Voltage Profile along the Permeation Pathway of an Open Channel
Jorge E. Contreras,†* Jin Chen,†Albert Y. Lau,‡Vishwanath Jogini,‡Benoı ˆt Roux,‡and Miguel Holmgren†*
†Molecular Neurophysiology Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National
Institutes of Health, Bethesda, Maryland; and‡Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois
At the microscopic level, the transmembrane potential is thought to decay nonlinearly across the ion permeation pathway
because of the irregular three-dimensional shape of the channel’s pore. By taking advantage of the current structural and func-
tional understanding of cyclic nucleotide-gated channels, in this study we experimentally explore the transmembrane potential’s
distribution across the open pore. As a readout for the voltage drop, we engineered cysteine residues along the selectivity filter
and scanned the sensitivity of their modification rates by Agþto the transmembrane potential. The experimental data, which
indicate that the majority of the electric field drops across the selectivity filter, are in good agreement with continuum electrostatic
calculations using a homology model of an open CNG channel. By focusing the transmembrane potential across the selectivity
filter, the electromotive driving force is coupled with the movement of permeant ions in the filter, maximizing the efficiency of this
For ion channels, the transmembrane potential plays a critical role by acting as a driving force for permeant ions.
Ions cross biological membranes through specialized inte-
gral membrane proteins known as ion channels (1) and
transporters (2). Ion channels allow the passage of ions at
rates >107ion/s (1,3). These high throughput rates are
determined by a variety of physical factors, such as the
shape of the permeation pathway, the intrinsic electrostatic
potential of the protein along the permeation pathway,
ion-ion interactions within the pore, and the transmembrane
potential itself (1). In the last 10 years, the increasing avail-
ability of high-resolution ion-channel crystal structures,
together with functional and computational studies, has
permitted a more microscopic account of how some of these
parameters influence permeation (4–19). The transmem-
brane potential has been studied mostly using computational
approaches, remaining elusive to direct experimentation. A
continuum electrostatic approximationleadingto a modified
Poisson-Boltzmann equation was developed to describe the
influence of the transmembrane potential on transmembrane
proteins (i.e., the PB-V equation) (15). Calculations based
on crystal structures and homology models of potassium
channels suggested that the transmembrane voltage in an
open channel is mainly focused along the narrow selectivity
filter (11,12,20). Although this concept is in broad accord
with voltage-dependent current block by specific ions and
organic molecules (21–25), physical interpretations from
these studies have been difficult. In some instances, either
multiion occupancy and/or the intrinsic voltage dependence
of channel gating contribute to the voltage dependence of
blockade. Furthermore, the precise locations of where these
blockers act along the permeation pathway are difficult to
cyclic nucleotide-gated (CNG) channel by substituting
cysteine at three positions known to line the permeation
pathway (26,27): the intracellular cavity, and the inward and
outward facing ends of the selectivity filter. We then evaluate
thevoltage dependence of modification by positively charged
thiol reagents such as Agþ, Cd2þ, and trimethylaminoethyl
methanethiosulfonate (MTSET). Similar to permeant ions,
these probes will sense the transmembrane voltage as they
transverse the permeation pathway. Therefore, changes in
the reaction kinetics between these thiol reagents and the
substituted cysteines can be used to infer the voltage profile
along the pore. By comparing the voltage dependence of the
apparent modification rates along the permeation pathway of
CNG channels, we determined that, as hypothesized, the
voltage drops exclusively along the selectivity filter in an
model of an open CNG channel based on the high-resolution
structure of the NaK channel (28).
MATERIAL AND METHODS
Mutagenesis and expression
Cysteine mutations were introduced in a cysteine-free CNGA1 channel,
a kind gift from William Zagotta (University of Washington, Seattle,
WA). All mutations were generated by standard PCR techniques and
verified by DNA sequencing. In vitro cRNA synthesis was performed
with a T7 promoter-based kit from Ambion (Austin, TX). cRNA was in-
jected into Xenopus oocytes and allowed to express for 3–5 days before
Ionic currents were measured from inside-out excised patches (29) using an
Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Bath
Submitted April 15, 2010, and accepted for publication August 26, 2010.
*Correspondence: email@example.com or firstname.lastname@example.org
Editor: Richard W. Aldrich.
