Cooperative Activation of the T-type Ca_V3.2 Channel

Background: The low activation threshold of T-type Ca_V3.2 channels is central to neuronal rhythmogenesis.

Results: The S4-S5 linker of Domain II is functionally coupled with Domains II and III during channel activation.

Conclusion: Activation of Ca_V3.2 requires a specific interaction between adjacent domains.

Significance: Disrupting this protein interface could be a pharmacological strategy to decrease Ca²⁺ influx in neuronal pathologies.

Abstract

T-type Ca_V3 channels are important mediators of Ca²⁺ entry near the resting membrane potential. Little is known about the molecular mechanisms responsible for channel activation. Homology models based upon the high-resolution structure of bacterial Na_V channels predict interaction between the S4-S5 helix of Domain II (IIS4-S5) and the distal S6 pore region of Domain II (IIS6) and Domain III (IIIS6). Functional intra- and inter-domain interactions were investigated with a double mutant cycle analysis. Activation gating and channel kinetics were measured for 47 single mutants and 20 pairs of mutants. Significant coupling energies (ΔΔG_interact ≥ 1.5 kcal mol⁻¹) were measured for 4 specific pairs of mutants introduced between IIS4-S5 and IIS6 and between IIS4-S5 and IIIS6. In agreement with the computer based models, Thr-911 in IIS4-S5 was functionally coupled with Ile-1013 in IIS6 during channel activation. The interaction energy was, however, found to be stronger between Val-907 in IIS4-S5 and Ile-1013 in IIS6. In addition Val-907 was significantly coupled with Asn-1548 in IIIS6 but not with Asn-1853 in IVS6. Altogether, our results demonstrate that the S4-S5 and S6 helices from adjacent domains are energetically coupled during the activation of a low voltage-gated T-type Ca_V3 channel.

Introduction

Voltage-dependent Ca²⁺ channels (Ca_V)³ are membrane proteins that play a key role in promoting Ca²⁺ influx in response to membrane depolarization in excitable cells. Voltage-gated Ca²⁺ channels form oligomeric complexes that are classified according to the structural properties of the pore-forming Ca_Vα1 subunit. The Ca_Vα1 subunit bears high topological homology with the α subunit of Na_V channels with 24 transmembrane segments clustered in 4 homologous domains, each of which is analogous to a single subunit of Kv channels. The primary structures for 10 distinct Ca_Vα1 subunits (1–7) are classified into three main subfamilies according to their high-voltage activated gating (HVA Ca_V1 and HVA Ca_V2) or low-voltage activated gating (LVA Ca_V3). In HVA Ca_V1 and Ca_V2 channels, auxiliary subunits include a cytoplasmic Ca_Vβ subunit, a mostly extracellular Ca_Vα2δ subunit, and calmodulin constitutively bound to the C terminus of Ca_Vα1 (7–12) (for review, see Ref. 9). LVA T-type Ca_V3 channels are currently believed to achieve voltage-dependent activation and plasma membrane targeting without a significant contribution from Ca_Vβ and Ca_Vα2δ auxiliary subunits.

LVA T-type Ca_V3 channels open in response to a small membrane depolarization, making these channels the key mediators of Ca²⁺ entry near the resting membrane potential (14–17). They contribute to rhythmogenesis, sensory transmission, dendritic integration, cell proliferation, and differentiation. Alterations in T-type channel activity have been associated with pain signaling and a wide range of neuronal pathologies including idiopathic generalized epilepsies (18–21). Hence the T-type Ca_V3 channel has arisen as an important pharmacological target for the treatment of neurological and psychiatric disorders such as schizophrenia, mania, dementia, and epilepsy.

The molecular mechanisms controlling the voltage-dependent activation of T-type Ca_V3 channel remain, however, largely unknown. Many regions appear to participate in the channel gating. The loop between Domains I and II (I-II loop) appears to play a major role in this process (22–25) but molecular determinants are distributed in the IIIS6 pore domain (26, 27), on the C terminus (28) and the pore helix (29), and on S4 segments (30). Nonetheless, the series of events linking channel depolarization and pore opening have been only scarcely addressed.

The current model of activation postulates that the electrically driven outward movement and rotation of the S4 segment will lead to pore opening through a cascade of events that include pulling on the S4-S5 linker and bending the S6 segments to open the bundle crossing at their intracellular ends (31). In HVA Ca_V1.2, Ca_V2.1, and Ca_V2.3 channels, a cluster of hydrophobic residues in the distal S6 pore region were shown to stabilize the channel closed state (32–39). In particular, we have shown that the conserved isoleucine residue in distal S6 was functionally coupled with a leucine residue at position 596 in the S4-S5 helix during the activation of HVA Ca_V2.3 (39). Whether each of the four domains moves independently or in a concerted fashion remains to be established. By comparison, the opening of the Shaker K_V channel involves multiple activation steps as the S4 segments move from the resting state toward the activated state, which is followed by a concerted opening transition of the four identical S6 gates (40). This transition has been shown to occur through atomic interaction between adjacent subunits/domains (41).

In this current work, we have investigated the role of domain/domain interaction within the pore-forming Ca_Vα1 subunit during the activation of Ca_V3.2, a LVA Ca_V channel. Unlike HVA channels, the Ca_Vα1 subunit of Ca_V3.2 can be functionally expressed in the absence of Ca_Vβ and Ca_Vα2δ auxiliary subunits thus minimizing their effects on activation gating. To get structural clues regarding interaction interfaces, we produced three-dimensional models of the T-type Ca_V3.2 channel by homology with the recently published high-resolution three-dimensional structures of the bacterial Na_VRh (42) and Na_VAb channels in the closed and partially inactivated states (43, 44) (Fig. 1). The two homology models of Ca_V3.2 predict multiple points of interaction between adjacent domains. A total of 20 double mutants were herein functionally characterized using a double mutant cycle analysis. Strong interaction energies were measured for 4 pairs of mutants between the IIS4-S5 helix and the distal S6 helices of Domains II and III. Altogether our results confirm that the proximal IIS4-S5 helix is functionally coupled with the IIS6 region during channel activation. We also demonstrate that the distal IIS4-S5 helix is significantly coupled with residues in the IIIS6 region during the activation gating of Ca_V3.2. These results support of model of activation gating whereby opening of voltage-activated Ca²⁺ channels involves protein-protein interaction between adjacent domains and results in the concerted movement of the four domains.

FIGURE 1.

Modeling of Domains II and III was based upon the atomic coordinates of Na_VAb (PDB code 3RVY) (panels A and B) and Na_VRh (PDB code 4DXW) (panels C and D). Four different views of the computer-based molecular models of Domain II (panels A and C) and Domains II and III (panels B and D) of Ca_V3.2 are shown in a schematic representation. Modeling was achieved with Modeler 9v4. Panels A and C, the regions in red correspond to the IIS6 and the regions in blue correspond to the IIS4-S5. Panel B and D, the regions in red correspond to the predicted region in the IIIS6 could be interacting with the region in blue in the IIS4-S5. PD, pore domain; VSD, voltage sensor domain. The figures were produced using PyMol (DeLano Scientific).

EXPERIMENTAL PROCEDURES

Recombinant DNA Techniques

The human Ca_V3.2 (GenBank^TM AF051946) (5) was a gift from Edward Perez-Reyes (University of Virginia) and Leanne L. Cribbs (Loyola University). Point mutations were produced with the QuikChange XL-mutagenesis kit (Agilent Technologies, Santa Clara, CA) using 39-mer primers as described elsewhere (38, 39, 45). Constructs were verified by automated double-stranded sequence analysis (Genomics Platform, IRIC, Université de Montréal, QC, Canada). Run-off transcripts were prepared using the T7 RNA polymerase mMessage mMachine® transcription kit (Ambion, Invitrogen) and stored at −20 °C before use.

Functional Expression of Ca_V3.2

Oocytes were obtained from female Xenopus laevis clawed frogs as described previously (38, 39, 46). Oocytes were injected with 46 or 4.6 nl of a solution containing 1 μg/μl of cRNA coding for the Ca_V3.2 WT or mutant and incubated for 2 to 5 days after RNA injection.

Electrophysiological Recordings and Data Acquisition

Wild-type and mutant channels were screened at room temperature for macroscopic Ba²⁺ currents and for gating currents, with the cut-open oocyte voltage-clamp technique using a CA-1B amplifier (Dagan Corp., Minneapolis, MN) (47, 48) as described before (39). The membrane of the oocyte exposed to the bottom chamber was permeabilized by a brief treatment with 0.1% saponin. Data acquisition was performed with the Digidata 1322A 16-bit system (Molecular Devices, Sunnyvale, CA). The pCLAMP 10 software was used for on-line data acquisition and analysis.

