Pore stability and gating in voltage-activated calcium channels

Abstract

Calcium channel family members activate at different membrane potentials, which enables tissue specific calcium entry. Pore mutations affecting this voltage dependence are associated with channelopathies. In this review we analyze the link between voltage sensitivity and corresponding kinetic phenotypes of calcium channel activation. Systematic changes in hydrophobicity in the lower third of S6 segments gradually shift the activation curve thereby determining the voltage sensitivity. Homology modeling suggests that hydrophobic residues that are located in all four S6 segments close to the inner channel mouth might form adhesion points stabilizing the closed gate. Simulation studies support a scenario where voltage sensors and the pore are essentially independent structural units. We speculate that evolution designed the voltage sensing machinery as robust “all-or-non” device while the varietys of voltage sensitivities of different channel types was accomplished by shaping pore stability.

Introduction

Voltage-dependent gating involves the movement of a charged voltage sensor through the transmembrane electric field. Voltage sensor movements were first detected in sodium channels as “gating currents”.¹ Gating currents precede the ionic current and reflect the translocation of charged amino acid residues (mainly arginines) in segments S4 across the membrane. The opening of the channel pore can be considered as a multi-step process consisting of: (i) the movement of the voltage sensor; (ii) a transmission of the conformational change to the pore region and (iii) the opening of the pore itself.² These theoretical considerations are supported by structural data indicating that the transmembrane portion of voltage gated channels is composed of structurally distinct domains. Four voltage-sensing domains, each of which is composed of four transmembrane segments called S1–S4, are peripheral to a central pore domain formed by four sets of segments called S5, P and S6. The voltage sensors are essentially independent structural units.^3-5 Semi-independence of the major structural features of voltage sensors and the pore is apparent from crystal structures and from the fact that some K⁺ channels have a pore domain but no voltage-sensing domain while voltage sensing domains without a pore domain regulate phosphatase activity and proton permeation.⁶ Voltage-induced conformational changes within the voltage-sensing unit are transmitted to the pore through a segment linking the two units. Hence, some features of the pore may not involve the voltage sensor. For example, single channel studies indicate that the open probability is far from unity even at high depolarizations. Flickering between open and closed states reflects either fluctuations of the voltage-sensing machinery or, alternatively, fluctuations within the pore domain while the voltage sensors are steadily in an activated position. In the latter scenario, voltage sensor movements during a depolarization would just increase the probability of pore openings but not open the pore directly.^2,7-9

To understand the voltage dependence of ion channel openings and closures, one must answer several questions: How tightly are gating charge movements coupled to the pore region? What determinants stabilize the pore in the open or closed conformations? Which physico-chemical properties of pore residues stabilise the channel in the closed and/or open states? How do changes in pore stability modulate the kinetics of channel activation? Does the pore have multiple mechanisms by which it opens and closes?

No answers have yet been provided for voltage gated calcium channels (Ca_V). Calcium channel activation is affected by numerous mutations in the pore region. Diseases caused by mutations in ion channels are termed channelopathies.¹⁰ Many of these structural changes are associated with calcium channelopathies such as hemiplagic migraine, ataxia, stationary night blindness and other diseases.¹¹ Other residues determining Ca_V activation have been detected in pore forming S6 segments during structure activity studies.^12-15

In this review we focus on recent progress in analyzing the determinants of pore stability of Ca_V. Specifically, we analyze the impact of structural changes in the pore region of Ca_V (point mutations in pore forming S6 segments) on steady state channel activation and kinetics. We use homology modeling to illustrate why hydrophobic interactions in the lower third of S6 segments may have profound effects on the stability of the closed channel gate.