? 2010 by the Biophysical Society
Biophysical Journal Volume 99 November 2010 2863–28692863
solutions were rapidly exchanged by a computer-controlled system (RSC-
200; Biologic Science Instruments, Knoxville, TN). Bath and pipette
solutions containing 120 mM of either NaCl or NaNO3(when silver ions
were used), 10 mM HEPES, and EDTA (pH ¼ 7.4). For experiments using
MTSET the [EDTA] was 0.2 mM. EDTAwas omitted in experiments using
Cd2þand extracellular Mg2þ. Solutions used in experiments with Agþ
contained 10 mM EDTA to buffer free Agþto a desired concentration
(26). Cd2þmodification experiments in the presence of 40 mM external
Naþwere performed by reducing the concentration of Naþwithout
compensating for ionic strength or the change in osmolarity because we
have not been able to find a suitable replacement that is nonpermeant. Large
cations like NMDG or arginine, and osmotic substitutes like sucrose,
interact with the external mouth of CNG channels, causing substantial
block at all voltages (see Fig. S1 in the Supporting Material).
Sequence alignment of CNGA1 (GI: 27805875) with NaK (GI: 30018851)
was performed as previously described (27). The side chains of the CNG
channel were substituted onto the main chain of the open NaK
channel’s crystal structure (28) (PDB: 3E86) using SCWRL (30). Only
the M1-P-M2 region was modeled. The loop region between the M1 and
P helices (residues 359–365) was rebuilt using ModLoop (31) because
NaK has fewer residues in this region. Protein side chains and the rebuilt
loop were refined by energy minimization and molecular dynamics simula-
tions using CHARMM (32). All main chain atoms except residues 330–346
(the C-terminal end of the M1 helix and the connecting loop) and 365–369
(the loop between the filter and the M2 helix) were constrained during
Calculation of the transmembrane potential
The fraction of transmembrane potential was calculated from the CNG
model using the modified Poisson-Boltzmann (PB-V) equation (15) imple-
mented in the finite-difference PBEQ module (33) of CHARMM (32).
Similar calculations carried out for the KcsA channel have been previously
described (12,20) and experience shows that the results are not highly sensi-
tive to the details of the model. A dielectric of 2 was assigned to the protein.
The membrane was represented as a 25 A˚slab with a dielectric of 2. The
bulk solvent on either side of the membrane, and the aqueous crevices of
the pore, were assigned a dielectric of 80. A water probe of 1.4 A˚radius
was used to define the molecular surface corresponding to the dielectric
boundary, using a 0.5 A˚grid (33). It should be noted that all protein charges
were turned off for the calculation of the membrane potential profile, as
prescribed by the PB-V theory (15).
Voltage drop along the intracellular cavity
To estimate the voltage landscape along the permeation
pathway of an open channel, we used small probes (34–
36), which were driven by voltage toward specific sulfhy-
dryl side chains substituted along the pore (Fig. 1). This
approach allowed us to define the precise location of the
target sites. It is feasible only under conditions when the
channel’s gating is not sensitive to voltage, as for CNG
channels in the presence of saturating concentrations of
cGMP (37) (see also Fig. S2). Fig. 2 A shows the experi-
mental strategy used to examine channel activity before
and after chemical modification. In each experiment,
inside-out patches with multiple CNG channels were
positioned in front of a perfusion change system that al-
lowed rapid changes of the internal solution. The membrane
potential was held at 0 mV, except during treatments when
channels were exposed to a two-second application of
a specific cysteine reagent at a defined voltage, and with
a saturating concentration of agonist. The ionic current
through CNG channels before and after modification was
assessed with two brief 50-ms depolarizations to þ60 mV:
one in the absence, and one in the presence, of 2 mM
cGMP. Subtraction of these currents yielded the cGMP-acti-
vated current. Fig. 2 B shows leak-subtracted current records
before (black) and after (green) four successive MTSET
treatments on V391Cmutant channelsat a membrane poten-
tial of ?40 mV (green traces). At this position, located in
the middle of the intracellular cavity, chemical modification
by the positively charged MTS reagent produced a complete
and irreversible current reduction. The time course of
MTSET modification was similar at ?40 and 40 mV
(Fig. 2 C). The apparent second-order MTSET modification
rates were estimated from the reciprocal of the time constant
obtained by single exponential fits to the current reduction
data and the concentration of reagent applied. The apparent
modification rates for MTSET were similar for voltages
from ?60 to 60 mV (Fig. 2 D). Similarly, modification rates
did not differ with voltage when V391C channels were
exposed to Cd2þ(Fig. 2 E). These results suggest that posi-
tively charged probes sense no portion of the transmem-
brane potential as they move from the intracellular bulk
solution to the middle of the intracellular cavity.
cysteine reagent that can access the entire selectivity filter of
CNG channels, perhaps because it is a small monovalent
conformation. The pore model includes segments S5 and S6 of the
CNGA1 channel. For simplicity, only two opposite subunits are shown.
