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Review
. 2018 Sep;470(9):1291-1309.
doi: 10.1007/s00424-018-2163-7. Epub 2018 Jun 27.

Calcium channel gating

S Hering  1 , E-M Zangerl-Plessl  2 , S Beyl  2 , A Hohaus  2 , S Andranovits  2 , E N Timin  2
Affiliations
Review

Calcium channel gating

S Hering et al. Pflugers Arch. 2018 Sep.

Abstract

Tuned calcium entry through voltage-gated calcium channels is a key requirement for many cellular functions. This is ensured by channel gates which open during membrane depolarizations and seal the pore at rest. The gating process is determined by distinct sub-processes: movement of voltage-sensing domains (charged S4 segments) as well as opening and closure of S6 gates. Neutralization of S4 charges revealed that pore opening of CaV1.2 is triggered by a "gate releasing" movement of all four S4 segments with activation of IS4 (and IIIS4) being a rate-limiting stage. Segment IS4 additionally plays a crucial role in channel inactivation. Remarkably, S4 segments carrying only a single charged residue efficiently participate in gating. However, the complete set of S4 charges is required for stabilization of the open state. Voltage clamp fluorometry, the cryo-EM structure of a mammalian calcium channel, biophysical and pharmacological studies, and mathematical simulations have all contributed to a novel interpretation of the role of voltage sensors in channel opening, closure, and inactivation. We illustrate the role of the different methodologies in gating studies and discuss the key molecular events leading CaV channels to open and to close.

Keywords: Calcium channel; Gating; Molecular modeling; Voltage sensor.