Voltage Dependence of Calcium Channel Activation

Calcium entry through calcium channels during an action potential initiates and controls multiple cascades of intracellular events affecting a large variety of cellular functions such as generation and propagation of electrical impulses, sensory processes, muscle contraction, secretion of hormones and neurotransmitters, cell differentiation and gene expression.¹⁶ Mammalian Ca²⁺ channel α₁-subunits are encoded by at least 10 genes.¹⁷ The potential where voltage gated ion channels first open during a depolarisation (so called “threshold potential”) or, the more commonly estimated voltage where 50% of the channels are activated in steady-state (V_0.5) are hallmarks of the respective channel family members. It appears that the adjustment of the threshold potential during evolution represents an important mechanism for fine-tuning of voltage-dependent Ca²⁺ entry into cells of different tissues. Low voltage activated calcium channels (Ca_V3) open after small depolarisations of the plasma membrane and mediate low-threshold Ca²⁺ spikes.¹⁸ The V_0.5 of the Ca_V3 family is around −45 mV in 2 mM extracellular calcium.¹⁸ Larger membrane depolarisations are required for activation of high voltage-activated Ca²⁺ channels. Different isoforms of high threshold Ca²⁺ channels activate at significantly different potentials. The voltage for half-maximal activation of Cav1.1 lies between 8 and 14 mV (10 mM Ca²⁺), V_0.5 of Ca_V1.2 at −4 mV (15 mM Ba²⁺), V_0.5 of Ca_V1.3 at −18 mV (15 mM Ba²⁺), V_0.5 of Ca_V1.4 between −2.5 and −12 mV (15–20 mM Ba²⁺), V_0.5 of Ca_V2.1 between −5 and −11 mV (5 mM Ba²⁺), V_0.5 of Ca_V2.2 at 8 mV (15 mM Ba²⁺), V_0.5 of Ca_V2.3 at 3.5 mV (15 mM Ba²⁺).¹⁷ Ca_V1.1 channels activate slowly and serve as voltage sensors for the SR ryanodine receptor in skeletal muscle. Ca_V1.2 and Ca_V1.3 are distributed in neurons, sensory cells of the retina and the inner ear¹⁹ whereas the Ca_V1.4 is mainly found in the retina.²⁰ Ca_V2.1, Ca_V2.2 and Ca_V2.3 are predominantly found in presynaptic terminals, dendrites and cell bodies of neurons (Ca_V2.1 and Ca_V2.3 are also found in heart, testes and pituitary).¹⁷

Channelopathies are Associated with Changes in Voltage Dependence of Calcium Channel Activation

Structural determinants underlying the different voltage sensitivities of different calcium channel family members are currently unknown. The pore-forming α₁-subunits of all calcium channels are composed of four homologous repeats formed by six transmembrane segments (S1–S6) that are linked together on a single polypeptide.^17,18,21 In analogy to other voltage gated ion channels, it is assumed that the voltage-sensing machinery of calcium channels is formed by multiple charged residues located in S4 and interacting segments of each domain.

Mutations associated with a Ca²⁺ channelopathy called familial hemiplegic migraine (FHM) were identified on the α₁-subunit of Ca_V2.1.^22-25 Several of these mutations affect the voltage-dependence of channel activation. The voltage for half maximal activation of Ca_V2.1 (V_0.5) was shifted by −12 mV (W1684R in the IVS4-S5 linker),²⁵ −3 mV (K1336E in the IIIS3-S4 linker),²⁵ −7 mV (V1457L in the IIIS5-S6 loop),²⁴, −5 mV (I1815L in IVS6)²³ or by about −10 mV (V714A in IIS6).²³ A mutation associated with episodic ataxia (EA-2) was found to induce a shift of V_0.5 to the right by about 6 mV (G293R in the pore loop between IS5 and IS6).²⁶ A possible functional consequence of a leftward shift of the activation curves is an increased calcium entry at voltages near the resting potential and enhanced spiking activity leading to calcium overload and cellular dysfunction.

At least 48 mutations linked to X-linked recessive congenital stationary night blindness type 2 (CSNB2) have been identified in the CACNA1F gene encoding retinal L-type Ca²⁺ channels (Ca_V1.4).²⁷ Some of them are missense mutations affecting the voltage-dependence of channel activation. Hemara-Wahanui et al.²⁸ reported that a mutation of a conserved isoleucine residue in the pore-lining segment IIS6 of Ca_V1.4 causes severe visual impairment. Functional studies revealed that substitution I745T in the CACNA1F allele produced a remarkable shift of about −30 mV in the voltage-dependence of Ca_V1.4 channel activation and significantly slower inactivation kinetics in an expression system.²⁸ The corresponding mutation in Ca_V1.2 (I781T) induced a similar shift of the current voltage relation (Fig. 1). Hoda et al.²⁹ reported a shift of the activation curve in the hyperpolarising direction upon replacement of G369D in segment IS6 of Ca_V1.4 that was associated with slowed inactivation and removal of Ca²⁺-dependent inactivation.

Figure 1.