The homology model of the CNG pore was built using the structure of an
open NaK channel as template (28). This bacterial channel has been
proposed to resemble the ion permeation pathway of CNG channels (27).
Residues that were substituted by cysteine in CNG channels are highlighted
in colors that will be conserved throughout the article.
Homology model of the CNG channel pore in the open
Biophysical Journal 99(9) 2863–2869
2864 Contreras et al.
cation, like Naþor Kþ. At position T360C, located at the
intracellular end of the selectivity filter (i.e., at the top of
the intracellular cavity), the time courses for Agþmodifica-
tion were almost indistinguishable between ?40 and
40 mV (Fig. 3 A). The apparent modification rates for volt-
ages between ?60 and 60 mV were similar, being slightly
slower at the most negativevoltage (Fig. 3 B; solid symbols).
The lack of significant voltage dependence suggests that the
voltage drop along the intracellular cavity of the channel is
negligible. However, it is possible that a sizeable voltage
drop along this region is being masked by Agþpermeation
to the outside. In other words, occupancy at the site of modi-
fication appears voltage-independent because access from
the inside and exit to the outside, both voltage dependent
transitions, have similar magnitudes. To explore this possi-
bility, we performed Agþmodifications in the presence of
50 mM extracellular Mg2þ, which is known to block perme-
ation by interacting with the external end of the selectivity
filter in a voltage-dependent manner (27,38,39), a property
that is preserved in T360C mutant channels (Fig. S3).
The presence of external Mg2þslightly slowed the apparent
modification rates, without affecting their voltage indepen-
dence (Fig. 3 B; open symbols). These results suggest that
an intracellular permeant ion will not sense a significant
voltage drop as it approaches the intracellular end of the
Voltage drop across the selectivity filter
In contrast to the results presented thus far, access of intra-
cellularly applied Agþto T364C, a position at the outer end
of the selectivity filter (Fig. 1), showed substantial voltage
dependence. Fig. 4 A illustrates the time courses by Agþ
modification at ?80 (open circles), ?40 (semisolid circles),
and 40 mV (solid circles). Clearly, they are slower at more
negative potentials. A more extensive study of the voltage
dependence reveals that the apparent second-order rates
approach zero at negative potentials and reach a maximum
at positive voltages (Fig. 4 B). To analyze these data we
consider the following kinetic scheme:
The model assumes that modification at T364C is preceded
by intracellular Agþdwelling between two bound states:
lular cavity. (A) Experimental protocol to estimate modification rates at
different voltages. In each experiment, chemical modification was per-
formed using 2 s treatments at a define voltage and under saturating
[cGMP] (2 mM). Subsequently, cGMP-activated currents were monitored
by subtracting a 50-ms pulse to þ60 mV in the absence of cGMP from
a similar pulse in the presence of 2 mM cGMP. Treatments were applied
every 15 s. (B) cGMP-activated current traces before (black trace) and after
four consecutive MTSET treatments (green traces). (C) Time course of
MTSET modification. Plot shows normalized current upon MTSET
applications (arrow) at ?40 (open green circles) and 40 mV (solid green
circles), respectively. Dashed (?40 mV) and solid (40 mV) lines represent
single exponential fits to the cumulative modification data at each voltage.
(D) Voltage dependence of the modification rate for MTSET. The concen-
trations of MTSET used in these experiments were 50 or 100 mM. n ¼ 20
patches. (E) Voltage dependence of the modification rate for Cd2þ. These
apparent rates were estimated with [Cd2þ] between 0.5 and 2 mM. n ¼
Modification at position V391C: the middle of the intracel-
tivity filter facing the intracellular cavity. (A) Time course of Agþmodifi-
cation. (Open and solid blue symbols) Two experiments in which
modification was assessed at ?40 mV and 40 mV, respectively. (Lines)
Single exponential fits to the cumulative modification at ?40 (dashed)
and 40 mV (solid). (B) Voltage dependence of Agþmodification in the
absence (solid symbols; n ¼ 28 patches) and presence (open symbols;
n ¼ 23 patches) of 50 mM external Mg2þ. All apparent rates were estimated
with 35 nM Agþ.
