CaM-dependent modulation of human Ca_V1.3 whole-cell and single-channel currents by C-terminal CaMKII phosphorylation...

Phosphorylation enables rapid modulation of voltage-gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human Ca_V1.3 VGCC, however, is largely unexplored. We characterized modulation of Ca_V1.3 gating via S1475, the human equivalent of a phosphorylation site identified in the rat. S1475 is highly conserved in Ca_V1.3 but absent from all other high-voltage activating calcium channel types co-expressed with Ca_V1.3 in similar tissues. Further, it is located in the C-terminal EF-hand motif, which binds calmodulin (CaM). This is involved in calcium-dependent channel inactivation (CDI). We used amino acid exchanges that mimic either sustained phosphorylation (S1475D) or phosphorylation resistance (S1475A). Whole-cell and single-channel recordings of phosphorylation state imitating Ca_V1.3 variants in transiently transfected HEK-293 cells revealed functional relevance of S1475 in human Ca_V1.3. We obtained three main findings: (1) Ca_V1.3_S1475D, imitating sustained phosphorylation, displayed decreased current density, reduced CDI and (in-) activation kinetics shifted to more depolarized voltages compared with both wildtype Ca_V1.3 and the phosphorylation-resistant Ca_V1.3_S1475A variant. Corresponding to the decreased current density, we find a reduced open probability of Ca_V1.3_S1475D at the single-channel level. (2) Using CaM overexpression or depletion, we find that CaM is necessary for modulating Ca_V1.3 through S1475. (3) CaMKII activation led to Ca_V1.3_WT-current properties similar to those of Ca_V1.3_S1475D, but did not affect Ca_V1.3_S1475A, confirming that CaMKII modulates human Ca_V1.3 via S1475. Given the physiological and pathophysiological importance of Ca_V1.3, our findings on the S1475-mediated interplay of phosphorylation, CaM interaction and CDI provide hints for approaches on specific Ca_V1.3 modulation under physiological and pathophysiological conditions. KEY POINTS: Phosphorylation modulates activity of voltage-gated L-type calcium channels for specific cellular needs but is largely unexplored for human Ca_V1.3 channels. Here we report that S1475, a CaMKII phosphorylation site identified in rats, is functionally relevant in human Ca_V1.3. Imitating phosphorylation states at S1475 alters current density and inactivation in a calmodulin-dependent manner. In wildtype Ca_V1.3 but not in the phosphorylation-resistant variant S1475A, CaMKII activation elicits effects similar to constitutively mimicking phosphorylation at S1475. Our findings provide novel insights on the interplay of modulatory mechanisms of human Ca_V1.3 channels, and present a possible target for Ca_V1.3-specific gating modulation in physiological and pathophysiological conditions.