Functional analysis of I781T, the Ca_V1.2 mutant corresponding to I745T Ca_V1.4. (A) representative families of I_Ba through wild-type (top) and I781T mutant (bottom) channels during depolarizing test pulses from −100 mV (threshold and maximal voltages are indicated, 10-mV increments). Wild-type or I781T mutant Ca_V1.2 α₁-subunits were co-expressed together with β_1a and α₂-δ-subunits. (B) Averaged current-voltage relationships (normalized to maximal current) of the wild-type (n = 8, open circles) and I781T mutant (n = 7, filled circles) channels. Data reproduced from Hohaus et al.¹² with permission.

Kinetic Hallmarks of Pore Stability

Substitutions of I781 in α_1.2-subunit by residues of different hydrophobicity, size and polarity¹² all shifted channel activation in the hyperpolarising direction with I781P causing the most severe (−37 mV shift) effect (Fig. 2F). Mutations in position I781 slow deactivation at all potentials (Fig. 2C and E). The voltage dependence of the activation time constants of different mutants is illustrated in Figure 2B. The time courses of current activation at large depolarizations (>−20 mV) were similar, while channel activation near the footstep of the activation curve was slower than in wild-type (Fig. 2D, between −60 and −30 mV). Shifts to more hyperpolarized voltages correlated with slower activation. Additional mutational studies revealed an important role of neighboring residues. This motif of hydrophobic residues in the lower third of segment IIS6 is conserved in high voltage activated calcium channels (Fig. 2A). It was hypothesized that residues LAIA (779–782)¹² play an important role in activation gating. Replacement any of these IIS6 residues with helix breaking prolines induced similar changes in channel gating: a shift in the voltage-dependence of activation accompanied by a slowing of the activation kinetics near footstep of the activation curve, a slowing of deactivation at all potentials and decreased inactivation.

Figure 2.

Altered Gating of Ca_V1.2 by Substitution of Theronine and Proline for Isoleucine 781 in Segment II S6. (A) alignment of S6 segments (TM2 helices in KcsA), for Ca_V1.2, Ca_V2.3, NaChBac and KscA.). Adhesion points are highlighted gray, putative hydrophobic seals in Ca_V and NaChBac in analogy to KcsA are coloured green. (B and C) Activation (B) and deactivation (C) kinetics of I_Ba through wild-type, I781T and I781P channels (D and E) Voltage dependences of the time constants of activation (D) and deactivation (E) for wild-type, I781T and I781P mutant channels. (F) averaged voltage dependences of activation of wild-type (n = 8), I781P (n = 8), I781T (n = 7), I781A (n = 7), I781L (n = 5) and I781N (n = 4) channels. Data reproduced from Hohaus et al.¹² with permission.

Similar shifts of the activation curve and even more dramatic slowing of the activation and deactivation kinetics were observed in NaChBac. Mutation of the conserved glycine 219 in NaChBac (corresponding to a putative hinge glycine in S6 of most K⁺ channels, see Fig. 2A) to proline not only shifts the voltage dependence, but also slows deactivation (Fig. 3).¹⁵ A comparison of Figures 2 and 3 illustrates the common kinetic phenotypes in different channels: (i) shifted activation curve; (ii) slow activation and (iii) a deceleration of channel deactivation. Obviously these can be induced by structural changes in different positions of the S6 segments. Helix bending in the upper third of S6 in NaChBac induces even stronger effects than structural changes close to the inner channel mouth of L-type (Ca_V1.2) channels. A locus of conserved hydrophobic residues VAVIM (1718–1722) in pore forming segment IVS6 of Ca_V2.3 was systematically substituted by flexible glycines.¹⁴ Glycine mutations affected channel inactivation kinetics. Slow activation of mutant V1720G (IVS6, Fig. 4A) was accompanied by a −20 mV shift of the curve, suggesting a relative increase in open state stability. Different deactivation kinetics of glycine mutants V349G (IS6), I701G (IIS6), L1420G (IIIS6) and V1720G (IVS6) are illustrated in Figure 3. The most prominent effect in Ca_V2.3 was, however, induced by mutating I701 (corresponding to the I781 in IIS6 of Ca_V1.2) to glycine (Fig. 4B) which may indicate a particular role of IIS6 in calcium channel gating (Fig. 4). Different contributions of residues in analogous positions in segments IS6–IVS6 reflect the structural asymmetry of the channel pore.

Figure 3.

