Domain III regulates N-type (CaV2.2) calcium channel closing kinetics
Viktor Yarotskyy,1Guofeng Gao,2,4Blaise Z. Peterson,2and Keith S. Elmslie1,3
Departments of1Anesthesiology and2Cellular and Molecular Physiology, and4Medicine, Penn State University, Hershey,
Pennsylvania; and3Department of Pharmacology, Kirksville College of Osteopathic Medicine, AT Still University of Health
Sciences, Kirksville, Missouri
Submitted 28 October 2011; accepted in final form 26 December 2011
Yarotskyy V, Gao G, Peterson BZ, Elmslie KS. Domain III
regulates N-type (CaV2.2) calcium channel closing kinetics. J Neuro-
physiol 107: 1942–1951, 2012. First published December 28, 2011;
doi:10.1152/jn.00993.2011.—CaV2.2 (N-type) and CaV1.2 (L-type)
calcium channels gate differently in response to membrane depolar-
ization, which is critical to the unique physiological functions medi-
ated by these channels. We wondered if the source for these differ-
ences could be identified. As a first step, we examined the effect of
domain exchange between N-type and L-type channels on activation-
deactivation kinetics, which were significantly different between these
channels. Kinetic analysis of chimeric channels revealed N-channel-
like deactivation for all chimeric channels containing N-channel
domain III, while activation appeared to be a more distributed func-
tion across domains. This led us to hypothesize that domain III was an
important regulator of N-channel closing. This idea was further
examined with R-roscovitine, which is a trisubstituted purine that
slows N-channel deactivation by exclusively binding to activated
N-channels. L-channels lack this response to roscovitine, which al-
lowed us to use N-L chimeras to test the role of domain III in
roscovitine modulation of N-channel deactivation. In support of our
hypothesis, all chimeric channels containing the N-channel domain III
responded to roscovitine with slowed deactivation, while those chi-
meric channels with L-channel domain III did not. Thus a combina-
tion of kinetic and pharmacological evidence supports the hypothesis
that domain III is an important regulator of N-channel closing. Our
results support specialization of gating functions among calcium
CaV1.2; chimera; deactivation; L-type calcium channel; roscovitine
N-TYPE (CaV2.2) calcium channels are complex membrane
proteins that consist of one major pore-forming ?1B- plus
auxiliary ?- and ?2?-subunits (Catterall et al. 2005). These
channels play a crucial role in central and peripheral nervous
systems by controlling neuronal excitability and the release of
neurotransmitters and hormones (Harsing et al. 1992; Hirning
et al. 1988; Shimosawa et al. 2004; Wheeler et al. 1994).
N-channel activity mediates neuropathic pain (Saegusa et al.
2001, 2002), and N-channel-targeted drugs such as ?-cono-
toxin MVIIA (ziconotide, SNX-111, or Prialt) and ?-conotoxin
CVID (AM336) are promising analgesics (Elmslie 2004; Mc-
Givern 2006; Snutch 2005; Wermeling and Berger 2006).
Despite the crucial function, our understanding of N-channels
largely comes from similarity comparison with other voltage-
By analogy with results from work on potassium and sodium
channels, it is assumed that the transmembrane (TM) segment
S4 moves in response to membrane depolarization to activate
voltage-dependent calcium channels (Bezanilla 2000). The
voltage sensors appear to be linked to S6, which, in elegant
experiments by the Yang group, has been shown to be involved
in calcium channel opening and is likely the intracellular
activation gate (Xie et al. 2005; Zhen et al. 2005). Thus we
have a general idea of how voltage-dependent calcium chan-
nels activate and deactivate. However, we do not know how
these channels achieve such gating diversity. L-type calcium
channels typically gate with brief open times and a low open
probability (Po) (maximum ? 0.2), while N-type and P/Q-type
channels gate with high Po(maximum ? 0.8–0.9) and long
open times (Colecraft et al. 2001; Elmslie 1997; Forti and
Pietrobon 1993; Hess et al. 1984; Lee and Elmslie 1999; Marks
and Jones 1992). In addition, L-channels show a voltage-
independent open state (Marks and Jones 1992), while the
N-channel open state is voltage dependent (Colecraft et al.
2001; Lee and Elmslie 1999). One possible explanation for
these gating differences is domain “specialization,” which has
been proposed for voltage-dependent sodium channels (Chen
et al. 1996; Horn et al. 2000). Sodium channel fast inactivation
has been isolated to the intracellular loop between domains III
and IV (Patton et al. 1992, 1993), and strong evidence supports
domain IV S4 as the specialized voltage sensor for fast inac-
tivation (Chen et al. 1996; Horn et al. 2000). Like sodium
channels, we postulate that calcium channels have evolved
domain specialization. Indeed, previous studies have demon-
strated that activation kinetics of the skeletal muscle L-channel
(CaV1.1) can be transferred to the cardiac L-channel (CaV1.2)
by CaV1.1 domain I, and vice versa (Tanabe et al. 1991). This
suggests that domain I could be an important controller of
We investigated the domain specialization hypothesis by
examining activation-deactivation kinetics of chimeric calcium
channels comprising domains from N-type and L-type chan-
nels, which showed that deactivation kinetics were correlated
with domain III. This result was further supported by studies
using R-roscovitine, which was recently discovered to slow
deactivation of CaV2 (P/Q-, N-, and R-type) channels (Buraei
et al. 2007; Yarotskyy and Elmslie 2007; Yarotskyy et al.
