Calmodulin regulation (calmodulation) of voltage-gated calcium channels

Abstract

Calmodulin regulation (calmodulation) of the family of voltage-gated CaV1-2 channels comprises a prominent prototype for ion channel regulation, remarkable for its powerful Ca(2+) sensing capabilities, deep in elegant mechanistic lessons, and rich in biological and therapeutic implications. This field thereby resides squarely at the epicenter of Ca(2+) signaling biology, ion channel biophysics, and therapeutic advance. This review summarizes the historical development of ideas in this field, the scope and richly patterned organization of Ca(2+) feedback behaviors encompassed by this system, and the long-standing challenges and recent developments in discerning a molecular basis for calmodulation. We conclude by highlighting the considerable synergy between mechanism, biological insight, and promising therapeutics.

Figure 1.

Classic U-shaped signature of CDI. (A–C) Early voltage-clamp recordings from a multicellular preparation of frog atrial trabeculae cells demonstrates CDI of Ca²⁺ currents. All listed voltages are relative to resting potential E_R (∼−80 mV) Adapted from Mentrard et al. (1984). (A) Ca²⁺ currents (shaded pink) evoked in response to an +80-mV depolarizing test pulse show robust inactivation. Fast capacitive transient was estimated by blocking these Ca²⁺ currents with 3 mM Co²⁺ solution (Co). Vertical bar corresponds to 0.5 µA of current; horizontal bar, 100 ms. (B) The Ca²⁺ current in response to the test pulse is sharply attenuated when preceded by an +80 mV prepulse. This reduction in current amplitude reflects the inactivation of Ca²⁺ channels. Format is as in A. (C) Further increase in the prepulse potential to +120 mV restores Ca²⁺ current amplitude (compare with B). Here, the diminished Ca²⁺ entry during the prepulse was insufficient to trigger CDI. The format is as in A. (D–F) Single channel recordings of L-type Ca²⁺ channels from adult rat ventricular myocytes also exhibit U-shaped dependence of Ca²⁺ channel inactivation. Adapted from Imredy and Yue (1994) with permission from Elsevier. (D) Depolarizing voltage pulse to +20 mV evokes representative elementary Ca²⁺ currents. Ensemble average currents are shown on the bottom. (E) When depolarizing pulse was preceded by +40 mV prepulse, the elementary Ca²⁺ currents are sparser and the first opening is delayed. This reduction in open probability parallels the sharp attenuation of macroscopic Ca²⁺ currents seen in B. The ensemble average is shown on the bottom. (F) Further increase in prepulse voltage to +120 mV led to a reversal of this gating pattern, once again highlighting the U-shaped dependence of CDI. Ensemble average is shown on the bottom.

Figure 2.

Calmodulation: the ingredients and the flavors. (A) Channel diagram illustrates overall arrangement of structural landmarks critical for CDI. The Ca²⁺-inactivation (CI) region, spanning ∼160 residues of the channel carboxy terminus, is highly conserved across Ca_V1/2 channel families and is elemental for CDI. The proximal segment (PCI) of the CI region includes the dual vestigial EF hand (EF) segments (shaded purple and blue-green). The IQ domain, a canonical CaM binding motif critical for CDI, is located just downstream of the PCI segment. The NSCaTE element in the amino terminus of Ca_V1.2/1.3 channels is an N-lobe Ca²⁺/CaM effector site. The CBD element (gray) in the carboxy terminus of Ca_V2 channels is a CaM binding segment thought to be critical for CDI. (B) The table outlines functional bipartition of CaM in Ca_V channels and the corresponding spatial Ca²⁺ selectivities. In Ca_V1.2 and Ca_V1.3 channels, both the C-lobe and N-lobe of CaM enable fast CDI with local Ca²⁺ selectivity. Latent CDI of Ca_V1.4 channels is revealed upon deletion of an autoinhibitory domain in the distal carboxy terminus. Throughout the Ca_V2 channel family, the N-lobe of CaM evokes slow CDI with global Ca²⁺ spatial selectivity. In Ca_V2.1 channels, the C-lobe of CaM supports ultra-fast facilitation (CDF) with local Ca²⁺ selectivity. No C-lobe triggered modulation has been reported for Ca_V2.2 and Ca_V2.3 channels.

Figure 3.