Altered Gating in NaChBac by Substitution of Proline for a Hinge Glycine Residue in the S6 Segment (see Fig. 2A for location of G219). (A and B) Homology model of the proposed change in the preferred position of the S6 segments of NaChBac due to substitution of a proline (in space-filling representation, orange) for glycine (G219P). Model based on Jiang et al. (2002b), as described in Experimental Procedures as the channels move from closed (blue helices) to open (magenta helices) conformations (opening movement shown by arrows). (A) Side view of two channel subunits. (B) View from the cytoplasmic channel surface. (C) Sodium current records from wt (top) and G219P (bottom) in response to a series of depolarized test-pulse potentials from −55 mV (wt) or −115 mV (G219P) with 10 mV steps from a holding potential of −120 mV. (D) Voltage dependence of activation from measurements of tail currents after 50 ms (wt) or 2 s (G219P) depolarization. Voltages to activate half of the maximum current (V_0.5) are −24.0 ± 1.6 mV and −75.2 ± 0.9 mV for wild-type (n = 7) and G219P (n = 7), respectively. The slope factors are 10.8 ± 1.1 mV and 6.8 ± 0.7 mV for wild-type and G219P, respectively. (E) Sodium current traces during depolarizations to 0 mV (top) and time constants (τ) of activation (bottom) obtained by fitting the final activation phase of currents with single exponentials for wild-type (open circles, n = 6) and G219P (filled circles, n = 6). (F) Tail currents at −120 mV (top) and time constants of channel closure (bottom) measured during repolarization to the indicated potentials following a test pulse to −10 mV from a holding potential of −120 mV for wild-type (open circles, n = 5) and following a test pulse to −60 mV for G219P (closed circles, n = 8). From Zhao et al.¹⁵ with permission.

Figure 4.

Deactivation Time Constants for the S6 Residues Facing Val1720 in Ca_V2.3. (A) representative tail currents for wild-type, V349G, L1420G and V1720G channels. Currents were activated during a 8-ms conditioning depolarization to 0 mV for Ca_V2.3 wild–type, V349G and L1420G; and −10 mV for V1720G. Deactivation was recorded during subsequent repolarizations with 10-mV increments starting from −120 mV (test potentials). Time constants were estimated by fitting current deactivation to a monoexponential function. (B) representative tail currents for I701G channels. Currents were activated during a 15-ms conditioning depolarization to −40 mV. Deactivation was recorded during subsequent repolarizations with 10-mV increments starting from −120 to −40 mV (test potentials). Time constants were estimated by fitting current deactivation to a mono- or a biexponential function. Monoexponential functions were found to fit reasonably well the tail currents of all channels and allow for a better comparison between the wild-type and other mutants. (C) mean time constants of channel deactivation (monoexponential functions) for Ca_V2.3 wild-type, V349G, I701G, L1420G and V1720G are plotted versus test potential. At −40 mV, the time constants of deactivation were: 1.7 ± 0.1 ms (n = 8) for wild-type; 2.0 ± 0.2 ms (n = 7) for V349G; 3.3 ± 0.06 ms (n = 6) for L1420G; 5 ± 1 ms (n = 6) for V1720G; and 130 ± 20 ms (n = 6) for I701G such that deactivation kinetics decreased from wild-type ≅ V349G < L1420G < V1720G << I701G. The pulse protocol is shown in the inset. From Raybaud et al.¹⁴ with permission.

Gating Hinges in Calcium Channel S6 Segments?

In analogy to KcsA^30,31 it is assumed that S6 segments of voltage-gated calcium channels line the channel pore with a bundle crossing region forming the channel gate. In K_V the inner part of S6 rotates about a glycine “gating hinge” during the closed to open transition.³² This glycine residue, in position G89 of the MthK and G99 in KcsA potassium channels,³³ is highly conserved in many ion channels. Mutating the analogous glycine to proline in NaChBac drastically alters gating properties (Fig. 3). An analogous glycine 770 in the corresponding position of segment IIS6 of Ca_V1.2 was mutated to proline by Hohaus et al.¹² G770P had, however, neither significant effects on the current kinetics nor on the voltage-dependence of channel activation and inactivation. This finding suggests that the mechanism of Ca_V1.2 activation is different from NaChBac and MthK. Furthermore, in Ca_V1 and Ca_V2 the analogous S6 glycine is present only in IS6 and IIS6 (Fig. 2A) suggesting different structural changes during activation than in Kv and NaChBac. It is interesting to note that lethal arrhythmias are associated with mutations of glycine residues (G406R) in the human Ca_V1.2.^34,35

Sealing Points and Adhesion Points

From the crystal structure of the KcsA it was concluded that three amino acids in the pore lining transmembrane helices (TM2) (Thr107, Ala111 and Val115)³² are likely to form “hydrophobic seals” that prevent permeation through closed channels. A “sealing point” is characterized by convergence of the methylene groups from all amino acids of the symmetric TM2 segments. The methylene groups seal tight enough to prevent the passage of dehydrated potassium ions.³² It is currently unknown, however, whether hydrophobic interactions in these positions contribute to stabilization of KcsA in the closed state.