2009). R-roscovitine is a trisubstituted purine that was origi-
nally developed as a cyclin-dependent kinase (CDK) inhibitor
(Meijer 1996). However, the roscovitine-induced slowed de-
activation of N-type channels provides previously unavailable
opportunities to achieve insights into structures that control
channel closing. Since L-channel deactivation is not affected
by roscovitine, we used L-N chimeric channels to demonstrate
that the N-channel domain III is required for roscovitine-
induced slowed deactivation. Our kinetic and pharmacological
evidence supports domain III as a primary controller of cal-
Address for reprint requests and other correspondence: K. Elmslie, Dept. of
Pharmacology, Kirksville Coll. of Osteopathic Medicine, AT Still Univ., 800
W. Jefferson St., Kirksville, MO 63501 (e-mail: email@example.com).
J Neurophysiol 107: 1942–1951, 2012.
First published December 28, 2011; doi:10.1152/jn.00993.2011.
19420022-3077/12 Copyright © 2012 the American Physiological Society www.jn.org
cium channel deactivation and suggests that this domain may
play a dominant role in generating the high-POgating charac-
teristic of N-type channels (Colecraft et al. 2001; Lee and
MATERIALS AND METHODS
Construction of chimeric channels. Chimeric calcium channels
were constructed with cDNAs encoding the rabbit cardiac L-channel
(CaV1.2, GenBank Accession No. X15539) and rat N-channel
(CaV2.2, GenBank Accession No. AF055477; generously provided by
Dr. Leslie Parent) ?1-subunits. Chimeric channels were generated as
previously described (Yarotskyy et al. 2010). The overall integrity of
each chimera was confirmed by qualitative restriction enzyme digests
and DNA sequencing. For convenience, we termed ?1-domains by
channel type (L or N) and position (I–IV). For example, L-DIII refers
to L-type calcium channel domain III. Figure 1 shows a schematic of
the chimeric channels used for this study.
HEK cell transfection. We utilized the calcium phosphate precip-
itation method to exogenously express channels in HEK293 cells
(Wang et al. 2005; Yarotskyy et al. 2010). HEK293 cells were
maintained in standard DMEM-GlutaMAX medium containing 10%
fetal bovine serum (FBS) and 1% antibiotic-antimycotic (regular
medium) at 37°C in a 5% CO2incubator. Cells were transfected with
11 ?g of ?1-subunit (L-, N-, or chimeric channel), 8.5 ?g of ?2?, 5.5
?g of ?1b, 2.15 ?g of TAG (SV40 large T-antigen, to increase
expression efficiency), and 1 ?g of green fluorescent protein (GFP; to
visualize transfected cells). The transfected cells were split into
35-mm dishes that served as the recording chamber.
Measurement of ionic currents. Cells were voltage clamped with
the whole cell configuration of the patch-clamp technique. Pipettes
were pulled from Schott 8250 glass (King Precision Glass, Claremont,
CA) on a Sutter P-97 puller (Sutter Instruments, Novato, CA).
Currents were recorded with an Axopatch 200A amplifier (Molecular
Devices, Sunnyvale, CA) and digitized with ITC-18 data acquisition
interface (Instrutech, Port Washington, NY). Experiments were con-
trolled by a Power Macintosh G3 computer (Apple Computer, Cuper-
tino, CA) running S5 data acquisition software written by Dr. Stephen
Ikeda (National Institute on Alcohol Abuse and Alcoholism, National
Institutes of Health, Bethesda, MD). Leak current was subtracted
online with a ?P/4 protocol. The impact of series resistance (correc-
Fig. 1. Chimeric channels construction scheme. Domains I
through IV are shown, and each domain is shown as a set of 6
transmembrane segments 1 through 6 (S1–S6). The “?” in each
S4 indicates that this segment serves as the voltage sensor. The
loops between the segments and domains are shown as lines.
N-channel structures are shown in black, while L-channels are
in gray. Numbers at the chimeric connections show the number
of amino acid residues within that loop contributed by a given
channel type. To highlight the connection, the N-channel part of
the loop is shown as a thicker line. For the NN*LN chimera,
transmembrane S1–S4 in domain II are from the N-channel,
while S5–S6 are from the L-channel. The loop between S4 and
S5 is from the L-channel.
1943DOMAIN III REGULATES N-CHANNEL KINETICS
J Neurophysiol • doi:10.1152/jn.00993.2011 • www.jn.org
tive) and whole cell capacitance (predictive) on the whole cell voltage
clamp was compensated by at least 80% with circuitry of the Axo-
patch 200A. All recordings were carried out at room temperature, and
the holding potential was ?120 mV. Whole cell currents were
digitized at 50 kHz after analog filtering at 5–10 kHz.
Data analysis. Data were analyzed with IgorPro (WaveMetrics,
Lake Oswego, OR) running on a Macintosh computer. The voltage
dependence of channel activation was measured from tail currents by
averaging for 0.3 ms beginning 0.3 ms after repolarization to ?60 mV
from voltages ranging from ?80 to 80 mV. The tail current amplitude
was plotted versus step voltage and fitted by a Boltzmann function,
which yielded half-activation voltage (V1/2) and slope factor. Activa-
tion and deactivation kinetics were determined with single-exponen-
tial fitting as previously described (Buraei et al. 2005). Group data are
calculated as means ? SD throughout this report. Paired t-test was
used for within-cell comparisons. One-way ANOVA with Tukey
honestly significant difference (HSD) post hoc test was used to test for
differences among three or more independent groups. Significant
differences required P ? 0.05.
