inactivation
It has previously been suggested that the S4 domain
moves close to, and even interacts with, the S5-P linker
in an adjacent subunit of the channel (Blaustein et al.,
2000; Elinder et al., 2001; Gandhi et al., 2000; Larsson
and Elinder, 2000; Li-Smerin et al., 2000; Loots and
Page 16
Claydon et al. 453
Isacoff, 2000; Ortega-Saenz et al., 2000; Lainè et al.,
2003; Long et al., 2005) and that TMRM attached at
A359C at the top of the voltage sensor reports inactiva-
tion gating rearrangements (Loots and Isacoff, 1998,
2000; Gandhi et al., 2000; Long et al., 2005; Claydon
et al., 2006). It is clear from the present study that A359C
and R362C are unique amongst sites in the S3–S4 linker
and at the top of S4 in their ability to sense the pH-
induced enhancement of inactivation (Fig. 4). In a pre-
vious comprehensive scan of the extracellular domains
of the Shaker channel (Gandhi et al., 2000) it was dem-
onstrated that all sites from M356C to R362C report on
inactivation of the pore and that TMRM at sites within
the outer portion of the helical S4 domain can sense in-
activation rearrangements regardless of their orienta-
tion (Loots and Isacoff, 2000). Our results are generally
consistent with these (except that we could not record
de ections from either I360C or L361C); however, the
data in Fig. 4 suggest that only two of these sites, which
are adjacent to each other on the same side of the S4
-helix that faces the pore, sense the pH-induced changes
(although we cannot exclude that I360C and L361C
might sense such changes). It is unclear why we were
unable to record  uorescence de ections from I360C
and L361C (even with oocytes expressing >200 A of
current) that were reported by Loots and Isacoff (2000).
Since these authors suggest that these two sites report
only on inactivation (because the  uorescence signal is
dominated by a slow component and there is little evi-
dence of a fast component on the same time scale as ac-
tivation), it is possible that their inclusion of the
mutation C462A is the reason for the discrepancy. This
mutation is known to speed inactivation 20-fold (Loots
and Isacoff, 1998) and the report of inactivation in this
background would therefore be expected to be much
more pronounced.
Our data suggest either that the inactivation gating
rearrangements that are induced at low pH are local-
ized so that they are only sensed by this region or that
protons alter the interaction of S4 with the pore and, as
a consequence, the in uence of inactivation on TMRM
attached in S4. Further studies are clearly required to
address the effect of pH on the dynamic interaction of
S4 with the pore.
It has been suggested that the extent of  uorescence
quenching of TMRM attached at A359C is increased
with acidic pH (Cha and Bezanilla, 1997; Sorensen
et al., 2000), an effect that was attributed to the proton-
ation of a titratable group within the S3–S4 linker. In
the present study, we observed a trend for the  uores-
cence de ection to increase in amplitude during appli-
cation of acidic pH, although the data did not reach
statistical signi cance (Fig. 1, D and E). However, the
key observation was that the  uorescence amplitude
from Shaker A359C was not reduced during acidic pH
(Fig. 1, D and E). The lack of reduction of the signal
strongly suggests that the total gating charge movement
was not altered by external protons and is consistent
with the observation that acidic pH did not alter the
amplitude of on-gating charge movement (Fig. 2 C).
Clearly, the kinetics of the report of channel gating are
dramatically altered during acidic pH (Fig. 1, D and E),
but we do not interpret this as a reduction in the gating
charge movement, given that the total signal amplitude
is unchanged, but rather that the enhancement of the
inactivation report masks the report of activation. This
suggests that although the pore reports a loss of chan-
nel availability at low pH, the number of activatable
channels is not reduced.
Other Channels and Other Mechanisms
In Kv1.5 channels, although the pKa for the inhibition
by protons is pH 6.2 with 2–5 mM external K
(Steidl
and Yool, 1999; Kehl et al., 2002), lowering the pH to
5.5 results in a loss of conductance of channels in which
the histidine, which acts as a pH sensor in the outer pore,
has been mutated to glutamine (H463Q; Kehl et al.,
2002). This suggests that there are two titratable sites in
the Kv1.5 pore, one that has a pKa of pH 6.2 and,
when protonated, enhances inactivation, and a second
that has a lower pKa, which, when protonated, causes a
loss of macroscopic conductance. The Shaker channel
lacks the histidine pH sensor, and current is inhibited
with a pKa of pH 4.0–5.0 (Perez-Cornejo, 1999; Starkus
et al., 2003; see also Fig. 1 A), leading to the suggestion
that D447 in the outer pore may act as the pH sensor
(Starkus et al., 2003). Since this residue is conserved in
Kv1.5 channels, this site may also act as the low range
pH sensor that regulates availability in these channels.
