Residues Critical for Voltage-Sensor Transitions Determining Gating Properties of Cav1.1

Following membrane depolarization S4 segments of voltage-gated cation channels translocate through the membrane resulting in the opening of the channel pore. This mechanism has been extensively studied in potassium and sodium channels yet little is known about it in voltage-gated calcium channels. Here we used de-novo ROSETTA modelling and site-directed mutagenesis to study the transition of the IVth voltage-sensing-domain (VSD) of Cav1.1 calcium channel during gating. Previously we have shown that the embryonic Cav1.1e splice variant, lacking exon 29 in the IVS3-S4 linker, has an 8-fold higher current amplitude and 30mV left-shifted voltage-sensitivity compared to the adult Cav1.1a splice variant. Modeling 4 consecutive states of the VSD suggested that the number of H-bonds formed between the arginines of IVS4 and residues in IVS3 is higher in Cav1.1e than in Cav1.1a. Among them aspartate at position 1196 (D1196) shows the strongest difference between the two splice variants. Indeed, voltage clamp recordings show that neutralizing the negative charge in S3 (D1196N) has no effect on the voltage sensitivity of Cav1.1a but confers poor voltage sensitivity to Cav1.1e. Mutations of R1210, R1217, and R1220 of Cav1.1e are currently analyzed to identify the interaction partners of D1196 during gating. Furthermore, structure modeling revealed an extracellularly exposed hydrophobic cluster formed by two leucines in the middle of IVS3-S4 loop encoded by exon 29. Point mutations which eliminate this hydrophobic cluster partially recapitulate the effects of exon 29 deletion. Together these findings suggest a model according to which the extracellular loop S3-S4 controls the orientation of S3 and S4 relative to each other. This alters charge compensation of positive charges in S4 and dramatically reduces the voltage sensitivity of CaV1.1a calcium channels. Support: FWF W1101, P23479, LFU-P7400-027-011.