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Figures

Fig. 1
Fig. 1
Model of a CaV1.2 α1 subunit (based on the cryo-EM structure of CaV1.1 by Wu et al. [106, 107]). CaV1.1 and CaV1.2 are highly homologous (see sequence alignment in Supplementary Fig. 1). The helices are represented as cylinders. Domains I to IV are shown in green, gray, purple, and yellow, respectively. a The top view arrangement of the channel with the voltage-sensing domains (VSDI to VSDIV) and pore domain. b The side view shows the structural elements of opposing domains. The bundle-crossing region at the lower third of the S6 segments forms the activation gate. c The bottom view with a highlight on the S4–S5 linker ring (in red). The S4–S5 linker helices run in parallel to the intracellular side of the membrane. d One domain of the α subunit of the voltage-gated calcium channel. S1 to S3 (green) and S4 (blue) represent the voltage-sensing domain. Segment S5, the P-helices (green), the selectivity filter (dark blue), and the S6 (yellow) form the pore domain. S4 is connected to the S5 via the S4–S5 linker (red). The S4 is further in close proximity to the S5 of the adjacent subunit (light pink). The S6 as well as the P-helices of the adjacent subunit are shown in light gray. The G/A/G/A position is highlighted in orange (Depil et al. [36]), close to the loop between the S4–S5 linker and the S5 helix
Fig. 2
Fig. 2
Location of the activation gate: hydrophobic residues form a sealing region at the intracellular gate. a The permeation path of the pore domain (modified from Wu et al. [106]). The pore regions including S6 and selectivity filter between CaV1.1 and CaV1.2 are highly homologous (see Fig. S1 in supplemental materials). The figure displays the pore radii of CaV1.1 along the pore. The closest part of the hydrophobic area (a, position around 0) formed by V430, F778, F1191, and F1501 is emphasized with a red bar. The helix bundle crossing region is highlighted with a red dotted box. The blue dotted box marks the selectivity filter. b Cartoon representation of the pore domain. The third domain is omitted for clarity. Domain 2 (DII) is colored in gray, DIII in purple, and DIV in yellow. The dotted rectangles from a are extended to this figure to show the corresponding areas in the protein. The occluding residues V430, F778, F1191, and F1501 from the cavity facing the intracellular side are represented as spheres. c Effects of proline substitutions in positions F778–A782 and G770 on the voltage dependence of CaV1.2 activation. Solid lines represent fits to Boltzmann functions. Mutations causing large shifts and corresponding activation curves are shown in red. This research was originally published in the Journal of Biological Chemistry. Hohaus et al. [50]. d Detailed view on the helix bundle crossing region. The S6 helices are represented as cartoon with the third domain set transparent for clarity. Domain DI, DII, DIII, and DIV are colored in green, gray, purple, and yellow, respectively. The occluding residues V430, F778, F1191, and F1501 from the cavity side are represented as spheres. Please pay attention to the extended hydrophobic cluster of residues below the narrowest occluding positions that are represented as sticks. It is tempting to speculate that the hydrophobic cluster contributes to closed state stability to the activated not open state A (Fig. 3, see also del Camino et al. [24])
Fig. 3
Fig. 3
State transitions during activation (modified after Beyl et al. [16]) Activation gating is determined by two functionally separate processes: a voltage-sensing mechanism (++++) and the conducting pore. Each functional unit can dwell in two states: the VS in the resting (down) and activated (up) states 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, voltage-sensing mechanism is activated and releases the pore, which, however, remains closed; O, the pore is open; 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, δ) are assumed to be voltage-independent. Rate constants of voltage-sensing mechanism (x, y, u, and w) are voltage dependent
Fig. 4
Fig. 4
Closed channel gates trap a phenylalkylamine. a Left: Crystal structure of CaVAb in complex with Br-verapamil (pdb-code: 5kmh [98]). The P-helices and S6 helices are represented as cartoon. The Br-verapamil is shown as cyan sticks. The area spanned by the membrane is shown in gray. The region between the two blue dotted lines corresponds to 0.4–0.6 fraction of the membrane. Right: (−)qD888 docked into the cavity of the homology model of the CaV1.2 closed conformation. Domains 1 to 4 are colored in green, gray, purple, and yellow, respectively. Domains 1 and 2 are set transparent for better visibility. Compound (−)qD888 is shown as cyan and salmon sticks. The figure illustrates two high score poses. The previously identified putative binding determinants that are within 5 Å of the compound are represented as sticks [47]. The permanently charged nitrogen is located for both docking poses within the labeled fraction of 0.4–0.6 of the membrane potential. b Recovery of CaV1.2 from block by (−)qD888 (100 μM). (−)qD888 was applied to the intracellular side of the membrane and recovery from block was measured. Block was induced by a train of test pulses from − 80 to 10 mV and the holding potential subsequently switched to − 80, − 90, − 100, or − 110 mV. The fraction of recovered channels was measured after different time intervals. c Plot of the time constants of recovery versus voltage in semi-logarithmic coordinates revealed an exponential dependence with faster recovery at more negative voltages. Eyring analysis predicts a location of the charged locus of the PAA molecule close to the central pore region (the estimated fraction of the membrane potential affecting drug dissociation was 0.56, Beyl et al. [17]). For c and d: This research was originally published in the Journal of Biological Chemistry. Beyl et al. [17]. © the American Society for Biochemistry and Molecular Biology