Solutions. The internal pipette solution contained (in mM) 104
NMG-Cl, 14 creatine-PO4, 6 MgCl2, 10 NMG-HEPES, 5 Tris-ATP,
0.3 Tris-GTP, and 10 NMG-EGTA, with osmolarity of 280 mosM and
pH of 7.3. The external recording solution contained (in mM) 30
BaCl2, 100 NMG-Cl, and 10 NMG-HEPES, with osmolarity of 300
mosM and pH of 7.3. The mean liquid-liquid junction potential was
?2.7 mV, and we did not compensate for this potential in our
analysis. Importantly, the internal and external solutions were identi-
cal for all recordings so that the junction potential was the same for all
chimeric channels examined. The 30 mM Ba2?concentration in the
external solution was used to ensure that current could be measured
from all chimeric channels under the same conditions, since some
chimeras generate small currents. Higher divalent cation concentra-
tions induce a depolarizing shift in voltage-dependent properties
(Elmslie et al. 1994; Liang and Elmslie 2001), but channel activation
and deactivation kinetics are normal after adjusting for this voltage
shift (Zhou and Jones 1995). R-roscovitine was prepared as a 50 mM
stock solution in DMSO and stored at ?30°C. All external solutions
contained the same DMSO concentration so that the roscovitine
concentration was the sole variable when changing solutions. Test
solutions were applied from a gravity-fed perfusion system with an
exchange time of 1–2 s.
Chemicals. R-roscovitine was from LC Labs (Woburn, MA). DMEM,
FBS, and antibiotic/antimycotic were from Invitrogen (Carlsbad, CA).
Other chemicals were obtained from Sigma (St. Louis, MO).
N-channel-like deactivation follows domain III. It has long
been known that N-channels show several unique gating char-
acteristics relative to L-channels such as high Poand long open
times (Lee and Elmslie 1999; Marks and Jones 1992). In
addition, N-channels show voltage-dependent open times,
while L-channel open times do not change with voltage (Lee
and Elmslie 1999; Marks and Jones 1992). Since the mecha-
nisms that underlie these gating differences are not understood,
we wondered if N-L chimeras could be used to gain insights
into these differences. As a first step toward understanding the
root of these differences, we examined whole cell current
activation and deactivation kinetics to determine whether cer-
tain kinetic properties could be isolated to single domains. For
wild-type (wt) N- and L-channels, both activation and deacti-
vation time constants (?) were different at certain voltages, but
differences at single voltages are highly susceptible to many
factors that make them unreliable for our purposes. For this
reason, we compared these values over a range of voltages to
determine the e-fold change with voltage (Ve) (Buraei et al.
2005). The Vefor both activation and deactivation ? was
significantly smaller (i.e., steeper voltage dependence) for wt
N-type vs. L-type channels (Fig. 2). For activation ? averaged
from six cells for N-channels and seven cells for L-channels,
Ve(?ActVe) was 34 mV for N-channels versus 125 mV for
L-channels, whereas deactivation ? Ve(?DeactVe) from fitting
averaged results (n ? 5 for N-channels and n ? 5 for L-chan-
nels) yielded 28 mV for N-channels versus 47 mV for L-chan-
nels (Fig. 2). Statistical analysis of ?ActVeand ?DeactVe
(averaged from fitting single cells) showed that both values
significantly differed between N-type and L-type channels (see
Fig. 4). The steeper voltage dependence of N-channel ?Deact
likely reflects the voltage dependence of the N-channel open
state (Colecraft et al. 2001; Lee and Elmslie 1999).
LLNN was the first chimera we tested to determine whether
swapping half of each channel would produce gating changes
that would align with either wt N- or L-channels. The LLNN
chimera showed activation and deactivation kinetics that were
similar to those of the wt N-channel and were significantly
different from those of wt L-channels (see Figs. 1, 3, and 4),
which focused our attention on N-DIII and N-DIV. As we have
noted previously, chimeric channels that carried N-DII (includ-
ing NNLL) failed to generate current (Yarotskyy et al. 2010).
Fortunately, all chimeric channels with L-DII were functional,
which allowed us to test the role of N-DIII and/or N-DIV in
activation-deactivation. L-channel chimeras containing one or
two N-channel domains showed that ?DeactVewas statistically
different between chimeras containing N-DIII (NNNN, LLNN,
and LLNL) and L-DIII (LLLL, NLLL, and LLLN) (Fig. 3, 4B).
Thus the inclusion of N-DIII into L-channel backbone (LLNL)
made ?DeactVeN-channel-like, while the LLLN chimera
Fig. 2. Comparison of the whole cell kinetics of N- and L-current. N- and
L-channels were transiently expressed in HEK293 cells along with ?1b- and
?2?-subunits. A: example currents were recorded in 30 mM Ba2?from cells
expressing either L (LLLL)- or N (NNNN)-channels. Currents were activated
during 25-ms steps to ?20 mV, and tail currents were measured at ?60 mV.
B: the time constant of activation (?Act) was determined by fitting activation
with a single-exponential equation after a 0.3-ms delay and is plotted vs. step
voltage. The smooth lines are single-exponential fits to determine the e-fold
change in ? with voltage (Ve). NNNN ?Act(n ? 6) changed e-fold for 34 mV
(black line), and LLLL ?Act(n ? 7) changed e-fold for 125 mV (gray line).
C: the time constant of deactivation (?Deact) was determined by fitting tail
currents to a single-exponential equation and is plotted vs. tail voltage. The
smooth lines were generated as described in B. NNNN ?Deact(n ? 5) changed
e-fold for 28 mV (black line), and that for LLLL (n ? 5) changed e-fold for
47 mV (gray line).
