Intramolecular Ca v1.1 Chimeras Reveal the Molecular Mechanism Determining the Characteristic Gating Behaviour of the Skeletal Muscle Calcium Channel

Abstract

The Ca2+ channel CaV1.1 is the voltage sensor of skeletal muscle excitation-contraction coupling. The classical skeletal muscle CaV1.1 isoform has poor voltage sensitivity and conducts a small, slowly activating Ca2+ current. In contrast, a splice variant lacking exon 29 (α1S-ΔE29) (Tuluc et al.,2009) has an 8-fold higher current amplitude, fast activation-kinetics, and a 30mV left-shifted voltage-dependence of activation. Therefore, the extracellular loop in repeat IV (IVS3-IVS4) mainly coded by exon 29 is a critical determinant of the characteristic gating properties of CaV1.1. Here we used intramolecular chimeras between repeats I and IV to characterize the structural basis of the gating properties of CaV1.1. Inserting exon 29 (alone or in combination with IVS3) into the corresponding region of repeat I was ineffective. However, in combination with the voltage sensor (IVS4) it fully restored α1S-like amplitude and voltage-sensitivity to α1S-ΔE29. Interestingly, all three chimeras exhibit faster activation kinetics. Secondary structure predictions showed that the long IVS3-IVS4 loop contains a beta-sheet while the short loop forms a coil. Point mutations in exon 29 which abolish the beta-sheet fully mimic the effects of deleting exon 29 regarding the kinetic properties and increase the current amplitude by 3-fold and left-shift the voltage dependence by −15mV. Together with previous findings (Nakai et al., 1994) our data suggest that the S3-S4 loop of the first repeat determines activation kinetics, while the corresponding loop plus voltage sensor in the fourth repeat with its unique secondary structure dictate the voltage-dependence, amplitude, and kinetics of skeletal muscle Ca2+ currents. Grants: PT (MFI-2007-417), BEF (FWF-P20059-B05).