Toward an atomic-level understanding of CDI. (A) IQ-centric view of calmodulation of Ca_V channels. In this mechanistic scheme, ApoCaM is preassociated with the IQ domain (blue). Upon Ca²⁺ binding, CaM rebinds the same IQ domain with a higher affinity, and subtle conformational rearrangements are presumed to trigger CaM-mediated channel regulation. (B, left) Crystal structure of Ca_V1.2 IQ domain peptide in complex with Ca²⁺/CaM (Protein Data Bank accession no. 2BE6). The IQ domain is colored blue. CaM is show in cyan (N-lobe, pale cyan; C-lobe, cyan). Ca²⁺ ions are depicted as yellow spheres. The key isoleucine residue (red) serves as a hydrophobic anchor for CaM. Ca²⁺/CaM adopts a parallel arrangement with the IQ domain in which the N-lobe binds closer to the amino terminus of the IQ domain and the C-lobe binds further downstream. (B, right) Crystal structure of Ca²⁺/CaM bound to mutant Ca_V1.2 IQ domain with alanines substituted for the key isoleucine-glutamine residues (accession no. 2F3Z). This double mutation abolishes CDI. Structurally, however, Ca²⁺/CaM hugs the mutant IQ domain in a similar conformation as to its interaction with the wild-type (left). (C) Crystal structure of Ca²⁺/CaM bound to Ca_V2.1 IQ domain (left, accession no. 3BXK; right, accession no. 3DVM). The IQ domain is shaded green, CaM in cyan. Ca²⁺/CaM was reported to bind to the Ca_V2.1 IQ domain in both parallel (left) and antiparallel (right) arrangements. The antiparallel arrangement in which the C-lobe of CaM binds upstream of IQ domain has led to speculation that CDF may result from this inverted polarity of Ca²⁺/CaM association. (D) Simplified configurations of the CaM/channel complex relevant for calmodulation (E, A, I_C, I_N, and I_CN). In configuration E, channels lack preassociated CaM and therefore cannot undergo CDI. Configuration A corresponds to channels bound to apoCaM, and thus capable of undergoing robust CDI. Ca²⁺ binding to the C-lobe and N-lobe of CaM leads to the inactivated configurations I_C and I_N, respectively. Ca²⁺ binding to both lobes of CaM then yields configuration I_CN, with both C and N lobes of CaM engaged toward CDI. This final transition may exhibit positive cooperativity as specified by the constant λ >> 1. Under endogenous levels of CaM, channels may reside in any of the five configurations. Strong coexpression of wild-type or mutant CaM restricts accessibility to various states. Reproduced with permission from Ben Johny et al., 2013.

Figure 4.

Residue-level roadmap for calmodulation of Ca_V1.3 channels. Systematic alanine scanning mutagenesis of the entire CI domain of Ca_V1.3 channels. (A) CDI of wild-type and mutant Ca_V1.3 channels recombinantly expressed in HEK293 cells was measured with only endogenous CaM present. CDI observed here reflects properties of the entire system (stick figure) diagrammed in Fig. 3 D. Exemplar whole-cell current shows robust CDI for wild-type (WT) Ca_V1.3, reflecting their high affinity for apoCaM. Here and throughout, the vertical bar pertains to 0.2 nA of Ca²⁺ current (black); the Ba²⁺ current (gray) has been scaled ∼3-fold downward to aid comparison of decay kinetics. Horizontal bar, 100 ms. CDI is measured as the fractional reduction of Ca²⁺ current in comparison to Ba²⁺ at 300 ms (Ben Johny et al., 2013). Bottom, bar graph summary of CDI for alanine substitutions for indicated residues. CDI for WT channels, blue-green vertical line. Alanine substitutions in both EF hand regions and IQ domain resulted in diminished CDI. (B) Overexpression of CaM_WT isolates the behavior of the diamond-shaped subsystem. Such overexpression of CaM_WT should rescue CDI for mutations that weakened apoCaM preassociation. For WT Ca_V1.3, exemplar current shows that CDI is unaltered, as expected for a channel with high affinity for apoCaM. Bottom, bar graph summary of CDI for various mutant channels with CaM_WT overexpressed. Format is as in A. CDI is rescued for several mutations in the EF hand region (EF2) and the IQ domain, reflecting the preassociation of N-lobe and C-lobe of apoCaM. (C) Overexpression of CaM₁₂ isolates C-lobe CDI. Exemplar currents for WT channels show the fast C-lobe form of CDI. Bottom, bar graph summary shows the footprint of C-lobe CDI. Mutants in both the first EF hand and the IQ domain disrupted the C-lobe CDI (see the LGF loci in the EF hand regions and TFL and IQD loci in the IQ domain). The format is as in A. (D) Overexpression of CaM₃₄ isolates N-lobe CDI. N-lobe CDI was largely preserved by mutations in the CI region. Mutations that weakened N-lobe apoCaM preassociation exhibited enhanced N-lobe CDI (see EF2 segment). Reassuringly, alanine scanning mutagenesis of NSCaTE element resulted in specific disruption of N-lobe CDI of Ca_V1.3 channels (Tadross et al., 2008). Adapted with permission from Ben Johny et al. (2013) and Bazzazi et al. (2013).

Figure 5.

Next-generation blueprint for CaM/CaBP regulation of Ca_V channels. (A) De novo molecular model of Ca_V1.3 CI region docked to apoCaM. The N-lobe of apoCaM is thought to preassociate with the PCI region (green), whereas the C-lobe binds to the IQ domain (blue). The NSCaTE segment (tan) is unoccupied. This model corresponds to configuration A in Fig. 3 D, where the channels are charged with an apoCaM and ready to undergo CDI. (B) Proposed model for the Ca²⁺ inactivated state of the channel. The N-lobe of Ca²⁺/CaM is shown binding to the NSCaTE segment based on a recent NMR structure (Protein Data Bank accession no. 2LQC). This configuration is believed to result in N-lobe CDI. The de novo molecular model shows C-lobe of Ca²⁺/CaM, the EF hand segment, and the IQ domain forming a tripartite complex resulting in C-lobe CDI. Overall, this model corresponds to the inactive configuration I_CN in Fig. 3 D. (C) Proposed allosteric mechanism of CaBP action. CaBP and apoCaM may dually bind the Ca_V1 channel. However, CaBP may allosterically inhibit CDI of Ca_V1 channels by interacting with the NT, III-IV loop, or PCI segment, thereby preventing Ca²⁺/CaM from reaching its effector configuration. (A–C) Adapted with permission from Yang et al. (2014).