1944DOMAIN III REGULATES N-CHANNEL KINETICS
J Neurophysiol • doi:10.1152/jn.00993.2011 • www.jn.org
showed L-channel-like ?DeactVe. Interestingly, ?ActVewas
significantly different from that of the wt L-channel for all
chimeric channels tested (Fig. 4A). Activation kinetics are
more influenced by closed-closed transition rates compared
with deactivation kinetics (Yarotskyy and Elmslie 2009),
which suggests that the insertion of any N-channel domain can
increase transition rates among closed states. However, another
measure of activation was strongly affected by N-DIII and
N-DIV. N-channels (NNNN) show a significantly steeper ac-
tivation voltage dependence (Boltzmann slope factor) that was
only reproduced in the LLNN chimera (Fig. 4D). The Boltz-
mann half-activation voltage (V1/2) was not correlated with any
single domain switch between N-type and L-type channels,
which likely results from this parameter being sensitive to both
closed state vs. open state stability and the voltage dependence
of activation/deactivation (Logothetis et al. 1993).
These results supported N-DIII as the mediator of the steeper
voltage dependence of N-channel closing. We wanted to fur-
ther test this idea by measuring the kinetic parameters of the
NNLN chimera, which we expected to show L-type ?DeactVe.
However, the NNLN chimera contained N-DII, which pre-
vented the expression of functional channels (Yarotskyy et al.
Fig. 3. Typical currents recorded from chimeric channels. All
panels comprise the current example (top) and a voltage record
(bottom). Vertical and horizontal lines show scales for current
and time, respectively. Currents are shown for LLNN (A),
LLNL (B), NN*LN (C), LLLN (D), and NLLL (E) channels.
Fig. 4. The N-channel domain III dictates deactivation
gating. A: ?ActVewas determined as described in Fig. 2.
All channels containing N-channel domains showed a
significantly different ?ActVecompared with that from
wild-type (wt) L-type channels (LLLL). B: ?DeactVe
values for NNNN, LLNN, and LLNL were significantly
different from LLLN, NLLL, NN*LN, and LLLL chan-
nels. Half-activation voltage (V1/2, C) and slope (D)
were determined from single Boltzmann equation fits to
the activation current-voltage relationship (I-V) as de-
scribed in Fig. 5. V1/2was similar for all channels except
the LLNL and LLLN chimeras. Significant differences
for all comparisons were determined by ANOVA with
Tukey honestly significant difference (HSD) post hoc
test (P ? 0.05). Lowercase letters above or below each
column indicate the data that differ significantly with
NNNN (a), LLNN (b), LLNL (c), LLLN (d), NLLL (e),
NN*LN (f), and LLLL (g). The number of cells tested
is shown for each column.
1945 DOMAIN III REGULATES N-CHANNEL KINETICS
J Neurophysiol • doi:10.1152/jn.00993.2011 • www.jn.org
2010). In an effort to isolate the N-DII structures involved in
channel dysfunction, we generated hemidomain channels con-
taining domain II with N-type S1–S4 and L-type S5–S6 (Fig.
1), which we found could generate functional channels (Yar-
otskyy et al. 2010). This hemidomain construct is designated
N* throughout, and it allowed us to make NN*LN to test our
prediction. Indeed, we found that the ?DeactVeof this chimera
was statistically similar to that of wt L-channels, which sup-
ports the idea that domain III may be specialized to control
calcium channel closing. ?ActVefor this chimera was statisti-
cally similar to N-channels (Fig. 4A), which further supports
the idea that the control of activation may be more distributed
among the domains. The Boltzmann slope factor for NN*LN
was similar to that of the L-type channels, which further
supports a role for both N-DIII and N-DIV in establishing the
steeper voltage dependence of activation observed for wt
N-channels (Fig. 4D). Together our results support the idea that
domain III may be specialized to control calcium channel
closing and, thus, contribute to the gating differences between
L-type and N-type channels.
The ?2?-subunit fails to modulate N-channel closing. Under
control conditions, N-channels rapidly deactivate upon hyper-
polarization. However, roscovitine, a trisubstituted purine, can
slow deactivation of CaV2 channels by stabilizing high-Po
open state (Buraei et al. 2005, 2007). This effect was accom-
panied by an increase in the ?DeactVe, left shift in the activation
vs. voltage relationship (?V1/2) (Buraei et al. 2005, 2007), and
slowed gating charge relaxation (Yarotskyy and Elmslie 2009).
Thus roscovitine provides us with another method to probe
structures that control N-channel closing. The requirement of
CaV2 channel activation for the roscovitine-induced slowed
deactivation, as well as the fact that externally but not inter-
nally applied roscovitine induced the effect, suggested an
externally oriented binding site (Buraei et al. 2005, 2007).
While we had previously proposed that the binding site resided
on the ?1B-subunit, the associated ?2?-subunit is extracellu-
larly exposed, and is thought to be the target for clinically
relevant compounds such as gabapentin to affect channel
activity (Brown and Gee 1998). Thus, before testing roscovi-
tine on chimeric calcium channels, we needed to show that the
relevant site was not on the ?2?-subunit, which was tested by
expressing ?1B- and ?1b-subunits in HEK293 cells with and
without ?2?. The absence of the ?2?-subunit failed to affect
any of the monitored N-channel gating parameters. The acti-
vation vs. voltage relationship V1/2was 19.8 ? 7.1 mV (n ?
14) vs. 22.7 ? 4.1 [n ? 4, not significantly different (ns)] and
the Boltzmann slope factor was 10.9 ? 2.3 vs. 12.3 ? 3.1 (ns)
with or without ?2?, respectively. The ?ActVewas 31.2 ? 12.3
(n ? 12) vs. 38.8 ? 14.6 (n ? 5, ns) and ?DeactVewas 28.7 ?
