A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function

L-type voltage-gated Ca2+ channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I–IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca2+ levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca2+ channel function. Background: L-type Ca2+ channels (LTCCs) are fine-tuned by different molecular mechanisms. Results: Pore-forming α1-subunits of LTCCs contain a polybasic amino acid sequence within their I-II linkers that binds to the plasma membrane. This polybasic motif is required for normal channel gating and modulation. Conclusion: The polybasic cluster stabilizes normal channel activity. Significance: We discovered a new modulatory domain of LTCCs within their pore-forming α1-subunit.