Whole cell currents were recorded in the presence of an external solution containing 10 mm BaOH, 110 mm NaOH, 1 mm KOH, and 20 mm Hepes, titrated to pH 7.0 with methanesulfonic acid (49). The internal solution in contact with the oocyte cytoplasm was 120 mm N-methyl-d-glucamine, 10 mm EGTA, and 10 mm Hepes, titrated to pH 7.0 with methanesulfonic acid. Functional expression of mutants was deemed significant with whole cell Ba²⁺ currents larger than −50 nA. Current-voltage relationships were measured using a series of 150-ms voltage pulses (2 mV steps) applied at a frequency of 0.2 Hz from a holding potential of −100 mV. The holding potential was decreased to −120 mV for mutants displaying a negative shift in the voltage dependence of inactivation. Leak currents were subtracted off-line. Steady-state inactivation was determined using a one-pulse protocol with a 2-s conditioning pulse of varying amplitude followed by a test pulse at the voltage yielding the peak current and was applied at a frequency of 0.02 Hz. Normalized currents obtained at the test pulse (I/I_max) were fitted with a standard Boltzmann equation as described elsewhere (38, 46, 50). Recovery from inactivation was studied by using a paired-pulse protocol. Briefly, whole cell Ba²⁺ currents were inactivated with 1-s pulses to −20 mV and recovery was examined at a test pulse of −20 mV after a series of variable intervals at −100 mV.

Gating currents were recorded in the presence of an external solution containing 2 mm CoCl₂, 110 mm NaOH, and 10 mm Hepes, titrated to pH 7.0 with methanesulfonic acid (49). Hence the voltage dependence of gating charge movement and activation of macroscopic currents are measured with different divalent ion concentrations. Others have shown that this substitution left the charge movement unaltered and caused no changes in the surface potential (49). The internal solution in contact with the oocyte cytoplasm was 110 mm potassium glutamate and 10 mm Hepes, titrated to pH 7.0 with KOH. Gating currents were filtered at 1 kHz and digitized at 10 kHz. Holding potential was −120 mV and 30-ms voltage steps were evoked from −120 to 0 mV by 2-mV increments. Gating charge versus voltage (Q-V) relationships were obtained by integrating the OFF gating currents and then fitting single Boltzmann functions (Equation 1) according to,

where the parameter e is the elementary electric charge; E_0.5,Q is the mid-potential of charge displacement, k is the Boltzmann constant; z is the effective valence.

Data Analysis

Activation curves were obtained from normalized current-voltage (I-V) relationships. The estimation of E_0.5,act using nonstationary measurements rests upon the assumption that the transition to the open state is much faster than the transition to the inactivated state. The voltage dependence of activation was estimated using the normalized conductance-voltage (G-V) data with G = I/(V_m − E_r), where I is the normalized peak whole cell current, V_m is the voltage command, and E_r is the reversal potential. Normalized conductance values were fitted with a standard Boltzmann equation,

where E_0.5,act is the mid-potential of activation, and z is the effective valence. Each data point was reported as the mean of individual measurements ± S.E. as described elsewhere (38, 46, 50). The free energy of activation was calculated using the mid-potential of activation.

Activation, inactivation, and deactivation time constants of whole cell current traces were estimated with the predefined equations in Clampfit 10 that uses the Chebyshev routine and a 4-point smoothing filter with a single exponential function. Statistical analyses were performed using the one-way analysis of variance fitting routine for two independent populations included in OriginPro 8.0. Data were considered statistically significant at p < 0.05.

Double Mutant Cycle Analysis

The coupling or interaction energy ΔΔG_act,interact during channel activation was calculated as follows,

where ΔG_act,wt is the free energy of activation of the wild-type channel, ΔG_act,double is the free energy of activation of the double glycine mutant, ΔG_act,S4-S5 is the free energy of activation of the single glycine mutant in the S4-S5 linker, and ΔG_act,S6 is the free energy of activation of the single glycine mutant in S6. ΔΔG_act,interact = 0 suggests that the activation energies are purely additive hence that the residues are not functionally coupled. A value of ΔΔG_interact more positive than +1 kcal mol⁻¹ or more negative than −1 kcal mol⁻¹ (|ΔΔG_interact |> 1) was considered significantly different in similar studies (39, 51, 52). The coupling or interaction energy ΔΔG_{inact,interact} during channel inactivation was calculated as follows,

where ΔG_inact,wt is the free energy of inactivation of the wild-type channel, ΔG_inact,double is the free energy of inactivation of the double glycine mutant, ΔG_inact,S4-S5 is the free energy of inactivation of the single glycine mutant in the S4-S5 linker, and ΔG_inact,S6 is the free energy of inactivation of the single glycine mutant in S6.

Homology Modeling of Ca_V3.2

The primary sequence of Ca_V3.2 was aligned with Na_VAb and Na_VRh by using T-COFFEE. The computer-based molecular models of Ca_V3.2 were obtained with the stand-alone Modeler 9v4 software using the molecular coordinates of Na_VAb (PDB 3RVY and 4EKW) (43, 44) and Na_VRh (PDB 4DXW) (42) (Fig. 1). The first Na_VAb structure that was reported (PDB 3RVY) was estimated to depict the channel into an intermediate conformation along the channel activation pathway (53), whereas the second structure (PDB 4EKW) captured the channel in the inactivated state. The activation gate of Na_VRh also appears to be in the closed state. Although the structure of Na_VMs is reported to be captured in the open state, it lacks the S4-S5 linkers (54). Fifty models were generated and the models with the lowest molpdf scoring function (as provided in PyMol) were selected. The modeling was restricted to Domains II, III, and IV because Domain I of Ca_V3.2 displays a stretch of 100 residues in the loop preceding the pore region that could not be modeled with the current templates. In Domain II, Ca_V3.2 displays a series of 12 contiguous residues after transmembrane domain S5 that are not modeled, whereas in Domain III, the stretch of residues not accounted for by either template amounts to 27 residues at the same position. The gaps in the structural model imply a certain degree of uncertainty in the final model, especially in regard to the orientation of the S4-S5 linker with respect to the S6 residues. Despite these gaps, the structural homology for the S4-S5 to S6 region in Domains II and III is conclusive with an average root mean square deviation of 1.6 Å for the Na_VRh-based model and 1.3 Å for the Na_VAb-based model.

RESULTS

Activation Gating of Glycine Mutants in IIS6

The T-type Ca_V3.2 channel was functionally expressed in Xenopus oocytes and its electrophysiological properties were measured with the cut-open oocyte technique in the presence of 10 mm Ba²⁺ as the charge carrier. As seen in Fig. 2, its biophysical properties were comparable with the properties reported in the original cloning paper (5) and after recombinant expression in HEK cells (55). Under our conditions, Ca_V3.2 was activated with E_0.5,act = −48 ± 1 mV (n = 37), which is similar to E_0.5,act = −45.8 ± 0.7 mV reported in HEK cells in the presence of 1.25 mm Ca²⁺ in the external medium (55). This negative activation potential translated into a ΔG_act = −4.3 ± 0.2 kcal mol⁻¹ (n = 37). Ca_V3.2 activates at significantly more negative voltages than HVA Ca_V2.3 with E_0.5,act = −7 mV for the latter (39). The channel kinetics were typical of current-dependent inactivation with activation and inactivation kinetics becoming increasingly faster after the threshold potential (55). Other properties include a relatively slow deactivation time constant of 12 ± 1 ms (n = 9) at −40 mV and a time constant for recovery (τ_recovery) from inactivation of 480 ± 1 ms (n = 10), which is similar to the τ_recovery = 395 ms reported in HEK cells (55). Channels were inactivated with E_0.5,inact = −65 ± 1 mV (n = 10). These activation and inactivation parameters suggest a window current between −65 and −50 mV as shown for native T-type currents in vestibular neurons (56). The complete set of biophysical parameters is shown in Table 1.

FIGURE 2.