3.3 (n ? 11) vs. 31.0 ? 6.3 (n ? 5, ns) with and without ?2?,
respectively. The absence of the ?2?-subunit also had no effect
on roscovitine-induced slowed deactivation (Fig. 5). Roscovi-
tine at 100 ?M induced a shift in V½,with the average ?V1/2?
?8.2 ? 5.5 mV with ?2? (n ? 14) and ?6.5 ? 3.2 without ?2?
(n ? 4, ns), and ?DeactVewas significantly increased with
roscovitine relative to control, with the percent change in ?Deact
Ve? 152 ? 56% with ?2? (n ? 11) and 196 ? 56% without
?2? (n ? 5, ns). These results show that the ?2?-subunit does
not impact the roscovitine-induced slowed deactivation and
support the idea that roscovitine binds to the N-channel ?1-
subunit to affect deactivation. Thus testing roscovitine on the
chimeric channels will provide further insights into the do-
main(s) that control N-channel closing.
N-DIII mediates roscovitine-induced slowed deactivation.
We have previously established that roscovitine failed to slow
L-channel deactivation (Buraei et al. 2007), which allowed us
to use our chimera strategy to determine the N-channel do-
main(s) that mediate slowed deactivation. To facilitate com-
parisons with the chimeric channels, the effect of roscovitine
on L-channel gating is shown in Fig. 6A, and Fig. 5 shows the
effect on wt N-channel gating. The LLNN chimera was used to
localize the agonist effect to half of the channel. The current
generated by this chimera clearly shows roscovitine-induced
slowed deactivation along with an increased ?DeactVe, which
completely contrasts with the effect of roscovitine on the wt
L-channel (Fig. 6). The roscovitine-induced change in ?DeactVe
of the LLNN chimera was significantly different from that of
wt L-channels but statistically similar to that of wt N-channels
Fig. 5. Roscovitine-induced slowed deactivation results from interaction with
the ?1B-subunit. Data are shown from HEK293 cells expressing ?1B-, ?2?-,
and ?1b-subunits (??2?, left) and ?1B- and ?1b-subunits (??2?, right). A: 100
?M roscovitine induced a left shift in the activation I–V for both groups. The
data were fitted by a single Boltzmann function (smooth line) to determine V1/2
and Boltzmann slope factor. Tail current (ITail) amplitudes were scaled (nor-
malized) by the maximum value from the Boltzmann equation fit. Boltzmann
fitting values for control (Cntl), 100 ?M roscovitine (Rosc), and washout
(WO) were as follows: ??2?: V1/2? 19.7, 4.7, 20.6 mV and slope ? 12.8, 8.0,
12.5; ??2?: V1/2? 27.6, 8.7, 29.5 mV and slope ? 11.5, 8.7, 11.2, respec-
tively. B: roscovitine slowed deactivation of tail currents and increased the
?DeactVe. Tail currents were fit by a single-exponential function to obtain ?Deact
for each tail voltage. The plot of ?Deactvs. voltage was fit by using a
single-exponential equation to determine the e-fold change in ?Deactwith
voltage (Ve). ?DeactVefor Cntl, Rosc, and WO were as follows: ??2?: ?25.9,
?77.4, ?26.2 mV; ??2?: ?23.8, ?80.9, ?24.6 mV, respectively. C: repre-
sentative traces show the roscovitine-induced slowed deactivation (black
traces) compared with control and washout (gray traces) for HEK293 cells
expressing ?lB-subunits with or without ?2?-subunits.
1946 DOMAIN III REGULATES N-CHANNEL KINETICS
J Neurophysiol • doi:10.1152/jn.00993.2011 • www.jn.org
(see Fig. 8A). Interestingly, roscovitine failed to shift the
activation-voltage relationship in the LLNN chimera (Fig. 6B;
see Fig. 8B). As discussed below, this likely results from the
additional effects of roscovitine on the L-channel half of the
chimera (Yarotskyy et al. 2010). These results localize the struc-
tures involved in roscovitine-induced slowed deactivation to
N-DIII and/or N-DIV.
Single domain substitutions were used to further localize the
structures involved with slowed deactivation. Figures 6 and 7
clearly illustrate that the LLNL chimera shows an N-channel-
like response to roscovitine, while the LLLN chimera does not.
Deactivation of LLNL channels was slowed by application of
100 ?M roscovitine, which resulted in a significant increase in
?DeactVefrom 24.8 ? 1.9 mV (average of control and washout)
to 55.3 ? 5.2 mV (P ? 0.01, n ? 5; Fig. 7A). The roscovitine-
induced change in ?DeactVefor the LLNL chimera was signif-
icantly larger than that for wt L-channels and chimeric chan-
nels that lacked N-III (Fig. 8A). However, the percent change
in ?DeactVewas significantly smaller than that for either wt
N-channels or the LLNN chimera (Fig. 8A). In contrast, ros-
covitine failed to affect deactivation of the LLLN chimera (Fig.
6, Fig. 8A) and ?DeactVewas not significantly different between
control (43.3 ? 9.0 mV; average of control and washout) and
roscovitine (45.4 ? 7.7 mV; n ? 6) for this chimera. Surpris-
ingly, roscovitine significantly right-shifted the activation vs.
voltage relationship of the LLNL chimera (?V1/2was 14.8 ?
1.8 mV, P ? 0.001, n ? 4) (Fig. 7A, Fig. 8B), which was
unique among all the channels tested. This effect likely results
from the effect of roscovitine on the L-channel domains within
the chimera, namely, slowed activation (Yarotskyy et al. 2010).