Fig. 5
Fig. 5
Hydrophobic interactions stabilize “activated not open” conformation: Midpoint shifts of the activation curve (ΔVact) correlate with changes in hydrophobicity in position I781 on IIS6. a The side view of the CaV1.2 homology model (see Fig. 1) shows the structural elements of opposing elements of the domains with the helices represented as cylinders. The VSDs of domain I and III are colored in green and purple, respectively. The pore-forming domains of domain 2 and 4 are colored in gray and yellow, respectively. Positions of I781 (as well as A780 and A782 from the L/A/I/A motive) are highlighted as orange and gray balls. b Amino acid substitutions in position I781 (corresponding to the channelopathy mutation I745T in CaV1.4, [44]) destabilize the closed conformation and stabilize the open conformation of CaV1.2. In other words, changes in hydrophobicity in position I781 predict the shifts of the activation curve (see also Beyl et al. [14] for the role of other amino acid descriptors). Figure from Pflügers Archiv – European Journal of Physiology. Beyl et al. [14]. © The Authors. c A leftward shift of the activation curve is accompanied by deceleration of kinetics. Left panel shows representative families of IBa through wild type (upper traces) and I781P mutant channel (lower traces). Membrane potentials (left) indicate the threshold of channel opening (− 40 mV in WT and − 80 mV in I781t) and voltages applied for tail current measurements (right). This research was originally published in the Journal of Biological Chemistry. Hohaus et al. [49]. © The American Society for Biochemistry and Molecular Biology
Fig. 6
Fig. 6
Fluorophore labeled voltage sensors exhibit distinct voltage dependence and kinetics. a Mean normalized conductance (G; black down-pointing triangle) and charge movement (Q; white right-pointing triangle) from WT channels and fluorescence (F) from VSDs I (blue circle), II (red up-pointing triangle), III (green diamond), and IV (yellow square). The curves are fits to single or (for G) the sum of two Boltzmann distributions. Error bars indicate ± SEM. b Representative membrane current (gray) from WT channels for a − 90– → 20–mV pulse, with superimposed F reported from VSD I (τ1 = 2.6 ms, 59%; τ2 = 8.1 ms), II (τ1 = 1.1 ms, 98%; τ2 = 20 ms), III (τ1 = 0.88 ms, 68%; τ2 = 9.2 ms), and IV (τ = 17 ms). The black dashed lines are exponential functions with the reported time constants. The sequence of activation for the CaV1.2 VSDs (half-time to maximum, t0.5) is VSD II (1.0 ms), III (1.4 ms), I (2.9 ms), and IV (11 ms). Figure and adapted subscript from Proceedings of the National Academy of Sciences. Pantazis et al. [70]. A gating scheme deduced from these experiments is illustrated in Fig. 12c
Fig. 7
Fig. 7
Role of IIS4 in activation of wild type CaV1.2: Neutralization of all charges (construct IIS4N) has no effects on activation gating. a Schematic representation of the α1 subunit. Domains are numbered from I to IV. The S4 helices of each domain are represented as small cylinders. Zeros stand for charge neutralization at the indicated positions (glutamine substitutions) and plus indicate presence of charges (Arg or Lys) on IS4, IIIS4 and IVS4. b Left panel shows representative families of IBa through wild type channel and through a channel construct where all IIS4 charges were neutralized (IIS4N). Barium currents were evoked during depolarization starting from − 40 with 10 mV increments from a holding potential of − 100 mV. Right panel shows representative tail currents. Currents were activated during a 20-ms conditioning depolarization to 0 mV. Deactivation was recorded during subsequent repolarizations with 10 mV increments starting from − 100 mV. c Left panel, averaged activation curves of wild type and IIS4N channels. Right panel, voltage-dependent time constants of channel activation/deactivation. Adapted from Pflügers Archiv – European Journal of Physiology. Beyl et al. [13]. © The Authors
Fig. 8
Fig. 8
Key role of IS4 in activation gating: Movement of IS4 represents a rate-limiting stage. The cylinders represent the S4 residues with the positions of the mutations (plus stands for Arg or Lys, zero stands for Gln). a–d Averaged activation curves of wild-type and charge neutralization in IS4 (a), IIS4 (b), IIIS4 (c) and IVS4 (d). The most prominent changes in the slope of were observed for IS4 (a) and IIIS4 (c). Figure modified from Pflügers Archiv – European Journal of Physiology. Beyl et al. [15]. License: http://creativecommons.org/licenses/by/4.0/
Fig. 9
Fig. 9
Slowly gating IS6 mutant G432W reveals role of segment IIS4: Neutralization of IIS4 accelerates current kinetics and shifts activation curve. a Schematic representation of α1 subunit domains I and II. The cylinders inside represent the S4 or S6 helices. Arg or Lys are shown as (+) while zeros (0) indicate charge neutralizations by Gln. Mutation G423W is highlighted on IS6. b Detailed view of position G432 of CaV1.2. The protein is represented as cartoon. Domain I is shown in green and domain IV in yellow. Glycine 432 is represented as orange spheres and the side chains of the closest residues (P297 and L298, both within 3 Å) are labeled and illustrated as green spheres. These two residues are located at the loop between the S4–S5 linker and the S5 helix. It shows tight packing of this gating sensitive area. This might indicate that even minor changes of the packing can lead to changes in the gating behavior due to necessary rearrangements within this region. For an overview on this position in other channels, see Supplementary Fig. 2. c–f Neutralization of all IIS4 charges (IIS4N) shifts the activation curve of a slowly gating IS6 mutant G432W in the depolarizing direction and accelerates current kinetics (arrows in c and d). Rightward shifts of the activation curves and acceleration of current kinetics by S4 neutralizations are exclusive for mutations in G/A/G/A positions (Beyl et al. [13]). c, d Representative currents through G432W and G432W/IIS4N highlighting slow activation (c) and deactivation of G432W and accelerated activation and tail currents in G432W/IIS4N (d). e, f Averaged activation curves (e) and voltage dependence of the activation/deactivation time constants (f) of WT, G432W, IIS4N, and G432W/IIS4N. c–f Adapted from Pflügers Archiv – European Journal of Physiology. Beyl et al. [13]. © The Authors
