Bilobal architecture is a requirement for calmodulin signaling to CaV1.3 channels

Abstract

Calmodulin (CaM) regulation of voltage-gated calcium (Ca_V) channels is a powerful Ca²⁺ feedback mechanism that adjusts Ca²⁺ influx, affording rich mechanistic insights into Ca²⁺ decoding. CaM possesses a dual-lobed architecture, a salient feature of the myriad Ca²⁺-sensing proteins, where two homologous lobes that recognize similar targets hint at redundant signaling mechanisms. Here, by tethering CaM lobes, we demonstrate that bilobal architecture is obligatory for signaling to Ca_V channels. With one lobe bound, Ca_V carboxy tail rearranges itself, resulting in a preinhibited configuration precluded from Ca²⁺ feedback. Reconstitution of two lobes, even as separate molecules, relieves preinhibition and restores Ca²⁺ feedback. Ca_V channels thus detect the coincident binding of two Ca²⁺-free lobes to promote channel opening, a molecular implementation of a logical NOR operation that processes spatiotemporal Ca²⁺ signals bifurcated by CaM lobes. Overall, a unified scheme of Ca_V channel regulation by CaM now emerges, and our findings highlight the versatility of CaM to perform exquisite Ca²⁺ computations.

Keywords: CaV1.3; calcium regulation; calmodulin; ion channels; voltage-gated Ca channels.

Fig. 1.

Bilobal architecture of CaM is necessary for Ca_V1.3 channel regulation. (A) CaM contains two globular domains each composed of two EF hand Ca²⁺-binding motifs. The two lobes of CaM share high sequence similarity. (B) Ca²⁺-dependent conformational changes of CaM. (Left) In the absence of Ca²⁺, both N and C lobes of CaM adopt a highly similar conformation (PDB ID: 1CDF). (Right) The structural similarity of the CaM lobes persists even when Ca²⁺-bound (PDB ID: 1CLL). (C) Schematic summarizes CaM-dependent changes in Ca_V1.3 gating. (Left) Devoid of CaM, channels adopt a low P_O gating configuration. (Middle) The apoCaM binding switches channels to a high P_O gating mode. (Right) Ca²⁺ binding to CaM relieves the enhancement in P_O and switches channels to a low P_O mode. (D) Ca_V1.3 channels with tethered wild-type CaM (Ca_V1.3−CaM_WT, Top) exhibit robust Ca²⁺-dependent regulation. (Middle) Exemplar currents. Scale bar pertains to Ca²⁺ currents (red). Ba²⁺ currents (black) are scaled down ∼3× for comparison of decay kinetics. (Bottom) Population data depict fraction of peak current after 300-ms depolarization (r₃₀₀) versus voltage; red, relation for Ca²⁺; black, relation for Ba²⁺. Each point is mean ± SEM. (E) Genetic fusion of dominant negative mutant CaM₁₂₃₄ to Ca_V1.3 CT (Ca_V1.3−CaM₁₂₃₄) abrogates Ca²⁺ regulation. (F) Ca_V1.3 fused to CaM_C fails to support Ca²⁺ regulation, suggesting that bilobal architecture of CaM is necessary for functional modulation of Ca_V channels. (G) Genetic fusion of Ca_V1.3 with mutant CaM₁₂ whose Ca²⁺ binding is restricted to C lobe alone also supports Ca²⁺ regulation, suggesting C lobe can trigger channel modulation, provided N lobe is also present. (H) Genetic fusion of Ca_V1.3 with mutant CaM₃₄ whose Ca²⁺ binding is restricted to N lobe alone also supports Ca²⁺ regulation. Format for E–H is as in D.

Fig. 2.

Channels bound to a single CaM lobe adopt a preinhibited configuration. For experiments in A–D, CaM sponge was coexpressed. (A) Ca_V1.3 fused to CaM_WT (Ca_V1.3−CaM_WT) exhibits high baseline P_O, with Ba²⁺ as charge carrier, consistent with channels in gating configuration A (Fig. 1D). (Top) Exemplary current records show response to a voltage-ramp protocol. (Bottom) P_O–voltage (V) relationship determined from the ensemble average of single-channel recordings. (B) Ca_V1.3 channels lacking prebound CaM exhibit diminished baseline P_O. Here, we used the Ca_V1.3 MQDY variant with a low CaM binding affinity. (C) When Ca_V1.3 is fused to CaM_C (Ca_V1.3−CaM_C) alone, the baseline P_O is substantially diminished, consistent with channels adopting a preinhibited configuration. (D) Reconstitution of N lobe of CaM via attachment to the auxiliary Ca_Vβ_2A subunit (β_2A−CaM_N) potently up-regulates the maximal P_O of Ca_V1.3−CaM_C channels (red curve). Format for B−D is as in A.

Fig. 3.