We also examined the NLLL chimera and found that rosco-
vitine failed to affect deactivation as expected from our results
from the LLNN chimera (Fig. 8A). To support the idea that
N-DIII is both necessary and sufficient for roscovitine-induced
slowed deactivation, we tested the NN*LN chimera (Fig. 7B).
As we predicted, 100 ?M roscovitine failed to affect deacti-
vation of the NN*LN channel (Figs. 7 and 8). Thus we
conclude that domain III controls N-channel closing.
As a first step toward understanding the structures that
generate the kinetics differences between N-type and L-type
channels, we utilized N-L chimeras to determine whether
certain kinetic parameters could be isolated to single domains.
Analysis of the kinetic properties of these channels demon-
strated that the N-channel characteristic of steeper ?Deactvolt-
age dependence was associated with N-DIII, whereas the
steeper voltage dependence of activation (Boltzmann slope
factor) appeared to require both N-DIII and N-DIV. The
difference in ?ActVebetween N-type and L-type channels was
not correlated with any domains and thus appears to be a more
distributed function. We and others have previously demon-
strated that roscovitine slows deactivation of P/Q-, N-, and
R-type calcium channels, which results in elevated action
potential-induced calcium influx (Buraei et al. 2005, 2007; Cho
and Meriney 2006; Yan et al. 2002). We show here that N-DIII
is both necessary and sufficient to mediate this effect. Together
these results suggest that the calcium channel DIII is an
important controller of deactivation and imparts at least some
of the unique kinetic characteristics observed between N-type
and L-type channels.
Domain specialization. Most of our knowledge of the struc-
tures involved in ion channel gating comes from potassium
channels, which comprise four independent ?-subunits to form
the channel (MacKinnon 1991). Thus each ?-subunit must
carry all gating functions (i.e., voltage sensing, activation
gating, and inactivation gating), and each subunit appears to
contribute to channel activation (Horn et al. 2000) and inacti-
vation (Hoshi et al. 1990; Zagotta et al. 1990). On the other
hand, sodium and calcium channels have all four domains
together in one protein (Catterall 2000), which could allow for
specialization. Over the past decade evidence has accumulated
Fig. 6. Roscovitine-induced slowed deactivation is medi-
ated by N-DIII. Left: L-channel data (LLLL). Center and
right: data from LLNN and LLLN chimeras, respectively.
Please see Fig. 5 for comparative wt N-channel data. A: the
activation I-V was not shifted by 100 ?M Rosc for either
LLLL or LLNN channels, while for LLLN there was a
small positive shift. V1/2and Boltzmann slope factor were
obtained as described in Fig. 5. Values for CNTL, 100 ?M
Rosc, and WO were as follows: LLLL: V1/2? 7.9, 6.6, 9.1
mV and slope ? 12.1, 9.9, 12.9; LLNN: V1/2? 9.1, 8.2, 8.3
mV and slope ? 9.9, 8.5, 8.0; LLLN: V1/2? 45.0, 48.3,
43.3 mV and slope ? 16.7, 16.1, 15.0, respectively. B: ros-
covitine slowed deactivation of the LLNN tail current but
not that of either wt L (LLLL) or LLLN channels. Mea-
surements were done as described in Fig. 5. ?DeactVefor
CNTL, Rosc and WO were as follows: LLLL: 31.1, 31.7,
and 35.0 mV; LLNN: 17.9, 56.8, and 18.1 mV; LLLN: 35.5,
42.0, and 39.8 mV, respectively. Symbols have the same
meaning as in A. C: current traces show the slowed activa-
tion of LLLL channels (left) and slowed deactivation of the
currents from the LLNN chimera (center), but neither of
these effects was observed for LLLN chimera. Current
traces in the presence of 100 ?M Rosc are shown in black,
while those of CNTL and WO are in gray.
1947 DOMAIN III REGULATES N-CHANNEL KINETICS
J Neurophysiol • doi:10.1152/jn.00993.2011 • www.jn.org
supporting domain specialization in voltage-dependent sodium
channels. Sodium channel fast inactivation has been isolated to
the intracellular loop between domains III and IV (Patton et al.
1992, 1993), and the sodium channel DIV-S4 appears to be the
voltage sensor for fast inactivation (Chanda and Bezanilla
2002; Chen et al. 1996; Horn et al. 2000; Yang and Kuo 2003).
Thus the domains of sodium and calcium channels may have
evolved to contribute unique functions, and the identification
of a voltage-dependent calcium channel in yeast (CCH1) sug-
gests that there has been sufficient time to undergo such
evolution (Paidhungat and Garrett 1997).
There are several differences between N-type and L-type
channel kinetics that we could discern from whole cell cur-
rents. We wanted robust measures of channel function and so
rejected measurements taken at a single voltage in favor of
measurements taken across a range of voltages, which we
believed would better reflect the underlying gating processes.
One such measurement was ?ActVe, which was significantly
smaller (i.e., exhibited steeper voltage dependence) for N-type
vs. L-type channels. However, we were unable to attribute this
difference to a single domain or even two domains, since any
domain change induced a significant decrease in ?ActVecom-
pared with that of wt L-channels. This is not surprising since
this measure is very sensitive to closed state transitions (Buraei
et al. 2005; Marks and Jones 1992). Voltage-dependent chan-
nels move through multiple closed states on the pathway to
opening, and each of these voltage-dependent steps can have
an impact on ?Act. It is surprising that replacing any one of the
L-channel domains resulted in a significant change in ?ActVe.
It seems that all L-channel domains are involved in establish-
ing the weaker voltage dependence of ?Act.
It was previously demonstrated that differences in activation
? were determined by DI of the skeletal muscle (CaV1.1) and
cardiac (CaV1.2) L-channels (Tanabe et al. 1991). Activation
of CaV1.1 is an order of magnitude slower than that of CaV1.2.