Modification at position T360C: the internal end of the selec-
Biophysical Journal 99(9) 2863–2869
Voltage Profile along an Open Channel2865
S1$Ag and S2$Ag. The state S1$Ag might represent a silver
ion occupying the intracellular cavity. The S2$Ag state
would occur in the immediate vicinity of 364C. The
S1$Ag4S2$Ag transition is at equilibrium and is the sole
source of voltage dependence. Finally, Agþinteracts irre-
versibly with the cysteine at position T364C (T364C-Ag)
in a voltage-independent manner, with rates that are slower
than the preceding transition. In this model, the rate of
modification becomes proportional to the occupancy of
S2$Ag, which is distributed with voltage following a Boltz-
mann relation (Fig. S4 shows a simulation of this model).
The solid line in Fig. 4 B represents a fit of the modification
data to this model. The steepness of the fit is consistent with
the movement of a single positive charge across the entire
transmembrane potential (i.e., a single Agþsenses the entire
voltage drop across the membrane when moving from
S1$Ag to S2$Ag).
Influence of external ions on modification
of cysteines at the intracellular end
of the selectivity filter
For almost 40 years it has been known that external ions can
cross the pore of voltage-activated potassium channels and
influence molecules, such as quaternary ammonium
blockers, bound at the intracellular side of the permeation
pathway (21,40). This phenomenon has also been observed
in CNG channels (41,42). Therefore, in principle, we would
expect that external ions permeating through the open CNG
channel pore could influence the modification rate of our
voltage sensing probes, particularly at negative potentials
where the net flux of cations is inward. It is puzzling then
that external ions did not appear to affect the voltage
dependence of chemical modification, particularly at the
internal entryway of the selectivity filter (position T360C).
A plausible explanation would be that an external permeant
ion (e.g., Naþ) would influence the voltage dependence of
modification by an intracellularly applied probe only if
that ion displaces the probe from a binding site within the
pore. Perhaps, over the voltage range that we explored, the
residence time of Agþwithin the CNG permeation pathway
is too short, rendering the interactions between external Naþ
Intracellular Cd2þcan also access the intracellular end of
the selectivity filter (position T360C) with modification
rates that are 3–4 orders-of-magnitude slower than those
measured for Agþ(26). We wondered if the residence times
of Cd2þin the permeation pathway would be sufficiently
slow to detect a voltage dependence to chemical modifica-
tion imparted from external permeant ions. Fig. 5 A shows
the time courses of modification by 50 mM internal Cd2þ
at ?60 (open circles), 0 (semisolid circles), and þ60 mV
(solid circles). In contrast to our observations with Agþ
(Fig. 3), modification rates with Cd2þat T360C were slowed
at negative potentials. A complete study of the apparent
tivity filter. (A) Time course of Agþmodification. Open, semisolid, and
solid orange symbols represent three experiments in which apparent modi-
fication rates were estimated at ?80 mV, ?40 mV, and 40 mV, respectively.
Lines represent single exponential fits to the cumulative modification at
?40 (dashed) and 40 mV (solid). (B) Voltage dependence of the modifica-
tion rates. The concentrations of Agþused in these experiments were
between 75 and 130 nM. Modification rate at ?80 mV was set to zero
because there was no apparent modification over the timescale used in
our protocol. (Solid line) Boltzmann fit to the apparent modification rates
with an apparent valence of 1.1 5 0.3 and a midpoint of ?14 5 6 mV.
n ¼ 32 patches.
Modification at position T364C: the external end of the selec-
of extracellular permeant ions. (A) Temporal course for Cd2þmodification
with 50 mM applications as indicated (arrow). (Open, semisolid, and solid
blue symbols) Three experiments where modification was assessed at ?60,
0, þ60 mV, respectively. (Lines) Single exponential fits to the cumulative
modification at each voltage. (B) Voltage dependence of modification rates
for T360C from ?60 mV to þ80 mV. (Solid line) Boltzmann equation fit of
the apparent modification rate with an apparent valence of 1.02 5 0.2 and
a midpoint of 31 5 8.3 mV. n ¼ 27 patches. (C) Voltage dependence of the
apparent modification rates with Cd2þin the presence of 50 mM external
Mg2þ(open diamonds). n ¼ 18 patches. (D) Voltage dependence of the
apparent modification rates with Cd2þin a 40 mM extracellular Naþ
solution(opentriangles).n ¼21patches.Forcomparison,thefitfrom panel
B is also shown in panels C and D.
Voltage-dependent access of Cd2þto position T360C: effect
Biophysical Journal 99(9) 2863–2869
2866Contreras et al.
Cd2þmodification rates’ voltage dependence is shown in
Fig. 5 B. These rates approach zero at negative voltages
and tend to a maximum at positive potentials.