A, whole cell current traces are shown for Ca_V3.2 WT. Current traces were obtained using the cut-open oocyte technique in 10 mm Ba²⁺ from a holding potential of −100 mV. Leak subtraction was carried out off-line. B, bar graphs of the mean time constants of activation and inactivation of the Ca_V3.2 WT are shown as a function of the test potential. Leak-subtracted whole cell current traces were fitted by a sum of two exponential equations, one for the activation and the other one for inactivation (55). C, normalized current-voltage relationships for Ca_V3.2 WT were obtained by fitting the experimental data to a modified Boltzmann equation. D, representative tail currents for Ca_V3.2 WT. Currents were activated during 20-ms conditioning depolarizing voltage pulses at −30 mV. Deactivation was recorded during subsequent repolarization to test potentials from −120 to −20 mV (10-mV increments) as illustrated on the pulse protocol. E, the mean time constants of deactivation were obtained from fitting current traces with monoexponential functions. The mean deactivation time constant was 8.4 ± 0.6 ms (n = 9) at −50 mV for Ca_V3.2 WT. F, normalized gating charge (Q/Q_max) and normalized conductance (G/G_max) were plotted against the test voltages for Ca_V3.2 WT. As seen, both curves overlapped in contrast to observations reported in Kv channels where the Q/Q_max is left-shifted as compared with the G/G_max curve. Mid-points were −44 ± 1 mV (n = 4) for Q/Q_max and −47 ± 1 mV (n = 37) for G/G_max. G, time course of recovery from inactivation for the Ca_V3.2 WT. Recovery was measured using a double pulse protocol. A 1-s prepulse to −20 mV induced inactivation and, after a variable period at −100 mV, a test pulse to the same voltage (−20 mV) was applied to assess the extent to which channels had recovered from inactivation. H, data points from panel G were fitted with a single exponential equation of 480 ± 1 ms (n = 10). I, voltage dependence of inactivation for Ca_V3.2 WT was estimated from isochronal inactivation data points measured after 2-s conditioning pulses applied between −110 and 0 mV from a holding potential of −100 mV. The fraction of the noninactivating current was fitted with a Boltzmann equation yielding E_0.5,inact = −65 ± 1 mV (n = 10). Pooled data points are plotted against test voltages and are shown alongside the normalized G/G_max curve. As seen, a non-negligible window current is predicted between −65 and −50 mV for Ca_V3.2 WT. Numerical values are found in Table 1.

TABLE 1.

Biophysical parameters of Ca_V3.2 wild-type and S6 mutant channels were estimated from recordings obtained with the cut-open oocyte technique in the presence of 10 mm Ba²⁺

Activation properties (E_0.5,act and z) were estimated from the mean I-V relationships and fitted to a Boltzmann equation, where z is the slope factor. Activation and inactivation energies were calculated as follows: ΔG_act = z · F · E_0.5,act; ΔΔG_act = ΔG_act,mut − ΔG_act,wt; ΔG_inact = z · F · E_0.5,inact; and ΔΔG_inact = ΔG_inact,mut − ΔG_inact,wt. The voltage dependence of inactivation was determined from the peak currents after 2-s depolarizing pulses from a holding potential of −120 mV. The data are shown with the mean ± S.E. of the individual experiments and the number of experiments appears in parentheses.

	E0.5, act	ΔGact	ΔΔGact	Ipeak	E0.5, inact	ΔGinact	τact, −20 mV	τinact, −20 mV	τdeact, −50 mV
		kcal mol−1		nA		kcal mol−1	ms
CaV3.2 WT	−47 ± 1 (37)	−4.3 ± 0.2	NAa	−1300 ± 200	−65 ± 1 (10)	−8.6 ± 0.5	3.2 ± 0.1 (18)	11.6 ± 0.2	8.4 ± 0.6 (9)
V1005A	−36 ± 1 (5)	−3.3 ± 0.2	1.0 ± 0.4	−160 ± 40	−58.2 ± 0.5 (5)	−7.3 ± 0.5	3.5 ± 0.2 (5)	13.3 ± 0.6b	3.9 ± 0.9b (5)
V1005G	−37 ± 1 (5)	−3.0 ± 0.3b	1.3 ± 0.5	−170 ± 40	−61 ± 1 (4)	−8.3 ± 0.6	2.62 ± 0.09 (5)	17.2 ± 0.4c	5 ± 1 (5)
L1006A	−43 ± 2 (5)	−4.5 ± 0.5	0.2 ± 0.7	−210 ± 70	−67 ± 1 (5)	−7.1 ± 0.7	3.0 ± 0.3 (5)	13 ± 1	4 ± 1b (5)
L1006G	−39 ± 2 (6)	−3.8 ± 0.3	0.5 ± 0.5	−300 ± 100	−63 ± 1 (6)	−7.3 ± 0.8	3.3 ± 0.1 (6)	12.5 ± 0.3	3.0 ± 0.9c (6)
F1007G	−48.6 ± 0.7 (7)	−4.7 ± 0.2	−0.4 ± 0.4	−140 ± 40	−65 ± 2 (7)	−6.7 ± 0.4	4.3 ± 0.3c (7)	43 ± 2c	7.5 ± 2 (8)
N1008A	−35 ± 2 (4)	−3.1 ± 0.3	1.2 ± 0.5	−120 ± 20	−62 ± 1 (5)	−6.6 ± 0.4	3.2 ± 0.6 (4)	8.2 ± 0.6c	3.0 ± 0.8c (5)
N1008G	−38.8 ± 0.9 (5)	−3.2 ± 0.2	1.1 ± 0.4	−200 ± 100	−65 ± 1 (4)	−6.8 ± 0.5	2.4 ± 0.4 (5)	7.1 ± 0.9c	2.5 ± 0.5c (5)
L1009A	−39 ± 2 (11)	−3.2 ± 0.4b	1.1 ± 0.6	−500 ± 200	−71 ± 3 (5)	−5.9 ± 0.4	5.4 ± 0.6 (7)	16 ± 3	3.0 ± 0.7c (6)
L1009G	−44 ± 2 (6)	−3.3 ± 0.3	1.0 ± 0.5	−900 ± 400	−63 ± 1 (6)	−7.9 ± 0.6	3.6 ± 0.2 (6)	15.6 ± 0.4c	7 ± 1 (6)
L1010A	−26 ± 1 (5)	−1.3 ± 0.1c	3.0 ± 0.3	−170 ± 30	−58 ± 3 (3)	−6.9 ± 0.4	4.0 ± 0.3c (5)	16.0 ± 0.8c	3.5 ± 0.9b (4)
L1010G	−34.9 ± 0.9 (9)	−2.6 ± 0.2c	1.7 ± 0.4	−600 ± 100	−67.5 ± 0.7 (3)	−8.0 ± 0.3	4.4 ± 0.6 (9)	10.6 ± 0.6	6.7 ± 0.5 (4)
V1011A	−45 ± 2 (6)	−4.2 ± 0.4	0.1 ± 0.6	−800 ± 200	−63 ± 2 (3)	−8.4 ± 0.4	4.2 ± 0.6b (6)	28.4 ± 0.7c	17 ± 1c (5)
V1011G	−50 ± 1 (9)	−5.0 ± 0.3	−0.7 ± 0.5	−900 ± 200	−67 ± 1 (11)	−7.8 ± 0.4	5.2 ± 0.5c (11)	115 ± 16c	8 ± 1 (4)
A1012G	−41 ± 3 (5)	−3.1 ± 0.4b	1.2 ± 0.6	−500 ± 200	−66.0 ± 0.4 (3)	−6.2 ± 0.4	3.1 ± 0.1 (5)	15.2 ± 0.6c	11.3 ± 0.7 (5)
I1013A	−41 ± 1 (10)	−3.6 ± 0.2	0.7 ± 0.4	−400 ± 100	−64 ± 2 (9)	−7.7 ± 0.6	4.8 ± 0.5**(10)	55 ± 1c	20 ± 4c (3)
I1013G	−54 ± 1 (10)	−5.4 ± 0.3b	−1.1 ± 0.5	−390 ± 90	−66.8 ± 0.7 (5)	−9 ± 1	8.5 ± 0.4c (10)	68 ± 4c	30 ± 9b (5)
I1013L	−41 ± 2 (5)	−3.2 ± 0.4	1.1 ± 0.3	−170 ± 60	−63.3 ± 0.7 (4)	−6.4 ± 0.1	3.7 ± 0.3 (5)	18.4 ± 0.3c	10.4 ± 0.5 (5)
I1013V	−45.5 ± 0.8 (7)	−4.2 ± 0.3	0.1 ± 0.5	−220 ± 60	−66 ± 1 (6)	−8.0 ± 0.3	2.21 ± 0.07c (7)	18.1 ± 0.5c	8 ± 2 (7)
L1014A	−39 ± 1 (6)	−3.1 ± 0.3b	1.2 ± 0.5	−700 ± 300	−66 ± 2 (6)	−7.8 ± 0.6	5.2 ± 0.4 (6)	17.7 ± 0.4c	11.0 ± 0.8 (6)
L1014G	−38 ± 1 (5)	−3.0 ± 0.2b	1.3 ± 0.4	−1100 ± 500	−67.0 ± 0.9 (5)	−9.9 ± 0.5	5.1 ± 0.4c (5)	20.8 ± 0.3c	11 ± 2 (5)
V1015A	−42 ± 1 (5)	−2.5 ± 0.1c	1.8 ± 0.3	−330 ± 50	NDd	ND	4.0 ± 0.3 (5)	12 ± 1	ND
V1015G	−43 ± 1 (5)	−2.7 ± 0.1b	1.4 ± 0.3	−330 ± 30	−75 ± 2 (4)	−7.0 ± 0.5	4.9 ± 0.4c (7)	24 ± 1c	3.6 ± 0.8c (5)
E1016A	−47 ± 1 (6)	−4.0 ± 0.2	0.3 ± 0.4	−400 ± 100	−67 ± 1 (6)	−8.8 ± 0.6	4.1 ± 0.2c (6)	15.9 ± 0.6c	10.7 ± 0.5 (6)
E1016G	−43 ± 3 (6)	−3.1 ± 0.4b	1.2 ± 0.6	−160 ± 30	ND	ND	5.9 ± 0.5c (6)	36 ± 3c	ND
N1548A	−42 ± 1 (5)	−3.9 ± 0.1	0.4 ± 0.3	−400 ± 300	−80 ± 1 (4)	−6.0 ± 0.2b	3.4 ± 0.2 (5)	9.5 ± 0.8c	3 ± 1 (5)
N1548G	−40.2 ± 0.9 (8)	−3.9 ± 0.2	0.4 ± 0.4	−400 ± 100	−66 ± 2 (8)	−8.1 ± 0.4	2.6 ± 0.2 (8)c	6.8 ± 0.8c	2.9 ± 0.5c (10)
M1549A	−42 ± 2 (7)	−3.1 ± 0.2b	1.2 ± 0.4	−400 ± 100	−68 ± 2 (4)	−6.2 ± 0.4	6.3 ± 0.5c (7)	29 ± 2c	11 ± 3 (5)
N1853A	−37 ± 1 (5)	−3.2 ± 0.3b	1.1 ± 0.5	−400 ± 200	ND	ND	5.0 ± 0.3c (5)	14.1 ± 0.3c	ND
N1853G	−37 ± 3 (5)	−2.2 ± 0.3c	2.1 ± 0.5	−500 ± 200	ND	ND	3.4 ± 0.2 (5)	11.6 ± 0.5	ND

