Towards a Unified Theory of Calmodulin Regulation (Calmodulation) of Voltage-Gated Calcium and Sodium Channels

Abstract

Voltage-gated Na and Ca(2+) channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca(2+) and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.

Figure (1)

A) Convenient experimental setup to examine CDI of Ca²⁺ channels. Left, no feedback regulation triggered upon Ba²⁺ influx through channel. Right, Ca²⁺ influx triggers Ca²⁺ regulation. B) Typical Ca²⁺ and Ba²⁺ current profiles through Ca_V1.3 channel. In response to a step depolarization, the Ca²⁺ current (red trace) activates and then sharply inactivates. Ba²⁺ current (black trace), does not inactivate. Adapted with permission [115]. C) Diagram depicting regions of Ca²⁺ channels critical for calmodulation, including dual vestigial EF hands (pink and green), IQ region (blue) on the C-terminus and NSCaTE (black) on the N-terminus of the channel. D) Mechanist scheme for CaM regulation of Ca²⁺ channels. In the E state, the channel is devoid of CaM and incapable of undergoing CDI. Following apoCaM binding (A state), the channel can undergo N- or C-lobe-driven CDI, state I_N and I_C, respectively. Configuration I_CN corresponds to a fully inactivated state where N-and C-lobe of CaM engages respective effector interfaces. E) Molecular model of apoCaM preassociation to Ca_V1.3. The N-lobe of apoCaM preassociates with the dual vestigial EF hand segments, and the C-lobe with the channel IQ domain. F) Proposed model of Ca²⁺/CaM interaction with channel interfaces. Upon Ca²⁺ binding, the N-lobe of Ca²⁺/CaM interacts with the NSCaTE segment. The C-lobe of Ca²⁺/CaM forms a tripartite complex with the channel dual vestigial EF hands and the IQ domain. Adapted with permission [41].

Figure (2)

A) Functional bipartition of CaM in Ca_V2.1 channel. Top, Ca_V2.1 currents evoked by a train of action potentials. Rapid Ca²⁺ dependent facilation of Ca²⁺ current (circle, bottom subpanel) is followed by Ca²⁺-dependent inactivation (square, bottom subpanel). Adapted with permission [70]. B) Distinct modes of spatial Ca²⁺ signaling. Under high Ca²⁺ buffering, Ca²⁺ elevations are restricted to the channel nanodomain (left). Low Ca²⁺ buffering permits global elevation of cytoplasmic Ca²⁺ (right). The resident CaM molecule can decode these distinct Ca²⁺ signals Adapted with permission [83]. C) Table summarizes dual regulation of Ca_V channels by the two lobes of CaM. Adapted with permission [16].

Figure (3)

A) Milestones in Na channel (left) and Ca²⁺ channel (right) calmodulation. B) Experimental scheme to study Ca²⁺ regulation of Na channels. Na current properties are evaluated from two population of cells dialyzed internal solutions containing either low and high Ca²⁺ concentrations C). Protocol used to determine steady-state inactivation of Na channels (left). Expected Ca²⁺ dependent effects for two well-studied Na channel families. Adapted with permission [115].

Figure (4)

A) Na channel regulation probed using rapid delivery of Ca²⁺ by photouncaging. Schematic shows simultaneous patch-fluorimetry and Ca²⁺ photouncaging. Ca²⁺ measured quantitatively using ratiometric fluorescence measurement. Ca²⁺ elevations are triggered using a brief UV pulse. B) Absence of Ca²⁺ regulation of cardiac Na channels (Na_V1.5) in a heterologous (HEK293) system. C) Ca²⁺ regulation of native Na_V1.5 current from ventricular myocytes. D) Robust Ca²⁺-dependent inactivation in heterologously expressed NaV1.4 channels. Here, intracellular Ca²⁺ elevation decreases peak Na current (second row). Population data shows a dose-dependent response of CDI to intracellular Ca²⁺ (bottom). E) Robust Ca²⁺-dependent inactivation of native NaV1.4 currents from skeletal myotubes (GLT cells). Adapted with permission [115].

Figure (5)

A) Table shows conservation of Ca²⁺/CaM regulation of Ca²⁺ and Na channels. B) Phylogenetic tree of the Ca²⁺ and Na channel superfamilies. HVA, high-voltage activated Ca²⁺ channels. LVA, low-voltage activated Ca²⁺ channels. C) Modularity of CI region. Transplanting CI region of Na_V1.4 to Ca_V1.3 supports CDI. Coexpression of mutant CaM₁₂₃₄ abolishes Ca²⁺ regulation of these chimeric channels. Adapted with permission [115].

Fig. (6)

A) Sequence alignment of the two lobes of CaM. The EF hand segments are shaded rose and green. LQTS-associated CaM mutations are highlighted. B) Reduction of CDI of Ca²⁺ currents in adult guinea pig ventricular myocytes (aGPVMs) expressing CaM_D96V (red), compared to CaM_WT (black). Ba²⁺ current through the same channels (gray) shows the extent of VDI in these cells. C) Averaged action potentials from aGPVMs expressing CaM_WT (black) and CaM_D96V (red). Shaded regions represent standard deviation. CaM_D96V significantly prolongs the APD. D) Averaged Ca²⁺ transient waveforms from aGPVMs expressing CaM_WT (black) and CaM_D96V (red). Shaded regions represent standard deviation. CaM_D96V significantly increases the Ca²⁺ transient magnitude. Adapted with permission [155].