If the voltage dependence of modification originates from
the effective displacement of internal Cd2þby external Naþ,
we would expect that changing external Naþaccess to the
permeation pathways should change thevoltage dependence
of Cd2þmodification. We tested this hypothesis by two
approaches: First, we used extracellular Mg2þto block
permeation (Fig. S3). Consistent with our hypothesis, modi-
fication by internal Cd2þbecame mostly voltage-insensitive
at voltages where Mg2þblockade was substantial (Fig. 5 C,
open diamonds). However, it sharply changed at potentials
where outwardly moving permeant ions effectively dis-
placed the bound external Mg2þ. Second, we shifted the
net flux of Naþalong the voltage axis by reducing the
external [Naþ] to 40 mM (Fig. S5). As expected, the entire
voltage dependence of modification shifted leftward along
the voltage axis (Fig. 5 D). Combined, these results strongly
suggest that the voltage dependence of chemical modifica-
tion by internal Cd2þat the internal entryway of the selec-
tivity filter (position T360C) results from incoming Naþ
expelling Cd2þfrom the permeation pathway before modi-
fication can occur. Interestingly, the solid line in Fig. 5 B
represents a fit of the modification data to a Boltzmann func-
tion. The steepness is consistent with one equivalent charge
moving from the external bulk solution to the site occupied
A homology model of the voltage profile along
an open CNG channel
The profile of the transmembrane potential along the axis of
the channel was calculated using the PB-V equation (15)
based on an atomic homology model of the CNG channel
in the open conformation (Fig. 6 A). In Fig. 6 B, the ordinate
represents the fraction of the transmembrane potential and
the abscissa corresponds to the axis of the pore. The center
of the bilayer is located at z ¼ 0 A˚. Consequently, the selec-
tivity filter (TTIGET) is located between z ¼ 0.8 and z ¼
16.1 (measured from the carbonyl oxygens of positions
T359 and T364). The fraction of the total transmembrane
potential measured at the side chains of positions T360
and T364 is 0.17 and 1, respectively. As with the experi-
mental data, these calculations predict that most of the trans-
membrane voltage drops along the selectivity filter.
Our functional studies indicate that the voltage drop along
an open CNG channel is focused within the selectivity filter.
We showed that modification of cysteine residues located in
the middle of the intracellular cavity, or at the inner-facing
end of the selectivity filter, by intracellularly applied
charged probes did not show any direct voltage dependence.
However, modification of cysteine residues located at the
external end of the selectivity filterby intracellularly applied
Agþexhibited pronounced voltage dependence. Consistent
with these data, theoretical calculations using a modified
PB equation on a homology model of an open CNG channel
showed aweak voltage drop along the inner cavity, and most
of the transmembrane potential focused at the selectivity
Quantitatively, our two approaches show remarkable
agreement. Experimentally, thevoltage dependence ofinter-
nally applied Agþmodification at position T364C is consis-
tent with a singlecharge traversing
transmembrane potential. By modeling, continuum electro-
static calculations indicate that the transmembrane potential
drop at the side chain of position 364C is ~1 (i.e., the entire
voltage drop along the permeation pathway). For positions
located at the inner end of the selectivity filter, or at the
middle of the intracellular cavity, our modification data
CNGchannel pore.(A) Valuesofthe fractional transmembranepotentialare
indicated along the permeation pathway of the CNG channel homology
model. (B) The curve represents the profile of the potential along the
pore of an open CNG channel homology model. The curve is drawn relative
to extracellular solution which is assumed to have a value of 1. The
selectivity filter is shown in dotted lines between z ¼ 0.8 and z ¼ 16.1.
Fractional transmembrane potential along the axis of an open
Biophysical Journal 99(9) 2863–2869
Voltage Profile along an Open Channel 2867
showed no detectable voltage dependence. Again, these
results are consistent with continuum calculations which
predict a mere 15% of the voltage drop along the intracel-
lular cavity. These results combined, strongly suggest that
the transmembrane potential in an open channel is indeed
focused across the narrowest part of the permeation
pathway, the selectivity filter. Using a purely theoretically
approach, this has been predicted for many open channel
structures or homology models of them (11,12). Modifica-
tion data at position T364C also suggest that Agþ, while
passing through the selectivity filter, interacts little with
other permeant ions. This result is not so surprising. As
long as ions move in a concerted fashion, strong ion-ion
interactions would be easily detected using electrophysio-
logical methods, as they have been observed in Kþchannels
(13). In CNG channels, on the other hand, many types of
permeant ions show no apparent interaction between them
(43–45), yet for other types of ions, ion-ion interactions
are readily observed (46,47).