Consistent with this idea, mutations of Shaker D447 ac-
celerate inactivation (D447E) or alter current level
(D447N) (Molina et al., 1998).
It is known that Kv channels, once inactivated, become
signi cantly more permeable to Na
(Starkus et al., 1997;
Wang et al., 2000), and this has been used as another
way to examine transitions between inactivated states at
low pH (Zhang et al., 2003). Many of the observations
made on Na
currents through inactivated Kv channels
support the conclusions made from the  uorescence
data obtained from Shaker channels described here. In
the Na
experiments there was a large decrease in open-
state current at low pH, but large slow inward Na
tail
currents were maintained upon repolarization. This
supports the idea that channels can still activate and de-
activate at low pH without passing through the open
state, and thus activation gating is expected to be main-
tained. This is supported by the gating current measure-
ments in Fig. 2 as well as the  uorescence experiments in
Figs. 1 and 3, which imply that low pH does not alter total
gating charge movement. As well, these Na
permeation
experiments showed an equivalent Na
conductance
through open-inactivated channels during sustained
Page 17
454 pH and Closed-State Inactivation
depolarizations at normal and acidic pH. These data sug-
gest the possibility that channels are closed-inactivated
at low pH, and are able to activate directly into Na
-
conductive depolarization-induced inactivated states
without actually opening. This idea is consistent with the
present  uorescence and single channel experiments,
which show directly that a population of channels be-
comes unavailable during acidic pH (Figs. 5, 6, and 10)
due to the stabilization of inactivation from resting
closed channel states (Figs. 10–13), making them unable
to open. A kinetic model to describe the Na
perme-
ation experiments suggested that channels accumulated
in the open-inactivated state during depolarization and
made the transition to closed-inactivated states via an
intermediate “R” state in the recovery pathway (Zhang
et al., 2003). Channels could not reopen from resting
closed-inactivated states in the model (Zhang et al., 2003),
but it is not clear whether or not Na
as the permeant ion
causes other structural changes within the selectivity  lter
that do not allow it to accurately model activation of
closed-inactivated channels with K
as the charge carrier
(Panyi and Deutsch, 2006).
It has previously been shown that Shaker channels may
undergo a type of closed-state inactivation called U-type
inactivation (Klemic et al., 2001) and it is helpful to
consider whether this is the closed state stabilized at low
pH. In U-type inactivation, maximal current reduction
occurs at intermediate potentials, where the dwell time
in intermediate states is longer, and less inactivation oc-
curs at more depolarized potentials when the open
probability is high, resulting in a characteristic U-shaped
inactivation–voltage relationship. However, although
the  uorescence data strongly support closed-state inac-
tivation, the effects of raising external K
reported here
are opposite to those expected for U-type inactivation.
U-type inactivation is enhanced by raising external K
(Klemic et al., 2001), whereas we show in Fig. 10 that
high external K
rescues channels from closed-state in-
activation, which suggests that the pH-induced closed-
state inactivation is unlikely to be U-type in nature.
This work was supported by grants from the Heart and Stroke
Foundation of British Columbia and Yukon and the Canadian In-
stitutes of Health Research to D. Fedida and S.J. Kehl. D. Fedida
is supported by a Career Investigator award from the Heart and
Stroke Foundation of British Columbia and Yukon. T.W. Claydon
was supported by a postdoctoral research fellowship funded by a
Focus on Stroke strategic initiative from The Canadian Stroke
Network, the Heart and Stroke Foundation, the CIHR Institute of
Circulatory and Respiratory Health, and the CIHR/Rx&D Pro-
gram along with AstraZeneca Canada. M. Vaid was supported by a
Michael Smith Foundation for Health Research Studentship. S.
Rezazadeh was supported by a Heart and Stroke Foundation of
British Columbia and Yukon Studentship and a University of Brit-
ish Columbia Graduate Fellowship.
Olaf S. Andersen served as editor.