In calcium channels changes in hydrophobicity in the inner pore region induce major changes in pore stability. Examples of how these changes in selected positions of S6 segments affect the kinetics and voltage sensitivity of Ca_V1.2 are shown in Figure 2 (see ref. 14 for Ca_V2.3).

Plotting the shifts of the activation curves (ΔV_0.5) versus the changes in hydrophobicity in position 781 reveals a strong correlation (Fig. 5B), whereas no correlation was observed for changes in molar mass and residue size (ΔVan der Waals volume). A similar approach for analysing the mutational data of Raybaud et al.,¹⁴ for substitutions in positions I701 (IIS6), L1420 (IIIS6) and V1720 (IVS6) revealed a similar strong correlation between ΔV_0.5 and changes in hydrophobicity for Ca_V2.3 (Fig. 6). The positions of these residues in Ca_V2.3 are highlighted in Figure 2A.

Figure 5.

Changes in Hydrophobicity Correlate with Shifts of the Ca_V1.2 Activation Curve. (A) Homology model of Ca_V1.2 in the closed channel conformation illustrating a putative “adhesion point” (residues L431 (IS6) + L779 (IIS6), I781 (IIS6) + V1192 (IIIS6), F1194 (IIIS6) + V1502 (IVS6) and V1504 (IVS6) + L431 (IS6). (B–D) The role of different amino acid properties in position I781 was analyzed by plotting changes in hydrophobicity (B), molar mass (C) and van der Waals volume (D) versus the estimated shifts of the activation curves (ΔV_0.5 = V_0.5,MUT - V_0.5,WT) (see Fig. 2F). A correlation between ΔV_0.5 and ΔHydrophobicity was observed. No correlation was evident for changes in molar mass or van der Waals volume.

Figure 6.

Changes in Hydrophobicity Correlate with Shifts of the Ca_V2.3 Activation Curve. (A) Illustration of the location of putative “adhesion point” (I701, L1420, V1720 residues) in a homology model of Ca_V2.3 in the closed channel conformation. (B-D) Changes in hydrophobicity (Δhydrophobicity) plotted versus the shifts of the activation curves (ΔV_0.5 = V_0.5,MUT - V_0.5,WT) for substitutions in position I701 (segment IIS6, B), L1420 (segment IIIS6, C) and V1720 (segment IIS6, D). Activation data (V_0.5) are taken from Raybaud et al.¹⁴

The correlation between activation and hydrophobicity suggests that the side chains are buried in a hydrophobic environment when the channel is closed but exposed to water when it is open. The homology models suggest that these residues interact with neighbouring hydrophobic residues in other S6 segments for the closed conformation. We hypothesise that the interaction between residues L347 (IS6) and V1720 (IVS6), I701 (IIS6) and V1418 (IIIS6), and L1420 (IIIS6) and V1718 (IVS6), provides stability to the closed channel pore. These interactions significantly contribute to pore stability and may therefore differ from the “sealing points” in KcsA described above. We prefer the term “adhesion point”. An “adhesion point” would thus more efficiently stabilize helix-helix interactions while prevention of calcium ion passage may occur at a different location in the pore.

Activation Determinants in Calcium Channel Families

The first attempt to localise structural elements in Ca²⁺ channel α₁-subunits involved in channel activation was made by Tanabe et al.,³⁶ who constructed chimeric channels in which sequence stretches of a slow-activating (“skeletal muscle-like”) Ca_V1.1 α₁-subunit were replaced by sequences from a fast-activating (“cardiac-like”) Ca_V1.2 α₁-subunit. The chimeras activated slowly if repeat I of the Ca_V1.2 α₁-subunit was replaced by the Ca_V1.1 α₁-sequence. Later studies of Nakai et al.³⁷ revealed that segment S3 and the S3-S4 linker of repeat I are critical for the difference in activation kinetics between cardiac (Ca_V1.2) and skeletal muscle (Ca_V1.1) calcium channels. An important role of the voltage sensors of domains I and III but not II and IV in voltage-dependence (V_0.5) and time-course of activation were reported by Garcia et al.,³⁸ who mutated the arginines in the S4 segments of all four domains of a chimeric channel to neutral or negative amino acids. The removal of prolines that are conserved in segments IS4 and IIIS4 of Ca²⁺ channels resulted in shorter channel open time, whereas introduction of extra prolines to corresponding positions of IIS4 and IVS4 lengthened the channel open time.³⁹