^a NA, not applicable.

^b p < 0.01 as compared with Ca_V3.2 WT.

^c p < 0.001 as compared with Ca_V3.2 WT.

^d ND, not determined.

Gating currents were isolated in 2 mm external Co²⁺ (49) and the voltage dependence of charge movement Q/Q_max was fitted with a single Boltzmann function. Q/Q_max and G/G_max curves of Ca_V3.2 WT displayed comparable voltage dependence in contrast to K_V2.1 (57) and HVA Ca_V2.3 (49) where the threshold for gating current activation was shown to be more negative than the threshold for activation of ion currents. Similar observations were reported for LVA Ca_V3.1 and Ca_V3.3 channels whether gating currents were measured with 1 mm La³⁺ or 30 μm Er³⁺ suggesting that this is an intrinsic property of LVA Ca_V3 channels and arguing against a role for a simple surface charge screening effect (25). These results suggest that charge movement of Ca_V3.2 is negligible in the pre-open state and support a model with a concerted opening of the four pore domains.

In HVA Ca_V2.3 channels, hydrophobic residues in distal S6 were shown to stabilize the channel closed state, whereas mutations to glycine residues promoted channel activation near the resting potential (38, 39). Given that LVA Ca_V3.2 channels open at negative membrane potentials, we investigated whether its closed state was also stabilized by hydrophobic residues in the distal S6 of Domain II (IIS6) (Fig. 3). We opted to perform a glycine scan of IIS6 as a way to significantly perturb the energies of gating transitions (40). Glycine is more hydrophilic than leucine, isoleucine, and valine residues (58). It thus appears to be suitable to study the role of hydrophobic interactions, whereas minimizing steric hindrance. Furthermore, the high conformational flexibility of side chain-free glycine residues appear to be compatible with the relatively loose α-helicoidal arrangement of the distal S6 region observed in high resolution of K⁺ channels (59). Point mutations were introduced into 10 residues from Val-1005 to Glu-1016 and their biophysical properties were measured. Current traces obtained for four mutations between L1010G and L1014G are shown in Fig. 4. As seen, the kinetics of L1010G, A1012G, and L1014G were not significantly different from Ca_V3.2 WT. In contrast, the activation, deactivation, and inactivation kinetics of V1011G and I1013G were significantly slower. Only I1013G activated at more negative potentials than Ca_V3.2 WT with a E_0.5,act = −54 ± 1 mV (n = 10). Introducing a glycine residue at other positions in the distal IIS6 either right-shifted the activation and inactivation potentials or else were without significant impact (p > 0.1) (Fig. 5, A and B). Similar results were reported when residues in IIS6 were mutated to alanine (Table 1). The slower kinetics of I1013G, its left-shift of the activation potential, and its lack of significant impact on the inactivation potential are qualitatively reminiscent of the IIS6 I701G mutant in Ca_V2.3 (38, 39). Multiple primary sequence alignments of S6 segments and homology models suggest that Ile-1013 is structurally equivalent to Ile-701 in Ca_V2.3 (39) and Ile-217 in Na_VAb (44), which were shown to play a role in activation gating. Although Ca_V3.2 I1013G activated at more negative potentials than Ca_V3.2 WT, the −10 mV negative shift was more modest than the ≈−35 mV shift in the voltage dependence of activation observed with Ca_V2.3 I701G (38, 39). In addition, glycine substitutions were the only ones to successfully left shift the activation potential because I1013A, I1013V, and I1013L either right shifted the activation curve or were without effect. Finally, none of the glycine mutations significantly affected the mid-potential of steady-state inactivation (Table 1).

FIGURE 3.

The primary sequences of IS6, IIS6, IIIS6, and IVS6 of Ca_V3.2 were aligned with S6 of Na_VAb, Na_VRh, K_V1.2, and the Ca_V2.3 Domain II using the T-coffee algorithm. Underlined residues have been identified in previous studies as determinants of activation gating: Ile-217 in Na_VAb, PVP locus in K_V channels, and Ile-701 in Ca_V2.3. The underlined residues in Ca_V3.2 designate the IIS6 residues studied herein.

FIGURE 4.

A, whole cell current traces are shown from left to right for Ca_V3.2 L1010G, V1011G, A1012G, I1013G, and L1014G. Current traces were obtained in 10 mm Ba²⁺ from a holding potential of −100 mV. B, bar graphs show the mean time constants of activation (dark gray bars) and inactivation (light gray bars) as a function of the test potential from left to right for Ca_V3.2 L1010G, V1011G, A1012G, I1013G, and L1014G. All graphs cover the same range but insets emphasize the details for the faster mutants (L1010G, A1012G, and L1014G). Inactivation kinetics were strongly decreased in V1011G and I1013G. C, the mean time constants of deactivation (obtained from fitting current traces from monoexponential functions) from left to right for Ca_V3.2 L1010G, V1011G, A1012G, I1013G, and L1014G are shown alongside Ca_V3.2 WT (circle). The data points for Ca_V3.2 WT are replotted from Fig. 1E in each graph to facilitate comparison. As seen the slower inactivating kinetics of V1011G and I1013G were accompanied by slower deactivation kinetics suggesting that the channel open state was stabilized by these mutations. D, normalized conductance-voltage relationships of G/G_max (shown from left to right for Ca_V3.2 L1010G, V1011G, A1012G, I1013G, and L1014G) were obtained by fitting the experimental data to a Boltzmann equation. The data obtained for the WT channel (dark circles) are shown alongside for comparison. Numerical values are found in Table 1.

FIGURE 5.

A, bar graph of the activation energies (ΔΔG_act) for the glycine mutants in IIS6 of Ca_V3.2. ΔΔG_act were obtained from the difference between the ΔG_act values for the mutant channels and the ΔG_act values for the WT channel such that ΔΔG_act = ΔG_act,mutant − ΔG_act,WT. As seen, only I1013G activated at voltages more negative than the WT channel. B, bar graph of the inactivation energies (ΔΔG_inact) for the glycine mutants in IIS6 of Ca_V3.2. ΔΔG_inact were obtained from the difference between the ΔG_inact values for the mutant channels and the ΔG_inact values for the WT channel such that ΔΔG_inact = ΔG_inact,mutant − ΔG_inact,WT. As seen, most of the mutations inactivated at voltages significantly more positive than the WT channel. Data are shown in kcal mol⁻¹. Numerical values are found in Table 1.