Submitted: 2 March 2007
Accepted: 6 April 2007
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  • Source
    • "Although P/C-type inactivation is classically viewed as an open channel inactivation mechanism (see Figures 1A and 2C), recent work has shown that the P-gate may also undergo inactivation when the channel is still closed. Claydon et al. (2007, 2008) have performed electrophysiological measurements combined with voltage-clamp fluorimetry to examine whether ShakerIR (N-type inactivation removed) channels may undergo P/C-type inactivation also from closed-states. Acidic pH, which promotes rearrangements at the P-gate, and the Shaker ILT triple-mutant (Smith-Maxwell et al., 1998), which segregates channel opening from voltage-dependent activation by shifting the respective curves apart, were exploited in this study. "
    [Show abstract] [Hide abstract] ABSTRACT: In voltage-gated potassium (Kv) channels membrane depolarization causes movement of a voltage sensor domain. This conformational change of the protein is transmitted to the pore domain and eventually leads to pore opening. However, the voltage sensor domain may interact with two distinct gates in the pore domain: the activation gate (A-gate), involving the cytoplasmic S6 bundle crossing, and the pore gate (P-gate), located externally in the selectivity filter. How the voltage sensor moves and how tightly it interacts with these two gates on its way to adopt a relaxed conformation when the membrane is depolarized may critically determine the mode of Kv channel inactivation. In certain Kv channels, voltage sensor movement leads to a tight interaction with the P-gate, which may cause conformational changes that render the selectivity filter non-conductive ("P/C-type inactivation"). Other Kv channels may preferably undergo inactivation from pre-open closed-states during voltage sensor movement, because the voltage sensor temporarily uncouples from the A-gate. For this behavior, known as "preferential" closed-state inactivation, we introduce the term "A/C-type inactivation". Mechanistically, P/C- and A/C-type inactivation represent two forms of "voltage sensor inactivation."
    Preview · Article · May 2012 · Frontiers in Pharmacology
  • Source
    • "The results presented herein indicate that regulation of the S4 transitions by pHo not only determines channel gating properties, but also determines the maximal conductance of the channel. The proton-induced channel blocking of Kvs is related to enhanced C-type inactivation (Zhang et al. 2003; Claydon et al. 2007). Since NaChBac inactivates via a C-type inactivation mechanism (Kuzmenkin et al. 2004), it would be expected that RCGmax values of NaChBac mutants would be correlated with channel steady-state inactivation. "
    [Show abstract] [Hide abstract] ABSTRACT: [Currently the author holds the copyright for this article. It was formerly accepted and published at the J MEMBRANE BIOL (see: http://sdrv.ms/PEu7sG) before it was pulled out by the author due to violation of the author's copyrighs. You can use it or any part of it as long as you indicate its source: Paldi T (2012) Deprotonation of arginines in S4 is involved in NaChBac gating. http://sdrv.ms/13hN4Vm] Voltage-gated ion channels participate in cell excitability by enabling selective ion flux in response to changes in the membrane potential. The channel senses voltage across the membrane via a voltage-sensing module composed of four membrane-spanning helices (S1-S4). A stretch of positively charged arginines in the fourth transmembrane segment (S4) that traverses the membrane's electric field is the principal sensing component of the module. Yet the driving forces behind S4 movement are not fully understood. The prevailing helical screw model, which describes the movement of S4 along its axis, suggests that salt bridges between positively charged residues on S4 and negatively charged residues on S1-S3 alternately break and reform in the course of S4 movement. However, the estimated energy needed to separate the charges in a low dielectric cavity is incompatible with experimental data (Green, J Theor Biol 193:475-483, 1998). Here, it is shown that extracellular titration of the three outermost arginines on S4 stabilizes the bacterial voltage-gated sodium channel (NaChBac) at different states and enhances the coupling of the outermost arginine on S4 with E43 on S1. It is suggested that salt bridges that stabilize S4 are impaired by arginine deprotonation during voltage-sensing module activity. It is also shown that acid-induced channel blocking is strongly affected by arginine substitutions at S4. The electrostatic interactions of these arginines with acidic residues, exemplified by a structural model of NaChBac, suggests that extracellular acidification induces the retraction of S4, thereby enhancing channel inactivation by reclosure of the channel's activation gate.
    Full-text · Article · Apr 2012 · Journal of Membrane Biology
  • Source
    • "It should be noted that sensing currents only capture movement of charges in the direction of the membrane electric field, whereas the fluorescence signal can report additional conformational transitions either directly reflecting movement of charged residues or conformational transitions that are triggered by the former. In this sense, the fluorescence signal of VSFP2.3 contains information similar to that obtained by voltage-clamp fluorometry (Claydon et al. 2007; Pathak et al. 2007; Villalba-Galea 2008a, b). For Ci-VSP it has been shown that the activated position of its VSD is not stable, thus causing the VSD to relax into a lower energy conformational state at prolonged depolarizations (Villalba-Galea et al. 2008b). "
    [Show abstract] [Hide abstract] ABSTRACT: A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy transfer) signal. Here we report sensing current measurements from VSFP2.3, and show that VSFP2.3 carries 1.2 e sensing charges, which are displaced within 1.5 ms. The sensing currents become faster at higher temperatures, and the voltage dependence of the decay time constants is temperature dependent. Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing mechanism of Ci-VSP, which will allow us to further improve the sensitivity and kinetics of the family of VSFP proteins.
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