Li et al.⁴⁰ constructed a series of chimeras between low voltage activated (Ca_V3.1) and high voltage activated (Ca_V1.2) channels. Their data suggest that domains I, III and IV (rather than domain II) are apparently critical for channel opening and, therefore, contribute strongly to the difference in voltage dependence of activation between Ca_V3.1 and Ca_V1.2. Determinants of the half-activation potential of low voltage activated Ca_V3 channels were identified in domains I and IV.⁴¹

Zhen et al.¹³ described substantial changes in Ca_V2.1 activation upon replacement of S6 residues and residues of the adjacent intracellular loops by cysteines. Their study suggests that important activation determinants of this channel type may be localised in intracellular loop segments. Cysteine accessibility by methanethiosulfonate ethyltrimethylammonium (MTSET) of the inner pore region suggests possible differences in the architecture of Ca_V2.1 compared to K⁺ channels.¹³

Modulation of Calcium Channel Activation by Auxiliary Subunits

It is well established that β-subunits modulate the gating of high-voltage-activated Ca²⁺ channels.^42-44 A significant hyperpolarising shift of the activation curve of the ionic current is observed upon coexpression of a β-subunit with the Ca_V1.2 α₁-subunit. The activation curve for the gating current was unaffected, suggesting that the β-subunit modulates activation exclusively by affecting pore stability (Fig. 7). The time-course of channel activation is also modulated in a subunit-specific manner.⁴² These findings suggest that β-subunits modulate not only the inactivation properties of the channels, but also affect activation by modulating pore stability. α₂-δ subunit causes a shift in the current-voltage and conductance-voltage curves toward more positive potentials and accelerates activation and deactivation kinetics.^45,46 Data of Obermair et al.⁴⁷ show, however, that α₂-δ depletion of reconstituted dysgenic α_1S-null myotubes significantly accelerated the current kinetics, suggesting a conversion of slowly activating into fast activating Ca²⁺ channels.

Figure 7.

Voltage dependence of charge movement (Q) and membrane conductance (G) in oocytes expressing α₁ (A) or α₁ plus β (B) subunits. For charge versus voltage plots (Q), gating currents were recorded in 2 mM external Co²⁺. Data from individual oocytes were fitted by Q_on = Q_max/{1 + exp[z_QF(V_0.5Q - V_m)/RT]} and normalized by Q_max. The average values after normalization from nine oocytes injected with α₁ cRNA and from 14 oocytes injected with α₁ plus β messages are shown as filled circles in (A and B), respectively. For values of G, inward currents (I_m) were evoked by 125-ms depolarization steps, filtered at 0.5 kHz and sampled at 4 kHz, and measured at the end of the pulse. For individual oocytes (19 with α₁ alone and 23 with α₁ plus β), normalized membrane conductances were calculated from the fit I_m = G_max(V_m - E_reV)/{1 + exp[z_GF(V_0.5G - V_m)/RT]}. Dotted lines indicate the half activation potential conductance. Voltage dependences of charge movement (Q) are similar for oocytes injected with α₁ alone (V_0.5Q = −19.8 ± 3.7 mV) and with α₁ plus β (V_0.5Q = −18.1 ± 3.1 mV). In contrast, membrane conductances (G) are significantly different for oocytes injected with α₁ alone (V_0.5G = 15.1 ± 2.4 mV) and α₁ plus β (V_0.5G = −1.5 ± 0.7 mV). Figure from Neely et al.⁵⁰ with permission.

The Link Between Pore Stability and Activation Kinetics

A major challenge for biophysicists and molecular biologists is the interpretation of the kinetic changes shown in Figures 2-4. Yifrach and MacKinnon⁴⁸ discussed that the leftward shift of the activation curve in “mutational perturbation” studies on Shaker potassium channels could reflect either the stabilization of the open state or the destabilization of the closed state. The authors reasoned, however, that the shifts towards negative potentials are more likely result from a destabilization of the closed channel pore than from a stabilized open state. Some of the “kinetic fingerprints” suggest, however, a stabilization of the open conformation (e.g., slow tail current kinetics, Figs. 2-4).