Functional Coupling within Domain II

The crystal structures of K_V and Na_V channels have shown that residues in the S6 activation gate are positioned within atomic distance of the S4-S5 α-helix. Mutational studies as well as statistical coupling analysis (60) have shown that the S4-S5 linker plays a major role in the activation gating of voltage-dependent ion channels and that compatibility across the S4-S5 linker to pore interface is required to couple voltage-sensor action to pore gating (60, 61). Previous studies have shown that Ile-701 in Ca_V2.3 (39) and Ile-217 in Na_VAb (44) are functionally coupled with a conserved leucine residue located in the S4-S5 linker of the same domain. This position is equivalent to Leu-908 in Ca_V3.2 (Fig. 6A). Both Na_VAb and Na_VRh homology models of Ca_V3.2, however, predict that Thr-911 in S4-S5 would be the closest residue to Ile-1013 in IIS6. Ca_V3.2 Leu-908 is predicted to lie further away with predicted distances ≥10 Å between their C-α atoms and/or C-β atoms. To investigate functional interaction between these residues, we performed a double-mutant cycle analysis. The double-mutant cycle analysis (62, 63) provides a way for isolating the energetics of specific pairwise interactions as shown in Na_V1.7 channels (64) and in our previous study with Ca_V2.3 (39). The initial glycine scan of IIS4-S5 was carried out in 11 individual positions from Leu-901 to Asp-913. Of all the residues tested, only V907G strongly shifted the ΔG_act toward negative voltages with ΔΔG_act = −3.3 ± 0.8 kcal mol⁻¹ (n = 11) (Fig. 6B). The Q/Q_max curve was shifted to the same extent and overlapped with the G/G_max curve as it was also shown for the WT channel. This tight coupling between the conductance and the charge means that electromechanical coupling was not altered, and suggests that V907G stabilized the pore in its open state (65). Remarkably, introduction of glycine residues at positions 902, 903, 904, 906, 908, 909, 911, 912, and 913 did not significantly alter ΔG_act (p > 0.01). T911G activated within the same voltage range as the WT channel although its activation and inactivation kinetics were significantly slower (Table 2).

FIGURE 6.

A, the primary sequences of IS4-S5, IIS4-S5, IIIS4-S5, and IVS4-S5 of Ca_V3.2 were aligned with S4-S5 of Na_VAb, Na_VRh, K_V1.2, and S4-S5 in domain II of Ca_V2.3 using the T-coffee algorithm. Underlined residues have been identified in previous studies as modulating the channel activation gating; Leu-123 in Na_VAb and Leu-596 in Ca_V2.3. The sequence of residues underlined in Ca_V3.2 designate the residues studied herein. B, bar graphs of the activation energies (ΔΔG_act) for the glycine mutants in IIS4-S5 of Ca_V3.2; ΔΔG_act = ΔG_act,mut − ΔG_act,WT. V907G activated at significantly more negative voltages than the WT channel with a ΔΔG_act = −3.3 ± 0.8 kcal mol⁻¹ (n = 11). The complete sets of numerical values are found in Table 2.

TABLE 2.

Biophysical parameters of Ca_V3.2 wild-type and S4-S5 mutant channels from Domain II (IIS4-S5) were estimated from recordings obtained with the cut-open oocyte technique in the presence of 10 mm Ba²⁺

Activation properties (E_0.5,act and z) were estimated from the mean I-V relationships and fitted to a Boltzmann equation where z is the slope factor. Activation and inactivation energies were calculated as follows: ΔGact = z · F · E_0.5,act; ΔΔG_act = ΔG_act,mut − ΔG_act,wt; ΔG_inact = z · F · E_0.5,inact; and ΔΔG_inact = ΔG_inact,mut − ΔG_inact,wt. The voltage-dependence of inactivation was determined from the peak currents after 2-s depolarizing pulses from a holding potential of −120 mV. The data are shown with the mean ± S.E. of the individual experiments and the number of experiments appears in parentheses.

	E0.5, act	ΔGact	ΔΔGact	Ipeak	E0.5, inact	ΔGinact	τact, −20 mV	τinact, −20 mV	τdeact, −50 mV
	mV	kcal mol−1		nA	mV	kcal mol−1	ms
CaV3.2 WT	−47 ± 1 (37)	−4.3 ± 0.2	NAa	−1300 ± 200	−65 ± 1 (10)	−8.6 ± 0.5	3.2 ± 0.1 (18)	11.6 ± 0.2	8.4 ± 0.6 (9)
L901G	−44 ± 1 (4)	−3.5 ± 0.2	0.8 ± 0.4	−260 ± 30	NDb	ND	5.7 ± 0.2c (4)	25.7 ± 0.4c	ND
R902G	−47.9 ± 0.5 (5)	−4.9 ± 0.5	−0.6 ± 0.7	−500 ± 100	−71 ± 1 (3)	−5.8 ± 0.5	5.6 ± 0.4c (5)	29 ± 3c	16 ± 3d (5)
R903G	−41 ± 1 (5)	−4.0 ± 0.5	0.3 ± 0.7	−180 ± 30	−60 ± 2 (4)	−6.2 ± 0.7	2.8 ± 0.2 (5)	10.6 ± 0.4	5.3 ± 0.7 (4)
Q904G	−54.6 ± 0.8 (7)	−5.3 ± 0.3	−1.0 ± 0.6	−230 ± 20	−74 ± 2 (6)	−6.7 ± 0.4	5.5 ± 0.2c (7)	46 ± 2c	19 ± 1c (4)
L905G	−51 ± 1 (5)	−5.5 ± 0.4d	−1.2 ± 0.5	−500 ± 100	−70 ± 2 (4)	−7.7 ± 0.7	2.8 ± 0.2 (5)	13 ± 0.7	5 ± 1d (5)
V906G	−43 ± 2 (5)	−4.2 ± 0.6	0.1 ± 0.6	−120 ± 20	−68 ± 4 (5)	−5.4 ± 0.4d	4.0 ± 0.6 (5)	14.1 ± 0.6c	3.4 ± 0.6c (5)
V907A	−41 ± 1 (6)	−4.8 ± 0.3	−0.5 ± 0.5	−240 ± 70	−74.2 ± 0.9 (5)	−7.2 ± 0.4	2.6 ± 0.3 (6)	11.9 ± 0.3	2.8 ± 0.5c (6)
V907G	−54 ± 1 (11)	−7.6 ± 0.6c	−3.3 ± 0.8	−310 ± 80	−74.6 ± 0.9 (9)	−7.7 ± 0.1	3.2 ± 0.2 (11)	11.1 ± 0.4	3.8 ± 0.6c (11)
L908A	−41 ± 1 (5)	−4.8 ± 0.4	−0.5 ± 0.6	−240 ± 70	−63.0 ± 0.8 (6)	−9.1 ± 0.8	2.7 ± 0.4 (5)	14.0 ± 0.9d	7 ± 1 (7)
L908G	−51 ± 2 (11)	−4.9 ± 0.3	−0.6 ± 0.5	−800 ± 200	−73 ± 2 (9)	−7.4 ± 0.4	2.6 ± 0.1 (6)	12.5 ± 0.3	8 ± 2 (5)
V909G	−41 ± 1 (5)	−4.2 ± 0.4	0.1 ± 0.6	−200 ± 50	−62 ± 1 (4)	−7.6 ± 0.3	5.0 ± 0.2 (5)c	11.9 ± 0.9	6 ± 1 (5)
K910G	−36.9 ± 0.6 (5)	−3.0 ± 0.2d	1.3 ± 0.4	−290 ± 80	−68 ± 2 (6)	−6.9 ± 0.4	3.8 ± 0.3 (5)	13.7 ± 0.6c	4.0 ± 0.7d (6)
T911A	−45 ± 2 (6)	−3.5 ± 0.3	0.8 ± 0.5	−290 ± 50	ND	ND	7.4 ± 0.4c (7)	77 ± 4c	ND
T911G	−52 ± 1 (11)	−4.3 ± 0.2	0.0 ± 0.4	−200 ± 30	−62 ± 2 (5)	−8.5 ± 0.6	4.2 ± 0.3d (11)	39 ± 5c	10 ± 1 (5)
M912G	−44 ± 1 (4)	−4.7 ± 0.5	−0.4 ± 0.7	−400 ± 300	−69.1 ± 0.7 (5)	−10 ± 1	2.7 ± 0.3 (5)	11.8 ± 0.8	8 ± 2 (5)
D913G	−47 ± 2 (6)	−4.9 ± 0.5	−0.6 ± 0.7	−1700 ± 400	−66 ± 2 (6)	−8.4 ± 0.4	3.2 ± 0.3 (6)	10.0 ± 0.3d	6.7 ± 0.4 (6)
N914A	−35 ± 2 (5)	−3.0 ± 0.4d	1.3 ± 0.6	−500 ± 100	−63 ± 2 (4)	−8.0 ± 0.3	4.5 ± 0.2c (5)	14 ± 1d	6 ± 1 (5)
N914G	−38 ± 2 (8)	−3.2 ± 0.4d	1.1 ± 0.6	−300 ± 70	−62.0 ± 0.6 (5)	−6.9 ± 0.5	3.4 ± 0.4 (8)	13.0 ± 0.7	6 ± 2 (5)

^a NA, not applicable.

