Peering into the Molecular Machinery for regulation of Cav1.2 Channel Clusters

There is growing appreciation that ion channels do not function in isolation but rather as part of a complex and dynamic microenvironment, comprised of accessory subunits, adapter/cytoskeletal proteins and regulatory molecules. More recently, our understanding of ion channel biology has continued to develop with increasing recognition and characterization of ‘channel clustering’, or the observation that individual ion channels can form organized colocalization with one another, leading to events such as cooperative gating and/or biophysical coupling, current augmentation, and physiologic function. In this issue of Circulation Research, Baudel et al. elegantly illustrate this phenomenon occurring in the L-type voltage-gated Ca²⁺ channel, Ca_v1.2 of arterial myocytes.¹ Channel clustering is identified as the mechanistic outcome of PKA-dependent phosphorylation of Ca_v1.2 at serine residue 1928 in the channel C-terminus leading ultimately to enhanced Ca²⁺ entry and vasoconstriction. Notably, this work not only reinforces previous findings of Ca_v1.2 clustering events, but also provides mechanistic understanding as well as physiologic significance to channel clustering events.

Voltage-gated Ca²⁺ channels (Ca_v) play important physiological roles in both excitable and non-excitable cells including neurons, atrial and ventricular myocytes, and smooth muscle.² Ca_v family members respond rapidly to changes in cell membrane potential effectively gating the influx of Ca²⁺ ions important for ubiquitous cell functions ranging from contraction, hormone and neurotransmitter release, and activation of gene expression pathways.³ There are at least 10 known Ca_v isoforms across 3 major subfamilies (Ca_v1, Ca_v2, and Ca_v3) with unique voltage sensitivities and gating kinetics. Conserved across family members is a pore forming α-subunit comprised of a single peptide chain made up of four homologous repeats (I-IV), each containing six transmembrane regions (S1-S6). The voltage sensing domain is derived from segments S1-S4, while S5, S6, and their linker region make up the channel pore, including the selectivity filter and activation gate. The movement of the voltage sensor, in response to changes in cell membrane potential, induces a conformational change that enables channel opening and Ca²⁺ flux. Additional variability in channel activity is satisfied by diverse heteromeric complexing with accessory subunits β, α₂δ, or γ, contributing to cell specific channel kinetics, trafficking, and regulation.⁴

Diversity of channel isoforms and accessory subunits promotes functional heterogeneity in the Cav family giving rise ultimately to a broad spectrum of behavior across cell types. However, even at the level of the single cell, heterogeneity has been observed within specific Ca_v channel populations with important implications for physiological function.^{5, 6} An important example of how heterogeneity arises from a single Ca_v populations comes from variability in the spatial distribution of Ca_v within the cell membrane. Specifically, organization of Ca_v1.2 channels in clusters at the t-tubule membrane in close apposition to ryanodine receptor Ca²⁺ release channels (RyR) in the sarcoplasmic reticulum (SR) underlie the phenomenon of “graded release” in cardiac myocytes whereby the amount of Ca²⁺ released from the SR is related to the amount of Ca²⁺ that comes into the myocytes through Ca_v1.2. Mathematical analysis reveals that in areas of channel clustering, the rate of simultaneous multi-channel openings exceeds the level predicted by binomial distributions, indicating the occurrence of cooperativity. Indeed, many investigations have suggested that if only independent channel gating occurred, then under physiologic conditions there would be an insufficient threshold of Ca²⁺ entry to enact physiologic processes such as excitation-contraction coupling in cardiac myocytes or tonic firing of neurons for memory and plasticity.^{7, 8} For Ca_v1.2, it is suggested that as many as 6–10 channels are capable of opening and closing simultaneously through cooperative gating, overall resulting in significantly amplified Ca²⁺ entry and the necessary tight spatiotemporal control needed for proper physiologic function.⁹

In this issue of Circulation Research, Baudel et al. apply an impressive battery of state-of-the-art imaging, electrophysiology and biochemistry techniques to comprehensively elucidate the mechanism underlying Ca_v1.2 clustering and cooperative gating in arteriole smooth muscle cells (Figure)¹. Using a mouse model of diabetic hyperglycemia with documented alterations in Ca_v1.2 channel activity, the authors elegantly demonstrate how local AKAP5-dependent protein complexes¹⁰ recruit PKA activity through the nucleotide stimulated G_S-coupled P2Y₁₁ receptor resulting in the targeted phosphorylation of serine residue 1928 within the Ca_v1.2 C-terminus (Figure). The resulting effect led to enhanced Ca²⁺ entry and vasoconstriction, which could be eliminated by the phosphoablation of S1928 to alanine, the removal of AKAP5, or the pharmacologic inhibition of PKA. Through multiple superresolution imaging modalities, the authors associate the changes in augmented Ca²⁺ entry to the inducible and dynamic clustering of Ca_v1.2 channels. Consequently, these inhibitory states abrogate vasoconstriction and reduce arterial resistance in diabetic mice, demonstrating the significance of Ca_v1.2 cluster signaling under pathophysiologic conditions and a potent illustration of how these mechanisms might be modulated in the setting of disease.