Changes in pore stability of Ca_V in terms of rate constants can be simulated making use of a simplified version of the model of Zagotta et al.,⁸ by assuming that Ca_V consist of two functionally distinct parts: (i) a voltage-sensing mechanism and (ii) a pore that opens and closes independently of voltage. As illustrated in Figure 8, the voltage sensor may dwell in the resting (locking) and activated (releasing) states and the pore in the open or closed states. The molecule therefore dwells in 2 × 2 = 4 states: locked/closed (R), released/closed (A), open (O) and locked/open (D). Rate constants of the pore opening and closing are assumed to be dependent on the voltage sensor position: rate constants α and β describing the pore opening and closure at an activated voltage sensing machinery (A ↔ O) accordingly differ from γ and δ (D ↔ R). The rate constants of the voltage sensor transitions between locking and “releasing” positions depend on the membrane potential. All rate constants are linked by the thermodynamic reversibility condition:

$$\frac{x}{y}\cdot \frac{\alpha }{\beta }\cdot \frac{w}{u}\cdot \frac{\delta }{\gamma }=1$$

at all potentials.

Figure 8.

Schematic representation of channel state transitions. Activation gating is assumed to be determined by two functionally separate processes: a voltage sensing mechanism (cylinders marked by ++) and the conducting pore. Each functional unit may dwell in two states: the voltage sensor may dwell in the resting (down) and activated (up) and the pore in the open or closed states. The entire molecule therefore dwells in 2 × 2 = 4 states: (R) pore is closed and voltage sensing mechanism locks the pore; (A) activated voltage sensing mechanism releases the pore remaining closed; (O) the pore is open and (D) the deactivated voltage sensing mechanism is in the down position while the pore is still open. Rate constants of the pore opening and closure (α, β, γ and δ) are assumed to be independent of voltage. Rate constants of voltage sensing mechanism (x, y, u and w) are voltage dependent.

This approach can be used to analyze kinetic effects of mutations in the calcium channel pore if we lump together possible closed state transitions and described the rate constants of the voltage sensing subsystem (R ↔ A) by simple exponential functions with V- membrane voltage, x and y rate constants of voltage sensor movements, x_o and y_o amplitude coefficients and k_x and k_y the inverses of the steepness of voltage dependences:

$$x=x_0\cdot \exp \left(\frac{V}{k_x}\right)\text{and}y=y_0\cdot \exp \left(-\frac{V}{k_y}\right)$$

It can be easily deduced from this model that the midpoint of the activation curve is:

$$V_m\approx V_X-k_S\cdot \ln \left(1+\frac{\alpha }{\beta }\right)$$

As discussed previously, shifts of the activation curves might be explained in terms of stabilization or destabilization of closed or open states (stabilization/destabilization are defined as decrease/increase of rate constants of the leaving the states). Shifts of the activation curves can be solely described as changes in α/β (pore opening and closure) without changes in the slope factor:

$$kS=\frac{k_x{k}_y}{k_x+k_y}$$

and midpoint V_x =k_sln(y₀/x₀) of the R ↔ A transition.

The transition from the locked-closed state (R) into the open state (O) under a depolarization occurs predominantly via the A state. Transitions R ↔ D ↔ O are very rare because the first transition R ↔ D (Fig. 8) is slow and negligible, which enabled the analysis of a simplified activation pathway:

This scheme reflects voltage sensor activation (transition R ↔ A) with the voltage-dependent rate constants x and y and the subsequent pore opening (transition A ↔ O) with the voltage-independent rate constants α and β. During deactivation a strong hyperpolarization will first induce a movement of the voltage sensors from the active to the “locking” position. In other words, the transition from open (O) to the resting state (R) under a hyperpolarization occurs predominantly via a deactivated but still open conformation (D), which enabled the analysis of the simplified model:

The scheme describes the voltage sensor deactivation (transition O ↔ D) with the voltage-dependent rate constants w and u and the subsequent pore closure (transition D ↔ R) with the voltage-independent rate constants δ and γ.