^b ND, not determined.

^c p < 0.001 as compared with Ca_V3.2 WT.

^d p < 0.01 as compared with Ca_V3.2 WT.

The properties of the double mutant T911G/I1013G are reported in Fig. 7. Inactivation kinetics of T911G/I1013G were slower than the WT and T911G but remained faster than I1013G (Fig. 7). More interestingly T911G/I1013G activated at slightly more depolarized voltages, whereas both single mutants activated at voltages more negative than the WT channel. Altogether, this yielded a ΔΔG_act,interact = 1.8 ± 0.9 kcal mol⁻¹ (n = 6), a value that is significantly different from 0. With a ΔΔG_interact ≥1 kcal mol⁻¹, this result suggests that Thr-911 is functionally coupled with Ile-1013 during channel activation. There seems to be a functional coupling during inactivation although the larger experimental error associated with this measure (ΔΔG_{inact,interact} = 2.9 ± 2.6 kcal mol⁻¹, n = 6) precludes its statistical significance. The ΔΔG_act,interact for the double mutant T911A/I1013A was −1.6 ± 1.2 kcal mol⁻¹ (n = 6) again suggestive albeit not conclusively of a possible interaction between these two residues during channel activation. Of note, Thr-911 and Ile-1013 in Ca_V3.2 are, respectively, equivalent to Ser-241 in IS4-S5 and Val-400 in IS6 that were shown to be functionally coupled during activation of Na_V1.7 channels (64).

FIGURE 7.

A, computer-based molecular models of Domain II of Ca_V3.2 are shown in a schematic representation. Modeling of Domain II was based upon the atomic coordinates of Na_VAb (PDB code 3RVY) (left panel) and Na_VRh (PDB code 4DXW) (right panel). Thr-911 (IIS4-S5) is shown in stick representation in blue and Ile-1013 (IIS6) is shown in red. B, whole cell current traces are shown from left to right for Ca_V3.2 T911G, I1013G, and the double mutant T911G/I1013G. Current traces were obtained in 10 mm Ba²⁺ from a holding potential of −100 mV. C, normalized conductance (G/G_max) for Ca_V3.2 WT (black circles), T911G (blue squares), I1013G (red triangles), and T9117G/I1013G (black stars) are plotted against the test voltages. D, bar graphs of the mean time constants of activation for Ca_V3.2 WT (black), T911G (pale gray), I1013G (white), and T911G/I1013G (dark gray) as a function of the test potential. E, bar graphs of the mean time constants of inactivation for Ca_V3.2 WT (black), T911G (pale gray), I1013G (white), and T911G/I1013G (dark gray) as a function of the test potential. Numerical values are found in Tables 1–3.

To assess for the specificity of interaction, biophysical properties were characterized for 8 double glycine mutants pairing IIS4-S5 residues (from Val-906 to Met-912) to I1013G or the neighboring L1014G residue. All these double glycine mutants were activated in a voltage-dependent manner albeit with significantly lower peak currents. Most double mutants inactivated with slower kinetics than the WT channel evoking the parent I1013G channel (Table 3). Interaction energies for activation and inactivation of the V906G/I1013G, V909G/I1013G, K910G/I1013G, and T911G/L1014G pairs were not significantly different from 0. Robust interaction energies were, however, measured between Val-907 or Leu-908 in IIS4-S5 and Ile-1013 in IIS6 during channel activation. With a value for ΔΔG_act,interact = 4.6 ± 1.3 kcal mol⁻¹ (n = 6), the interaction energy for V907G/I1013G was in fact stronger than for any other pair of mutants tested in Domain II. As seen, the activation curve (G/G_max) of the double mutant V907G/I1013G was superimposed with those of single mutants V907G and I1013G and significantly left-shifted as compared with the WT channel (Fig. 8). The significant alteration in the electromechanical coupling is supported by the observation that the Q/Q_max and the G/G_max curves of the double V907G/I1013G mutant are shifted in similar directions.

TABLE 3.

Biophysical parameters of Ca_V3.2 wild-type and double mutant channels were estimated from recordings obtained with the cut-open oocyte technique in the presence of 10 mm Ba²⁺

Double mutants are shown with the IIS4-S5 mutant always to the left of the pair. Most double mutants were made between IIS4-S5 and IIS6. Double mutants with Asn-1548 in IIIS6 and Asn-1853 in IVS6 are shown below and are separated by a line. Activation properties (E_0.5,act and z) were estimated from the mean I-V relationships and fitted to a Boltzmann equation where z is the slope factor. Activation and inactivation energies were calculated as follows: ΔG_act = z · F · E_0.5,act; ΔΔG_act = ΔG_act,mut − ΔG_act,wt; ΔG_inact = z · F · E_0.5,inact; and ΔΔG_inact = ΔG_inact,mut − ΔG_inact,wt. The voltage dependence of inactivation was determined from the peak currents after 2-s depolarizing pulses from a holding potential of −120 mV. The data are shown with the mean ± S.E. of the individual experiments and the number of experiments appears in parentheses.