Chimeric channels containing CaV1.2 DI activated with a ? that
was statistically similar to that of wt CaV1.2, while those
channels with CaV1.1 DI activated significantly slower. Thus
DI of the L-channel appears to control activation kinetics
(Tanabe et al. 1991). However, the voltage dependence of ?Act
was not examined in that study. One possibility is that DI of
CaV1.1 introduces a rate-limiting step to activation, causing
channels with this domain to activate slowly. In our case,
activation kinetics were not different between N-type and
L-type channels over a large range of voltages, so we focused
on the voltage dependence of activation in our studies.
Another significant difference between N-type and L-type
channels was the steeper steady-state activation vs. voltage-
relationship for N-channels. One might expect that this param-
eter would be very sensitive to changes in both closed-closed
and closed-open gating, which would suggest that a single
domain would have little impact. While our data showed that
introduction of a single N-channel domain into the L-channel
backbone failed to significantly alter the Boltzmann slope
factor, the introduction of L-DIII into the N-channel (NN*LN)
had a significant effect to decrease the steepness of activation
to that of the L-channel, which suggests that N-DIII is impor-
tant for this critical channel gating parameter. However, N-DIII
appears to require N-DIV to mediate steeper activation, since
only the LLNN chimera showed a slope factor that was
significantly different from that of the wt L-channels and
statistically similar to that of the wt N-channel. Thus these two
Fig. 7. The N-channel domain III is necessary and sufficient for
roscovitine-induced slowed deactivation. LLNL chimera data
are presented on left, whereas those from the NN*LN chi-
mera are shown on right. A: the activation I–V was right-shifted
by application of 100 ?M roscovitine for the LLNL chimera.
V1/2and Boltzmann slope factor were determined as descri-
bed in Fig. 5 for control (Cntl), 100 ?M roscovitine (Rosc),
and washout (WO): LLNL: V1/2? ?17.6, ?2.6, ?21.1 mV and
slope ? 12.1, 13, 10.5; NN*LN: V1/2? 6.2, 4.5, 5.4 mV and
slope ? 16.5, 13.1, 16.2, respectively. B: roscovitine slowed
deactivation of the LLNL chimera but not that of NN*LN
channels. ?DeactVein control, roscovitine, and washout were
24.9, 52.3, 25.8 mV for the LLNL chimera and 35.4, 36.9, and
31.8 for the NN*LN chimera, respectively. C: current traces in
the presence of 100 ?M roscovitine are shown in black, while
those of control and washout are in gray. Note that roscovitine
induces both slowed activation and slowed deactivation of
currents from the LLNL chimera.
1948 DOMAIN III REGULATES N-CHANNEL KINETICS
J Neurophysiol • doi:10.1152/jn.00993.2011 • www.jn.org
domains may together contribute to increased sensitivity of
N-channel activation to membrane depolarization. Interest-
ingly, each of these two N-channel domains carries one addi-
tional positively charged amino acid compared with the corre-
sponding L-channel domains, which could explain the steeper
voltage dependence of N-channel activation.
Our chimera data show that DIII is crucial to establish the
voltage dependence of calcium channel deactivation, since the
N-like ?DeactVerequired N-DIII. The steeper voltage depen-
dence of N-channel deactivation very likely reflects the volt-
age-dependent N-channel open state. Our modeling shows that
open state transitions have a strong control over N-channel
deactivation kinetics (Buraei et al. 2005; Yarotskyy and
Elmslie 2009). This suggests that DIII may be a primary
controller of calcium channel Po, which is highly dependent on
the transition rate from the open to the closed state (Wonderlin
et al. 1990).
One potential mediator of this voltage dependence to the
N-channel open state is charged amino acids within N-DIII-S4,
which has the highest number of positively charged amino
acids (6) relative to any L-channel domain (4–5) or other
N-channel domains (5). Perhaps the extra charge mediates the
increased ?DeactVeobserved in channels with N-DIII. Further
investigation of domain III could uncover the structural mech-
anisms that distinguish the gating of N-type from L-type
N-DIII is critical for roscovitine-induced slowed deactivation.
Roscovitine was initially developed as a CDK inhibitor (Meijer
1996) that has more recently been used to provide unique
insights into N-channel gating (Buraei et al. 2005; Buraei and
Elmslie 2008; DeStefino et al. 2010; Yarotskyy and Elmslie
2009, 2010). The profound slowing of N-channel closing
(Buraei et al. 2005) induced by this drug provided a good test
for our hypothesis that DIII controls N-channel closing.
Experiments on the N-L chimeric channels showed that ros-
covitine slowed deactivation in all channels containing N-DIII
(NNNN, LLNN, and LLNL). The LLNL and NN*LN chimeras
are particularly important since LLNL shows that N-DIII is
sufficient to transfer the roscovitine-induced slowed deactiva-
tion to L-type channels, while NN*LN demonstrates that
N-DIII is necessary for slowed deactivation of N-type chan-
nels. The simplest explanation that accounts for our kinetic and
roscovitine results is that DIII contains both the roscovitine
binding site and the gating machinery required for channel
closing. However, it is also possible that the roscovitine bind-
ing site is located elsewhere on the channel and is conserved
between L- and N-channels. One interesting difference we
observed is that the roscovitine-induced %??DeactVewas
significantly smaller for LLNL relative to either LLNN or the
wt N-channel, suggesting that N-DIV is required for a full
roscovitine response. However, it is clear from our data that
N-DIV does not mediate roscovitine responsiveness. This ob-
servation suggests that the gating machinery in N-DIII is
energetically coupled to molecular determinants in N-DIV, but
N-DIII alone is necessary and sufficient for roscovitine-depen-
dent changes in deactivation. Together our kinetic analysis of
the N-L chimeric channels and of the roscovitine effect on
those channels supports the hypothesis that DIII is a critical
controller of N-channel deactivation, and reveals for the first
time domain specialization for the gating of N-type calcium
We have previously demonstrated that roscovitine can in-
duce N-channel inhibition as well as slowed deactivation
(Buraei et al. 2005, 2007). This N-channel inhibition results
from enhancement of closed-state inactivation (Buraei and
Elmslie 2008), and the inhibitory effect appears to require
N-DI (Yarotskyy et al. 2010). Thus it is likely that two
roscovitine binding sites exist on the N-channel. One site in
N-DIII regulates N-channel closing, and another in N-DI
regulates closed-state inactivation.