Time constants of activation and deactivation of wild-type Ca_V1.2 and mutants I781P and I781T are given in Figure 2. Traces in Figure 9A represent a simulation with the rate constants given in Tables 1 and 2. It appears that the description of the activation processes with unchanged x(V) and y(V) (see also Fig. 7) requires simultaneous changes in α and β. In other words, in such a scenario an increase in α (destabilization of the open conformation) goes in parallel with a decrease in β (stabilization of the open conformation). The activation time constant for wild-type channels appears to be almost voltage-independent, suggesting that the pore opening is the rate-limiting stage at all potentials. Destabilization of the pore accentuated voltage dependence of activation gating in Ca_V1.2 (Fig. 4).

Figure 9.

Model simulations. (A) Current simulations of activation (left column) and deactivation (right column) kinetics of I_Ba through wild-type, I781T and I781P channels from −80 mV to indicated voltages (B) Simulated (solid lines) voltage dependences of activation of wild-type, I781T and I781P mutant channels. (C) Simulated (solid lines) of the time constants of activation (open) and deactivation (closed) for wild-type, I781T and I781P mutant channels. Solid lines represent a simulation with the rate constants given in Table 1. Compare the simulated current kinetics and steady state activation curves with Figure 2.

Table 1. Rate constants of pore opening and closure.

	α	β	γ	δ
WT	0.2	0.25	0.08	1.2
I781T	8.3	0.07	0.011	0.6
I781P	22	0.035	0.007	0.3

α,β,γ and δ represent the rate constants of the model illustrated in Figure 8. The corresponding simulations are illustrated in Figure 9.

Table 2. Parameters for voltage dependent rate constants x(V) and y(V).

x0	kx	y0	ky
0.38	55	0.17	8

Note the steeper voltage-dependence of y(V) compared to x(V) (k_y < k_x). We speculate that the membrane voltage controls channel closure rather than the channel opening.

Conclusions and Outlook

Different calcium channel family members expressed in different tissues open at different membrane voltages. Their activation thresholds enable calcium entry to be fine-tuned with respect to resting potential and action potential firing for specific tissues. The molecular basis for differences in channel activation between calcium channel family members (e.g., Ca_V1 and Ca_V3) remains obscure.

Progress has been made, however, in understanding the impact of individual amino acids (e.g., mutations associated with channelopathies). Replacement of single amino acid may shift the activation threshold by more than 30 mV, as illustrated in Figures 2 and 3. The evidence shows that changes in hydrophobicity play an essential role (Figs. 5 and 6) for mutations within the activation gate, while other amino acid properties such as bulkiness (van der Waals volume) appear to be less important. The results of systematic changes in hydrophobicity led us to conclude that the activation gate in Ca_V1.2 and Ca_V2.3 is at least partially formed by hydrophobic interactions in the inner channel mouth of the closed conformation. Homology modeling suggests that there are more interactions between each pair of S6 helices in addition to the one corresponding to the I781 data described here. Future studies will reveal the relative impact of these interactions in closed state stability and/or pore occlusion. The molecular mechanism of the particular strong effect of mutations in position I781 of Ca_V1.2 (I701 in Ca_V2.3, Figs. 2 and 4) remains, however, to be elucidated. Putative hinge points known from potassium channels³² have yet to be identified in voltage-gated calcium channels. Figure 5 illustrates that changes in voltage sensitivity induced by mutations to flexible glycines show no particular phenotype and reflect at least partially changes in hydrophobicity. Other open questions concern the correlation between the shifted activation and inactivation curves observed in Hohaus et al.¹² (reviewed in ref. 49).

It is tempting to speculate that evolution designed the voltage sensing machinery as robust “all-or-non” device while the verity of voltage sensitivities was accomplished by shaping pore stability. The pore of α₁ subunits of voltage gated calcium channels is asymmetric. Mutations of amino acids in different S6 segments (I–IV) will correspondingly differentially affect pore stability and kinetics. In NaChBac a single point mutation (e.g., G219P)¹⁵ induces changes in all four pore lining helices, which may explain the stronger modulation. Progress in understanding of calcium channel activation will depend on the quantification of pore stability in terms of rate constants (e.g., Figs. 8 and 9). Previous attempts^8,15,48 revealed that steady-state and/or kinetic changes can be described by changes in the rate constants of pore transitions without affecting rate constants of voltage sensor movements. A similar scenario is illustrated in Figure 9 for Ca_V1.2. The best simulation was obtained by simultaneous changes in α and β, suggesting that changes in current kinetics and steady state activation reflect a concurrent destabilization of the closed conformation (increase in α) and a stabilization of the open conformation (decrease in β, see Table 1). Analyzing a larger set of mutations may help to understand this intriguing interrelationship.