	V0.5, act	ΔGact	ΔΔGact	Ipeak	V0.5, inact	ΔGinact	τact, −20 mV	τinact, −20 mV	ΔΔGinter (act)	ΔΔGinteract
	mV	kcal mol−1		nA	mV	kcal mol−1	ms		kcal mol−1
CaV3.2 WT	−47 ± 1 (37)	−4.3 ± 0.2	NAa	−1300 ± 200	−65 ± 1 (10)	−8.6 ± 0.5	3.2 ± 0.1 (18)	11.6 ± 0.2	NA	NA
V906G/I1013G	−54 ± 2 (5)	−6.1 ± 0.7b	−1.8 ± 0.9	−300 ± 100	−69.1 ± 0.6 (4)	−7.9 ± 0.6	4.2 ± 0.3c (5)	48 ± 2b	−0.8 ± 1.8	−2.1 ± 2.5
V907G/I1013G	−55 ± 1 (6)	−4.1 ± 0.2	0.2 ± 0.4	−190 ± 40	−78.2 ± 0.2 (3)	−7.8 ± 0.5	2.9 ± 0.3 (6)	40 ± 1b	4.6 ± 1.3	0.3 ± 2.1
L908G/N1008G	−50 ± 3 (5)	−5.0 ± 0.7	−0.7 ± 0.9	−80 ± 20	−76 ± 1 (4)	−6.9 ± 0.3	2.8 ± 0.3 (5)	11 ± 1	−1.3 ± 1.9	−0.5 ± 1.9
L908A/I1013A	−52 ± 1 (5)	−5.4 ± 0.3	−1.1 ± 0.5	−160 ± 40	−67 ± 1 (5)	−5.8 ± 0.6c	3.4 ± 0.3 (5)	72 ± 2b	−1.3 ± 1.2	2.4 ± 2.5
L908G/I1013G	−48 ± 1 (6)	−3.9 ± 0.3	0.4 ± 0.5	−120 ± 20	−72 ± 1 (4)	−7.2 ± 0.4	3.7 ± 0.5 (6)	25.0 ± 0.8b	2.1 ± 1.1	0.6 ± 2.5
V909G/I1013G	−48 ± 1 (4)	−4.9 ± 0.9	−0.6 ± 1.1	−170 ± 40	−65 ± 2 (3)	−6.9 ± 0.8	5.0 ± 0.5b (4)	53 ± 3b	0.4 ± 1.8	1.1 ± 2.6
K910G/I1013G	−46.7 ± 0.6 (5)	−3.9 ± 0.2	0.4 ± 0.4	−150 ± 30	−65 ± 1 (4)	−6.2 ± 0.2	3.6 ± 0.2 (5)	47 ± 4b	0.2 ± 0.9	1.1 ± 2.3
T911A/I1013A	−46 ± 2 (6)	−4.4 ± 0.4	−0.1 ± 0.6	−340 ± 80	−60.5 ± 0.8 (6)	−6.9 ± 0.5	6.1 ± 0.5b (6)	71 ± 5b	−1.6 ± 1.2	NDd
T911G/I1013G	−43.4 ± 0.8 (6)	−3.6 ± 0.2	0.7 ± 0.4	−260 ± 30	−66 ± 1 (6)	−6.0 ± 0.5c	6.7 ± 0.5b (6)	41 ± 2b	1.8 ± 0.9	2.9 ± 2.6
T911G/L1014G	−47 ± 2 (6)	−3.8 ± 0.4	0.5 ± 0.6	−200 ± 100	−75 ± 1 (4)	−8 ± 1	8 ± 1b (6)	26 ± 1b	−0.8 ± 1	1.8 ± 2.8
M912G/I1013G	−48.6 ± 0.7 (6)	−4.7 ± 0.1	−0.4 ± 0.3	−180 ± 60	−68 ± 1 (6)	−7.5 ± 0.4	5.0 ± 0.6b (6)	42 ± 1b	1.1 ± 1.1	2.3 ± 2.8
N914G/E1016G	−36.5 ± 0.9 (6)	−2.8 ± 0.2b	1.5 ± 0.4	−90 ± 10	−61.0 ± 0.7 (6)	−7.0 ± 0.4	3.9 ± 0.2c (6)	15.0 ± 0.3b	−0.8 ± 1.2	ND
V915G/L1010G	−27 ± 3 (6)	−1.6 ± 0.3b	2.7 ± 0.5	−110 ± 30	−58 ± 1 (3)	−8.6 ± 0.6	5.1 ± 0.6b (6)	14.9 ± 0.9b	−0.3 ± 0.6	ND
V907A/N1548A	−43 ± 3 (4)	−3.4 ± 0.6	0.9 ± 0.8	−50 ± 10	−90 ± 2 (2)	−6 ± 2	3.1 ± 0.2 (4)	12 ± 2	1 ± 1.2	−1.4 ± 3.1
V907G/N1548G	−47 ± 1 (10)	−3.8 ± 0.2	0.5 ± 0.4	−110 ± 17	−82 ± 2 (6)	−5.7 ± 0.6c	2.6 ± 0.1 (10)	15 ± 1b	3.4 ± 1.2	1.6 ± 1.6
L908A/N1548A	−39 ± 2 (6)	−3.6 ± 0.3	0.7 ± 0.5	−180 ± 30	−74.6 ± 0.9 (6)	−7.3 ± 0.2	4.0 ± 0.3c (6)	9.9 ± 0.3c	0.8 ± 1.0	−0.8 ± 1.7
L908G/N1548G	−35 ± 1 (11)	−3.3 ± 0.4c	1.0 ± 0.6	−220 ± 60	−74 ± 1 (9)	−7.2 ± 0.5	3.5 ± 0.2 (11)	16.5 ± 0.4b	1.2 ± 1.1	−0.3 ± 2.0
T911A/N1548A	−36 ± 2 (7)	−2.9 ± 0.4b	1.4 ± 0.6	−240 ± 70	−73 ± 2 (6)	−8.4 ± 0.5	4.0 ± 0.2c (7)	10.0 ± 0.7c	0.2 ± 1.0	ND
T911G/N1548G	−37 ± 2 (7)	−2.6 ± 0.2b	1.7 ± 0.4	−100 ± 20	−74 ± 2 (4)	−7.6 ± 0.4	3.4 ± 0.4 (7)	13 ± 1	1.3 ± 0.8	0.4 ± 1.9
L908G/N1853G	−43.4 ± 0.9 (9)	−3.8 ± 0.2	0.5 ± 0.4	−180 ± 30	−70 ± 2 (5)	−7.9 ± 0.6	3.5 ± 0.2 (9)	17.2 ± 0.4b	−1 ± 1	ND

^a NA, not applicable.

^b p < 0.01 as compared with Ca_V3.2 WT.

^c p < 0.001 as compared with Ca_V3.2 WT.

^d ND, not determined.

FIGURE 8.

A, computer-based molecular model and Domain II of Ca_V3.2 are shown in a schematic representation. Modeling of Domain II was based upon the atomic coordinates of Na_VAb (PDB code 3RVY) (left panel) and Na_VRh (PDB code 4DXW) (right panel). Val-907 (IIS4-S5) is shown in stick representation in blue and Ile-1013 (IIS6) is shown in red. B, whole cell current traces are shown from left to right for Ca_V3.2 V907G, I1013G, and the double mutant V907G/I1013G. Current traces were obtained in 10 mm Ba²⁺ from a holding potential of −100 mV. C, normalized conductance (G/G_max) for Ca_V3.2 WT (black circles), V907G (blue squares), I1013G (red triangles), and V907G/I1013G (black squares) are plotted against the test voltages. D, normalized gating charge (Q/Q_max) was plotted against voltage plot for Ca_V3.2 WT (black circles), V907G (blue squares), I1013G (red triangles), and V907G/I1013G (black squares). Mid-points of charge movement Q/Q_max were −44 ± 1 mV (n = 4) for WT, −57 ± 4 mV (n = 6) for V907G, −56 ± 2 mV (n = 5) for I1013G, and −57 ± 2 mV (n = 6) for V907G/I1013G. E, bar graphs of the mean time constants of inactivation for Ca_V3.2 WT (black), V907G (pale gray), I1013G (white), and V907G/I1013G (dark gray) as a function of the test potential. Numerical values are found in Tables 1–3.

Finally, a few other pairs of residues predicted by the three-dimensional structure of Na_VAb to be in close proximity were tested using the same approach. Asn-211 in S6 of Na_VAb is predicted to lie within the atomic distance of Leu-123 in S4-S5 (44). These residues are aligned with Asn-1008 (IIS6) and Leu-908 (IIS4-S5) in Ca_V3.2 (Figs. 3 and 6A). Double mutants L908G/N1008G, N914G/E1016G, and V915G/L1010G yielded voltage-activated currents with ΔΔG_act,interact values that were not significantly different from 0 although these residues might be coupled during inactivation (Table 3).

Interdomain Coupling during Activation

Homology models of Ca_V3.2 also predict numerous points of contact between S4-S5 and S6 of adjacent domains (Fig. 9). In particular, Val-907 and Leu-908 in IIS4-S5, shown to be functionally coupled with Ile-1013 in IIS6, are predicted to face Asn-1548 in IIIS6 in both homology models. Leu-908 in IIS4-S5 is predicted to be closer to Asn-1548 in IIIS6 than Ile-1013 in IIS6. To note, Asn-1548 is strictly conserved in the primary sequence of IIIS6 in all HVA and LVA Ca_V channels (26). Despite this predicted proximity (≤8 Å between the closest atoms in the lateral chains), the ΔΔG_act,interact = 1.2 ± 1.1 kcal mol⁻¹ (n = 6) was not significantly different from 0 (Table 3).

FIGURE 9.

Computer-based molecular models of Domains II and III (top center and bottom right panels) of Ca_V3.2 are shown in a schematic representation. Modeling of Domains II and III was based upon the atomic coordinates of Na_VAb (PDB code 3RVY) (bottom panels) and Na_VRh (PDB code 4DXW) (top panels). Top left and bottom left, Leu-908 (IIS4-S5) is shown in stick representation in blue and Ile-1013 (IIS6) and Asn-1548 (IIIS6) are shown in red. Top center panels, Leu-908 and Thr-911 (IIS4-S5) are shown in stick representation in blue and Ile-1013 (IIS6) and Asn-1548 (IIIS6) are shown in red. Right top and bottom panels, Val-907, Leu-908, and Thr-911 (IIS4-S5) are shown in stick representation in blue and Ile-1013 (IIS6) and Asn-1548 (IIIS6) are shown in red.

The neighboring residue Val-907 is predicted to protrude slightly further away from Asn-1548 with predicted distances ranging from 7 to 10 Å (Fig. 10). The double mutant V907G/N1548G activated in a voltage-dependent manner with ΔG_act = −3.7 ± 0.3 kcal mol⁻¹ (Table 3). Both single mutants activated at significantly more depolarized (N1548G) or significantly hyperpolarized (V907G) voltages than the WT channel yielding a large ΔΔG_act,interact = 3.5 ± 1.3 kcal mol⁻¹ (n = 6) (Tables 1–3). This interaction energy suggests significant coupling between Domains II and III during channel activation. We could not, however, estimate the change in Q/Q_max. Poor functional expression of V907G/N1548G, with peak currents in average 10 times lower than recorded for the WT channel (even when measured 7 days after injection) precluded the measure of gating currents. Qualitatively similar results were obtained for the V907A/N1548A double alanine mutant although the ΔΔG_act,interact value was smaller. This reduced interaction energy might be explained by the fact that alanine residues are more hydrophobic than glycine. The V907G/N1548G channel showed slightly slower inactivation kinetics and slightly faster deactivation kinetics. The coupling energy between Val-907 and Asn-1548 was stronger than for double mutants T911G/N1548G, T911A/N1548A, L908G/N1548G, L908A/N1548A, and L908G/N1853G, the latter being in IVS6 (Fig. 11 and Table 3). Nonetheless, interaction between these residues cannot be ruled out as the values of ΔΔG_act,interact we have measured remain on the verge of statistical significance.