In contrast to Agþ, modification by Cd2þat the inner end
of the selectivity filter (position T360C) showed strong
voltage dependence. Where does this voltage dependence
come from? We propose that it comes from external Naþ
permeating through the open channel and effectively
competing with internal Cd2þbefore modification occurs.
In Fig. 5, we presented results from two sets of experiments
that support this hypothesis. In both cases, reducing the
translocation of external permeant ions drastically changed
the voltage dependence of modification by Cd2þ. In addi-
tion, there is a long history of studies that describe how
ion permeation affects the voltage dependence of internal
blockers. For example, Armstrong and co-worker (21,48)
showed that blockade of squid Kv channels by intracellular
quaternary ammonium is voltage-dependent, and influenced
by external Kþ. They found that external Kþincreased
TEA’s rate of dissociation most significantly at membrane
potentials that favored inward currents. Based on these find-
ings, they hypothesized that the voltage dependence of
blockade originates from external permeant ions moving
across the transmembrane voltage and interacting with the
blockers inside of the pore. Furthermore, recent reports on
inward rectifying Kþchannels show a sharp voltage depen-
dence for polyamine blockade, with electrical distances
between 3 and 5. To explain these values, it has been
proposed that voltage-dependent block by polyamines
results primarily from the displacement of multiple Kþ
ions traversing the transmembrane electric field as the
blocker reaches its binding site (25,49–55). Finally, CNG
channels are also blocked by QA and polyamines in
a voltage-dependent-manner (41,42,56). In both cases,
voltage dependence of blockade may come from permeant
ions traversing the field rather than the charge carried by
the blocker. External Naþmay in fact influence Agþ
modification at the inner end of the selectivity filter (posi-
tion T360C), but only over a voltage range beyond our
experimental protocols. In fact, we did observe a consistent
reduction of modification rates at ?80 mV but poor patch
stability prevented us from examining more negative
Even though ion permeation is influenced by many
factors (1,12,13,19), focusing the field along the selectivity
filter in an open channel will optimize the coupling between
the movement of permeant ions within the filter and the
electromotive driving force, and consequently setting ion
access to the inner cavity to be the rate limiting step for
Five figures are available at http://www.biophysj.org/biophysj/supplemental/
The background CNGA1 cysteine-less construct was a gift from Dr.
William Zagotta. In addition, we thank Joe Mindell and Kenton Swartz
for helpful discussions, and Josh Rosenthal for carefully reading the
J.E.C. was supported by a Ruth L. Kirschstein Postdoctoral Fellowship.
This research was supported by the Intramural Research Program of the
National Institutes of Health, National Institute of Neurological Disorders
and Stroke. V.J., A.Y.L., and B.R. are supported by grant No. GM-62342
from the National Institutes of Health.
1. Hille, B. 2001. Ion Channels of Excitable Membranes. Sinauer
Associates, Sunderland, MA.
2. La ¨uger, P. 1991. Electrogenic Ion Pumps. Sinauer Associates,
3. Neher, E., and B. Sakmann. 1976. Single-channel currents recorded
from membrane of denervated frog muscle fibers. Nature. 260:
4. Berne `che, S., and B. Roux. 2001. Energetics of ion conduction through
the Kþchannel. Nature. 414:73–77.
5. Berne `che, S., and B. Roux. 2003. A microscopic view of ion conduc-
tion through the Kþchannel. Proc. Natl. Acad. Sci. USA. 100:
6. Brelidze, T. I., X. Niu, and K. L. Magleby. 2003. A ring of eight
conserved negatively charged amino acids doubles the conductance
of BK channels and prevents inward rectification. Proc. Natl. Acad.
Sci. USA. 100:9017–9022.
7. Carvacho, I., W. Gonzalez, ., R. Latorre. 2008. Intrinsic electrostatic
potential in the BK channel pore: role in determining single channel
conductance and block. J. Gen. Physiol. 131:147–161.
8. D’Avanzo, N., H. C. Cho, ., P. H. Backx. 2005. Conduction through
the inward rectifier potassium channel, Kir2.1, is increased by nega-
tively charged extracellular residues. J. Gen. Physiol. 125:493–503.
9. Doyle, D. A., J. Morais Cabral, ., R. MacKinnon. 1998. The structure
of the potassium channel: molecular basis of Kþconduction and
selectivity. Science (NY). 280:69–77.
10. Imoto, K., C. Busch, ., S. Numa. 1988. Rings of negatively charged
amino acids determine the acetylcholine receptor channel conductance.