Fig. 10
Fig. 10
Full or partial neutralization of IIS4 prevents stabilization of the open state. Various IIS4 charge neutralizations (with exception of IIS4N) have similar gating effects on the slowly gating construct A780T. a A780 (orange sphere), like other G/A/G/A residues (Figs. 9b and S2 supplemental materials), putatively interacts with the S4–S5 linker and S5 loop. Positions of the charged residues on S4 are represented as blue spheres. b Detailed view of the position A780 of CaV1.2. Domain I (green) and domain II (gray). The side chain of A780 and the closest residues (S680 and I681, both within 3 Å) are represented as orange and gray spheres, respectively. S680 and I681 are located at the loop between the S4–S5 linker and the S5 helix. Similar to Fig. 9b, this shows the tight packing of the residues at this critical location. Changes in size or property of the A780 are likely to lead to changes in the dynamic behavior of the channel since this perfect arrangement can be important for the stabilization of certain states. c Schematic representation of the mutations in domain II. The cylinders inside represent the IIS4 and IIS6 helices. Plus stands for Arg or Lys, zero stands for Gln. d Currents of the indicated channel constructs (colors correspond to the illustrations in c). Channels were activated by conditioning pulses from − 80 to 10 mV, deactivation was induced by hyperpolarizing steps to voltages between − 70 and − 100 mV. e Voltage dependence of time constants of activation/deactivation. f Bar graphs illustrating the maxima of the bell shaped curves (shown in e) reflecting the slowest kinetics of activation/deactivation
Fig. 11
Fig. 11
Key role of IS4 in inactivation gating: Neutralization of segment IS4 modulates CaV1.2 inactivation. a Steady state inactivation curves of WT and the indicated IS4 mutants. Slope of the Boltzmann curves ranged from 6.2 ± 0.7 mV in IS4N+R276 (diamond) to 17.4 ± 3.5 mV in K264Q (circle). The cylinders represent the S4 residues with the according mutations (plus stands for Arg or Lys, zero stands for Gln). b Superimposed typical normalized IBa through WT and mutant IS4N+R276. During 3s depolarizations from − 80 mV to the voltages of the maximum of the current–voltage relationship. Note the faster development of inactivation in IS4N+R276. Current decay was fitted to a monoexponential function yielding time constants of τinact(WT) = 393 ± 24 ms and τinact(IS4N+R276) = 235 ± 29 ms, respectively. Solid lines represent the fitted functions. Figure modified from Pflügers Archiv – European Journal of Physiology. Andranovits et al. [6]. License (http://creativecommons.org/licenses/by/4.0/)
Fig. 12
Fig. 12
Structural determinants (a) and models (b, c) of CaV1.2 activation. a The α1c subunit in top view, represented as gray transparent surface. The S4 and S6 helices are shown as cartoon. Blue spheres represent the charged residues on the S4 segments. Orange spheres represent the G/A/G/A positions on the S6 segments. The black dotted line indicates a cooperative unit. The outlined double arrows indicate interactions of S4 segments (IS4 - IVS4) with all four G/A/G/A positions. Two gating concepts have been proposed: In the cooperative gating model (Beyl et al., figure b) upward movement of S4 segments disengages the interlinked (illustrated as invaginations) S6 gates and channels enter the activated/not open conformation. Further separation of S6 gates results in concerted channel opening. In this way, a single VS can modulate all four pore-forming elements (as observed in experiments [13]). Upward movement of IS4 (and IIIS4) is a rate-limiting step for activation [15]. c In a gating model proposed by Pantazis et al. [71], the authors estimated the energy (W1 to W4) contributed by individual S4 segments to pore opening based on fluorescence changes of CaV1.2 constructs with individually labeled S4 segments during voltage clamp steps. Calculated different energies (W1 to W4) suggest that IIS4 and IIIS4 provide most of the energy for pore opening. This figure is modified from Proceedings of the National Academy of Sciences. Pantazis et al. [71]
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References

    1. Adams B, Tanabe T. Structural regions of the cardiac Ca channel alpha subunit involved in Ca-dependent inactivation. J Gen Physiol. 1997;110:379–389. - PMC - PubMed
    1. Aggarwal SK, MacKinnon R. Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron. 1996;16:1169–1177. - PubMed
    1. Ahern CA, Horn R. Focused electric field across the voltage sensor of potassium channels. Neuron. 2005;48:25–29. - PubMed
    1. Alam A, Jiang Y. High-resolution structure of the open NaK channel. Nat Struct Mol Biol. 2009;16:30–34. - PMC - PubMed
    1. SPH A, Kelly E, Marrion NV, Peters JA, Faccenda E, Harding SD, Pawson AJ, Sharman JL, Southan C, Buneman OP, Cidlowski JA, Christopoulos A, Davenport AP, Fabbro D, Spedding M, Striessnig J, Davies JA, CGTP Collaborators 0 The concise guide to pharmacology 2017/18: overview. Br J Pharmacol. 2017;174:S1–S16. - PubMed

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