FIGURE 10.

A, computer-based molecular models of Domains II and III of Ca_V3.2 are shown in a schematic representation. Modeling of Domains II and III was based upon the atomic coordinates of Na_VAb (PDB code 3RVY) (left panel) and Na_VRh (PDB code 4DXW) (right panel) and was achieved with Modeler 9v4. Val-907 (IIS4-S5) is shown in stick representation in blue and Asn-1548 (IIIS6) is shown in red. The shortest distance between Val-907 and Asn-1548 was estimated to be 6.3 Å for the model based with Na_VAb and 4.3 Å for the model based with Na_VRh. B, whole cell current traces are shown from left to right for Ca_V3.2 V907G, N1548G, and V907G/N1548G. Current traces were obtained in 10 mm Ba²⁺ from a holding potential of −100 mV. C, normalized conductance for Ca_V3.2 WT (black circles), V907G (blue squares), N1548G (red triangles), and V907G/N1548G (black stars) were obtained by fitting the experimental data to a Boltzmann equation. D, bar graphs of the mean time constants of activation for Ca_V3.2 WT (black), V907G (pale gray), N1548G (white), and V907G/N1548G (dark gray) as a function of the test potential. E, the mean time constants of deactivation were obtained from fitting tail currents with monoexponential functions. The deactivation time constants measured at −50 mV were 8.4 ± 0.6 ms (n = 9) for WT, 3.8 ± 0.6 ms (n = 11) for V907G, 2.5 ± 0.5 ms (n = 10) for N1548G, and 2.9 ± 0.3 ms (n = 6) for V907G/N1548G. Numerical values are found in Tables 1–3.

FIGURE 11.

Bar graph of the coupling energies (ΔΔG_act,interact) for the double glycine mutants of Ca_V3.2, ΔΔG_act,interact = (ΔG_act,WT + ΔG_act,double) − (ΔG_act,S4-S5 + ΔG_act,S6). As seen, the interaction energies were significantly stronger for V907G/I1013G and V907G/N1548G than for any other double mutant. Data are shown in kcal mol⁻¹. Symbols * and ** identify double mutants with interaction energies significantly larger than 1.5 kcal mol⁻¹ at p < 0.05 and p < 0.01, respectively. Numerical values are found in Table 3.

DISCUSSION

In this study we have analyzed the molecular mechanisms responsible for the voltage-dependent activation of the T-type Ca_V3.2 channel, and more specifically the role of the domain/domain interaction in this process. The asymmetrical nature of Ca_V3.2 is perfectly suited to study the nature of the inter-domain (or intersubunit in K_V channels) interaction during channel activation. Furthermore, the T-type Ca_V3.2 channel can be functionally expressed in the absence of auxiliary subunits unlike HVA Ca_V1.2 (66) and Ca_V2.3 (39, 45), thus minimizing indirect effects caused by modulation of gating by Ca_Vβ and Ca_Vα2δ subunits. Biophysical properties were characterized for 47 single mutants and 20 pairs of glycine mutants. Our data identified 4 unique sets of interaction suggesting that residues in the S4-S5 linker of Domain II are functionally coupled with key residues located on the S6 segments of Domains II and III during the activation of LVA Ca_V3 channels. Mutations of nearby residues in IIS4-S5 displayed milder if any coupling with other residues in IIS6, IIIS6, or IVS6. This is the first report of residues in the S4-S5 linker being coupled at once with S6 residues from 2 adjacent pore regions (Domains II and III). Interdomain (intersubunit) interaction between the voltage sensor S4 and pore residues in S6 has been inferred from the formation of disulfide bonds in Shaker Kv (41) and from double mutant cycle analysis in Ca_V1.2 (13), respectively.

These zones of interaction were in general agreement with predictions from bacterial Na_V crystal structures. Although the latter structures turned out to be superior templates for T-type Ca_V channels than K_V1.2 (59), the presence of large gaps in the region following S5 in Domains II and III of Ca_V3.2 in both computer-based three-dimensional models limits our predictions regarding the relative orientation of the residues between the S4-S5 linker and the S6 pore region. This constraint does not exclude possible physical interaction between residues in S4-S5 and distal S6 but precludes a conclusive statement. Nonetheless, the strong interaction energy measured with the V907G/I1013G mutant suggests that van der Waals interactions between Val-907 in II S4-S5 and Ile-1013 in IIS6 are compromised in the presence of a pair of glycine residues. Interactions at play during activation might not be identical to the ones determining inactivation. Pairs of residues showing functional coupling during channel activation (Val-907/Ile-1013, Leu-908/Ile-1013, Val-907/Asn-1548, and Thr-911/Ile-1013) did not show significant interaction energy during channel inactivation. These observations suggest either that activation and inactivation are structurally distinct processes or that inactivation engages a larger number of contact points between the pore region and the connecting linkers.

Limitations of Our Study

The ΔG_act and ΔΔG_act,interact reported herein for Ca_V3.2 with glycine mutants were significantly lower than the one estimated between L596G in IIS4-S5 and I701G in IIS6 in Ca_V2.3 (39). By comparison, introducing glycine residues in the IIS4-S5 linker or in IIS6 of Ca_V2.3 significantly decreased the activation potential with −2 < ΔΔG_act < −6 kcal mol⁻¹ (39). It remains to be seen whether the smaller changes in ΔΔG_act observed for Ca_V3.2 suggests that the channel structure is already optimized to open in response to depolarization.

Nonetheless, small ΔΔG_act values yield smaller ΔΔG_act,interact values. Because of the experimental error inherent to the measure of 4 independent experimental parameters, significant ΔΔG_act,interact values are more likely to be identified from coupling two single mutants that are displaying large ΔG_act. Point in case, ΔΔG_act,interact was smaller for the double V907A/N1548A than for V907G/N1548G despite the fact that the mutants positions are identical in the two mutants. One might speculate that mutations with residues more hydrophilic than glycine might increase the ΔΔG_act,interact values. Given the inherent limitations of the analysis, a small ΔΔG_act,interact value hence does not necessarily rule out functional coupling. Double-mutant cycle analysis carried out in Ca_V1.2 and Ca_V2.1 with residues predicted to be aligned with Leu-596 and Ile-701 failed to show any significant energy coupling (39) despite the high likelihood that the coupling between S4-S5 and S6 involves universally conserved pairs of residues. Caution is thus to be exercised in interpreting ΔΔG_act,interact as this parameter measures the relative coupling energy and not the absolute value of energy coupling. Despite these limitations, the thermodynamic mutant analysis remains undeniably a useful tool to qualitatively identify functional hot spots that may not be inferred from static crystal structures.

Model of Channel Activation

LVA Ca_V channels open in response to smaller depolarization than HVA Ca_V1.2 and Ca_V2.3 channels, yet their S4-S5 helices appear to be strongly coupled with S6 residues during channel activation. The electromechanical model of channel activation states that the channel closed state is stabilized by hydrophobic interactions in the distal S6 region and the upward movement of the voltage sensor is mechanically transduced to the channel pore S6 residues through the S4-S5 helices. The role of the S4-S5 linker in this process has yet to be elucidated. The S4-S5 linker can act as a high-energy bolt that prevents the pore S6 residues from opening. The outward movement of the voltage sensor would be sufficient to disrupt the interaction and the pore would then open passively in the absence of any physical hindrance (65). It is also possible that the S4-S5 linker and the pore S6 residues remain in close contact during activation and are being pulled away simultaneously as a single unit. In both cases, the interaction between S4-S5 and S6 has to occur in the closed state, a conclusion that is also borne out by the Na_V-based molecular models. The observation that the voltage dependence of the charge movement (Q/Q_max) and the conductance (G/G_max) were shifted similarly pleads in favor of a model where the energy deployed by the movement of the voltage sensor is directly transduced to the S6 pore residues. Finally, the strong interaction energies measured between Domains II and III suggests that activation of Ca_V3.2 occurs in a concerted manner across all 4 voltage sensors rather than by each S4 moving separately in a stochastic manner. It would be highly interesting to investigate whether the changes induced by the glycine mutations in IIS4-S5 or IIIS6 are caused or results from the movement of any S4 segment using voltage-clamp fluorimetry. These questions go beyond the scope of this current investigation and await further structural studies of Ca_V channels.