The BayK8644-induced slowing of L-channel deactivation
is superficially similar to the roscovitine effect on N-channels.
BayK8644 binds to a site comprising both L-DIII and L-DIV,
and the site appears to be accessible via the membrane (Hock-
erman et al. 1997). The available evidence supports the rosco-
vitine-binding site within N-DIII, and we have argued that the
site is extracellularly exposed (Buraei et al. 2005, 2007). Thus
it seems unlikely that the roscovitine site is evolutionarily
related to the BayK8644 site. In addition, there are significant
differences in the effects of BayK8644 and roscovitine that
further support this idea. First, roscovitine exclusively binds
activated N-channels to slow deactivation (Buraei et al. 2005),
while BayK8633 appears to bind to closed channels to affect
gating (Hockerman et al. 1997). Second, roscovitine slows
off-gating charge movement of N-type channels (Yarotskyy
and Elmslie 2009), while drugs that slow L-channel deactiva-
tion such as BayK8644 and FPL64176 have little or no effect
on gating charge movement (Artigas et al. 2003; Fan et al.
2000; McDonough et al. 2005). Thus the evidence supports a
strong distinction between the effects of roscovitine on N-
channels and BayK8644 on L-channels.
Fig. 8. Roscovitine-induced increase in ?DeactVedepends on N-channel domain
III. A: the 100 ?M roscovitine-induced % change in ?DeactVewas determined
by averaging the Vevalues from control and washout before calculating the %
change. B: ?V1/2was determined by averaging V1/2values from control and
washout before subtracting that measured in 100 ?M roscovitine. The signif-
icant differences for all comparisons were determined by ANOVA with Tukey
HSD post hoc test (P ? 0.05). Lowercase letters above or below each column
indicate the data that differ significantly from NNNN (a), LLNN (b), LLNL
(c), LLLN (d), NLLL (e), NN*LN (f), and LLLL (g). The number of cells
tested is shown for each column.
1949 DOMAIN III REGULATES N-CHANNEL KINETICS
J Neurophysiol • doi:10.1152/jn.00993.2011 • www.jn.org
Clinical relevance of slowed deactivation. CaV2 channels
are localized within nerve terminals in both the central and
peripheral nervous systems and serve as the major pathway for
Ca2?entry that triggers neurotransmitter release (Harsing et al.
1992; Hirning et al. 1988; Shimosawa et al. 2004; Wheeler et
al. 1994). Until the identification of R-roscovitine, there were
no drugs available to enhance Ca2?influx through presynaptic
CaV2 channels (Buraei et al. 2005, 2007; Yan et al. 2002).
Several studies have demonstrated that R-roscovitine can en-
hance neurotransmitter release (Cho and Meriney 2006; Yan et
al. 2002) and, thus, could be used as a treatment for diseases
that result from either reduced neurotransmitter release (e.g.,
Lambert-Eaton syndrome and Parkinson’s disease) or dimin-
ished neurotransmitter receptor levels (e.g., myasthenia gra-
vis). One potential problem with using R-roscovitine is cross-
reactivity with other proteins including kinases, voltage-depen-
dent potassium channels, and L-type calcium channels. Thus
identification of the binding site could provide the information
needed for the structure-directed synthesis of roscovitine ana-
logs that have enhanced specificity for the agonist-binding site
on CaV2 channels. The progress toward identification of this
site has highlighted a previous unknown functionality of DIII
as a regulator of N-channel deactivation.
We thank Lei Du and Yunhua Wang for superb technical assistance in
chimeric channel development and preparation.
Present addresses: V. Yarotskyy, Dept. of Pharmacology and Physiology,
University of Rochester, 601 Elmwood Ave., Rochester, NY 14642; Guofeng
Gao, Dept. of Medicine, Penn State College of Medicine, Penn State Univer-
sity, Hershey, PA 17033.
This work was supported in part by grants from the Pennsylvania (PA)
Department of Health using Tobacco Settlement Funds and National Institutes
of Health Grants AR-059397 (K. S. Elmslie) and HL-074143 (B. Z. Peterson).
The PA Department of Health specifically disclaims responsibility for analy-
ses, interpretations, and conclusions presented here.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: V.Y., G.G., B.Z.P., and K.S.E. conception and design
of research; V.Y., G.G., and K.S.E. performed experiments; V.Y., G.G., and
K.S.E. analyzed data; V.Y., G.G., B.Z.P., and K.S.E. interpreted results of
experiments; V.Y. and K.S.E. prepared figures; V.Y., B.Z.P., and K.S.E.
drafted manuscript; V.Y., G.G., B.Z.P., and K.S.E. edited and revised manu-
script; V.Y., G.G., B.Z.P., and K.S.E. approved final version of manuscript.
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