11. Jiang, Y., A. Lee, ., R. MacKinnon. 2002. The open pore conforma-
tion of potassium channels. Nature. 417:523–526.
12. Jogini, V., and B. Roux. 2005. Electrostatics of the intracellular
vestibule of Kþchannels. J. Mol. Biol. 354:272–288.
Biophysical Journal 99(9) 2863–2869
2868 Contreras et al.
13. Morais-Cabral, J. H., Y. Zhou, and R. MacKinnon. 2001. Energetic Download full-text
optimization of ion conduction rate by the Kþselectivity filter. Nature.
14. Nimigean, C. M., J. S. Chappie, and C. Miller. 2003. Electrostatic
tuning of ion conductance in potassium channels. Biochemistry.
15. Roux, B. 1997. Influence of the membrane potential on the free energy
of an intrinsic protein. Biophys. J. 73:2980–2989.
16. Roux, B., and R. MacKinnon. 1999. The cavity and pore helices in the
KcsA Kþchannel: electrostatic stabilization of monovalent cations.
Science (NY). 285:100–102.
17. Sobolevsky, A. I., M. V. Yelshansky, and L. P. Wollmuth. 2005. State-
dependent changes in the electrostatic potential in the pore of a GluR
channel. Biophys. J. 88:235–242.
18. Zhou, Y., and R. MacKinnon. 2003. The occupancy of ions in the Kþ
conformational change underlie high conduction rates. J. Mol. Biol.
19. Zhou, Y., J. H. Morais-Cabral, ., R. MacKinnon. 2001. Chemistry of
ion coordination and hydration revealed by a Kþchannel-Fab complex
at 2.0 A˚resolution. Nature. 414:43–48.
20. Roux, B., S. Berne `che, and W. Im. 2000. Ion channels, permeation, and
electrostatics: insight into the function of KcsA. Biochemistry.
21. Armstrong, C. M. 1971. Interaction of tetraethylammonium ion
derivatives with the potassium channels of giant axons. J. Gen. Physiol.
22. Neyton, J., and C. Miller. 1988. Discrete Ba2þblock as a probe of ion
occupancy and pore structure in the high-conductance Ca2þ-activated
Kþchannel. J. Gen. Physiol. 92:569–586.
23. Neyton, J., and C. Miller. 1988. Potassium blocks barium permeation
through a calcium-activated potassium channel. J. Gen. Physiol.
24. Kutluay, E., B. Roux, and L. Heginbotham. 2005. Rapid intracellular
TEA block of the KcsA potassium channel. Biophys. J. 88:1018–1029.
25. Lu, Z. 2004. Mechanism of rectification in inward-rectifier Kþ
channels. Annu. Rev. Physiol. 66:103–129.
26. Contreras, J. E., D. Srikumar, and M. Holmgren. 2008. Gating at the
selectivity filter in cyclic nucleotide-gated channels. Proc. Natl.
Acad. Sci. USA. 105:3310–3314.
27. Shi, N., S. Ye, ., Y. Jiang. 2006. Atomic structure of a Naþ- and Kþ-
conducting channel. Nature. 440:570–574.
28. Alam, A., and Y. Jiang. 2009. High-resolution structure of the open
NaK channel. Nat. Struct. Mol. Biol. 16:30–34.
29. Hamill, O. P., A. Marty, ., F. J. Sigworth. 1981. Improved patch-
clamp techniques for high-resolution current recording from cells
and cell-free membrane patches. Pflugers Arch. 391:85–100.
30. Canutescu, A. A., A. A. Shelenkov, and R. L. Dunbrack, Jr. 2003.
A graph-theory algorithm for rapid protein side-chain prediction.
Protein Sci. 12:2001–2014.
31. Fiser, A., and A. Sali. 2003. MODLOOP: automated modeling of loops
in protein structures. Bioinformatics. 19:2500–2501.
32. Brooks, B. R., R. E. Bruccoleri, ., K. Martin. 1983. CHARMM:
a program for macromolecular energy, minimization, and dynamics
calculations. J. Comput. Chem. 4:187–217.
33. Im, W., D. Beglov, and B. Roux. 1998. Continuum solvation model:
computation of electrostatics forces from numerical solutions to Pois-
son-Boltzmann equation. Comput. Phys. Commun. 111:59–75.
34. Akabas, M. H., D. A. Stauffer, ., A. Karlin. 1992. Acetylcholine
receptor channel structure probed in cysteine-substitution mutants.
Science (NY). 258:307–310.