Figure.

Schematic depicting the proposed pathway for regulation of channel clustering in arteriole myocytes via protein kinase A-dependent phosphorylation of S1928 in the Ca_v1.2 C-terminus. High glucose in the setting of diabetic hyperglycemia leads to activation of the P2Y₁₁ receptor, elevated intracellular cAMP, and protein kinase A activation via a signaling microdomain organized by the A kinase anchoring protein AKAP5. Ca_v1.2 phosphorylation at S1928 in turn promotes channel clustering, enhanced Ca²⁺ influx and vasoconstriction.

Studies on Ca_v1.2 have contributed greatly to our general understanding of ion channel clustering as a general phenomenon important for regulation of physiological function across biology¹¹. Evidence for channel clustering with cooperative gating and/or biophysical coupling has been observed in a host of other ion channels, including transient receptor potential (TRP) channels, multiple families of K⁺-channels, RyR, Inositol Trisphosphate (IP₃) receptors, Hyperpolarization-Cyclic Nucleotide-Activated (HCN) channels, and voltage-gated Na⁺ channels (Na_v).¹¹ Beyond Cav family members, another example may be found in Na_v isoforms, Na_v1.1, Na_v1.2, and Na_v1.5, which have been demonstrated to assemble, gate, and function as dimers through interaction with the regulatory protein 14-3-3.¹² This functional coupling leads to current augmentation but also allows for dominant-negative effects through coupling of WT and dysfunction (through inherited or acquired defect) channels. Although there are similarities between the type of clustering observed with Na_v and Ca_v1.2 channels, there are important differences as well including partner proteins (AKAP5 vs. 14-3-3), degree of clustering, mode of regulation and functional effects. Taken together, however, these examples support channel clustering as a more universal mechanism for regulation of channel activity and cell membrane excitability with implications for physiology and potential therapies.

Importantly, while the study from Baudel and colleagues depicts clustering as a physiologic state necessary for proper timing and thresholding of signal generation, it also illustrates that coupled channel interactions can promote pathologic states, and that uncoupling can be an opportunity to mitigate disease. Indeed, pharmacologic blockers used to attenuate over-active channels are often met with limited success and often promote arrhythmias when exposed to the heart. Therefore, it becomes even more paramount that novel therapeutic targets of channel coupling may represent more viable strategies for attenuating channel function going forward.

Indeed, proof-of-principle data support that phospho-ablation, knockout of AKAP5, or in vitro pharmacologic treatment with the PKA inhibitor rp-cAMP limit the vasoconstrictive effects of diabetes. While these models are not readily translated into a therapeutic intervention, they reveal a novel pathway for future investigation into translational targeting. A potential translational avenue to explore going forward is in peptide-based strategies. For example, peptide-based approaches to inhibit PKA using engineered versions of the endogenous PKA regulatory element PKI are currently being developed.¹³ Additionally, studies using artificial expression of just the C-terminal fragment of Ca_v1.2 showed its role as potent vasodilator, likely serving as a competitive decoy sufficient for interaction but without the corresponding contribution to cooperative gating.¹⁴ Certainly, like any therapeutic compound, potential challenges like effective delivery, tissue and cell specificity and off target effects will need to be addressed, particularly given the global expression for many of the players and occurrences of channel coupling.

Although the new findings from the Baudel group provide important insight into the mechanistic pathway linking posttranslational modification of Ca_v1.2, changes in channel clustering and function, they also raise a number of compelling unanswered questions. Given that functional channel coupling has been observed for many tissues where Ca_v1.2 is expressed, future directions will need to explore how universal this mechanism is throughout those tissues, as well as whether the other Ca_v isoforms utilize a similar signaling network to modulate channel clustering and cell function. Furthermore, PKA activation (and potentially Ca_v1.2 phosphorylation at S1928) resides downstream of a host of agonists and cell signaling pathways beyond glucose stimulation. It will be interesting going forward to determine the extent to which Ca_v1.2 phosphorylation at S1928 integrates stress stimuli upstream of PKA. Of course, perhaps the most pressing question for future work is to what extent can modulation of Cav1.2 S1928 phosphorylation and channel clustering mitigate vascular complications in the setting of diabetic hyperglycemia.