Binding of CaM lobes evoke discrete Ca_V1.3 gating modes. CaM sponge was present for A–L. (A) Sequential single-channel trials of Ca_V1.3−CaM_WT in response to a voltage ramp (187 records). Diary plot displays single-trial average P_O computed for −30 mV ≤ V ≤ +25 mV (${\overline{P}}_{\text{O}}$). Dashed line discriminates low (red area) and high P_O (gray area) traces. (B) Histogram shows number of sweeps with ${\overline{P}}_{\text{O}}$(−30 ≤ V ≤ 25) for given range. (C) Average P_O at each voltage calculated for high P_O traces estimates P_O−V relationship for mode A. (D) Cumulative open duration distribution [green bars; P(T_O > t)] follows a single-exponential decay (green fit) consistent with a single open state in mode A with an exit rate, k_OC|A = 3.3 ms⁻¹. (E–H) Ca_V1.3_S/MQDY with CaM sponge approximates behavior of CaM-less channels (117 records). Openings are brief and sparse. Single-trial ${\overline{P}}_{\text{O}}$ distribution is unimodal in low P_O range. Maximal P_O is reduced. Open-duration P(T_O > t) is single-exponential with k_OC|E = 9.3 ms⁻¹ > k_OC|A. Format is as in A–D. (I–L) Analysis of Ca_V1.3−CaM_C (141 records) shows similarity to CaM-less channels with brief and sparse openings and ${\overline{P}}_{\text{O}}$ histogram and P_O−V relation reminiscent of mode E. P(T_O > t) is single exponential with rate constant ≈ k_OC|E. (M–P) With β_2A−CaM_N, Ca_V1.3−CaM_C P_O is enhanced in a quantized manner (139 records). Channels switch between low and high activity epochs with bimodal ${\overline{P}}_{\text{O}}$ distribution. P(T_O > t) distribution is biexponential consistent with rate constants k_OC|E and k_OC|A.

Fig. 4.

Reconstitution of detached CaM hemilobes is sufficient to restore Ca_V regulation. (A) Coexpression of β_2A−CaM_N partially restores Ca²⁺ regulation to Ca_V1.3−CaM_C channels, suggesting that the presence of two lobes of CaM is sufficient to evoke channel Ca²⁺ feedback regulation. Format is as in Fig. 1A. (B) Reconstitution of CaM N lobe with its Ca²⁺ binding disabled (β_2A−CaM_N12) is also sufficient to partially rescue Ca²⁺ regulation of Ca_V1.3−CaM_C. Format is as in A. (C) Reconstitution of β_2A−CaM_N with Ca_V1.3−CaM_C34 results in small residual CDI. (D) Ca²⁺ regulation is absent following reconstitution of β_2A−CaM_N12 to Ca_V1.3−CaM_C34. (E) Exemplary current records show robust CDI of Ca_V1.3 mediated by CaM_CC, CaM variant with two identical C lobes. Population data confirms strong CDI of Ca_V1.3 when bound to CaM_CC. (F) Coexpression of CaM_CC1234, with Ca²⁺ binding disabled to its two C -lobes, strongly reduces CDI of Ca_V1.3.

Fig. 5.

Binding of CaM lobes evokes distinct conformational rearrangements for Ca_V1.3 CI. (A) MD simulation shows the relative stability of the Ca_V1.3 CI bound to CaM. (Top) C_α rmsd for Ca_V1.3 CI region for 100-ns trajectory. (Bottom) Structural model following 40-ns equilibration. The angle between the IQ domain and a helix within EF_1,2 is shown, to facilitate comparison of conformational changes. (B) MD simulation shows the dramatic conformational rearrangement of Ca_V1.3 CI module devoid of CaM. Format is as in A. (C) Ca_V1.3 CI module bound to CaM C lobe alone also undergoes a conformational rearrangement. Format is as in A. (D) Ca_V1.3 CI bound to CaM_CC undergoes minor structural reorientations. Format is as in A. (E) Small-angle X-ray solution scattering profile of the Ca_V1.3 CI module in complex with CaM_WT (black) or CaM_C (red). (Left) Scattering intensity is plotted as a function of momentum transfer. (Right) Radial pair-distribution function [P(r)] is computed for radial vectors (r) and describes the set of all paired distances within the structure. (F) (Left) Ab initio molecular envelope of Ca_V1.3 CI/CaM complex overlaid on a homology model (Fig. S4D). (Right) Ab initio molecular envelope of Ca_V1.3 CI/CaM_C complex overlaid on the MD-relaxed model. (G) Schematic shows a unifying model of CaM regulation of Ca_V1 channels. Devoid of CaM, the channel CI adopts a bent conformation, with the IQ reoriented toward the EF_1,2 domains, while openings are allosterically diminished. If only one apoCaM lobe is bound, the bent conformation of the CI persists, and channel openings remain diminished. Binding of two apoCaM lobes switches the CI into an extended conformation, and this rearrangement enhances channel openings. On Ca²⁺ binding, one or two lobes of CaM depart from its preassociation interface, and the CI module reverts to a bent conformation and channel openings are diminished.