35. Lu, Q., and C. Miller. 1995. Silver as a probe of pore-forming residues
in a potassium channel. Science (NY). 268:304–307.
36. Yellen, G., D. Sodickson, ., M. E. Jurman. 1994. An engineered
cysteine in the external mouth of a Kþchannel allows inactivation to
be modulated by metal binding. Biophys. J. 66:1068–1075.
37. Benndorf, K., R. Koopmann, ., U. B. Kaupp. 1999. Gating by cyclic
GMPand voltage in the alpha subunit of the cyclic GMP-gated channel
from rod photoreceptors. J. Gen. Physiol. 114:477–490.
38. Eismann, E., F. Mu ¨ller, ., U. B. Kaupp. 1994. A single negative
charge within the pore region of a cGMP-gated channel controls
rectification, Ca2þblockage, and ionic selectivity. Proc. Natl. Acad.
Sci. USA. 91:1109–1113.
39. Root, M. J., and R. MacKinnon. 1993. Identification of an external
divalent cation-binding site in the pore of a cGMP-activated channel.
40. Armstrong, C. M. 1969. Inactivation of the potassium conductance and
related phenomena caused by quaternary ammonium ion injection in
squid axons. J. Gen. Physiol. 54:553–575.
41. Contreras, J. E., and M. Holmgren. 2006. Access of quaternary ammo-
nium blockers to the internal pore of cyclic nucleotide-gated channels:
implications for the location of the gate. J. Gen. Physiol. 127:481–494.
42. Guo,D., andZ. Lu. 2000.MechanismofcGMP-gatedchannel blockby
intracellular polyamines. J. Gen. Physiol. 115:783–798.
43. Menini, A. 1990. Currents carried by monovalent cations through
cyclic GMP-activated channels in excised patches from salamander
rods. J. Physiol. 424:167–185.
44. Zimmerman, A. L., and D. A. Baylor. 1992. Cation interactions within
the cyclic GMP-activated channel of retinal rods from the tiger sala-
mander. J. Physiol. 449:759–783.
45. Frings, S., J. W. Lynch, and B. Lindemann. 1992. Properties of cyclic
nucleotide-gated channels mediating olfactory transduction. Activa-
tion, selectivity, and blockage. J. Gen. Physiol. 100:45–67.
46. Furman, R. E., and J. C. Tanaka. 1990. Monovalent selectivity of the
cyclic guanosine monophosphate-activated ion channel. J. Gen.
47. Qu, W., A. J. Moorhouse, ., P. H. Barry. 2001. Anomalous
mole-fraction effectsin recombinant and nativecyclic nucleotide-gated
channels in rat olfactory receptor neurons. Proc. Biol. Sci. 268:
48. Armstrong, C. M., and L. Binstock. 1965. Anomalous rectification in
the squid giant axon injected with tetraethylammonium chloride.
J. Gen. Physiol. 48:859–872.
49. Guo, D., and Z. Lu. 2003. Interaction mechanisms between polyamines
and IRK1 inward rectifier Kþchannels. J. Gen. Physiol. 122:485–500.
50. Guo, D., Y. Ramu, ., Z. Lu. 2003. Mechanism of rectification in
inward-rectifier Kþchannels. J. Gen. Physiol. 121:261–275.
51. Guo, D., and Z. Lu. 2001. Kinetics of inward-rectifier Kþchannel
block by quaternary alkylammonium ions. Dimension and properties
of the inner pore. J. Gen. Physiol. 117:395–406.
52. Guo, D., and Z. Lu. 2000. Mechanism of IRK1 channel block by
intracellular polyamines. J. Gen. Physiol. 115:799–814.
53. Shin, H. G., Y. Xu, and Z. Lu. 2005. Evidence for sequential ion-
binding loci along the inner pore of the IRK1 inward-rectifier Kþ
channel. J. Gen. Physiol. 126:123–135.
54. Shin, H. G., and Z. Lu. 2005. Mechanism of the voltage sensitivity of
IRK1 inward-rectifier Kþchannel block by the polyamine spermine.
J. Gen. Physiol. 125:413–426.
55. Xu, Y., H. G. Shin, ., Z. Lu. 2009. Physical determinants of strong
voltage sensitivity of Kþchannel block. Nat. Struct. Mol. Biol.
56. Martı ´nez-Franc ¸ois, J. R., and Z. Lu. 2010. Intrinsic versus extrinsic
voltage sensitivity of blocker interaction with an ion channel pore.
J. Gen. Physiol. 135:149–167.
Biophysical Journal 99(9) 2863–2869
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