Effects of Substituting Tryptophan for Basic Residues in the S4 Voltage-Sensing Helices of CaV1.1

In skeletal muscle, CaV1.1 serves dual functions as the voltage sensor for excitation-contraction (EC) coupling and as an L-type Ca2+ channel. It has been long established that L-type Ca2+ current activates at potentials ∼20 mV more depolarized than intramembrane charge movement or EC coupling. We recently characterized a CaV1.1 mutation (R174W) which affects the innermost basic residue of the voltage-sensing S4 helix of Repeat I and results in malignant hyperthermia susceptibility in humans. Interestingly, R174W abolishes activation of the L-type current in response to 200 ms depolarizations without affecting the voltage dependence of intramembrane charge movement or of SR Ca2+ release. In this study, we have made corresponding mutations in Repeats II (K537W), III (R906W) and IV (K1245W) and expressed these mutants in dysgenic (CaV1.1 null) myotubes. Confocal imaging of affixed YFP tags indicated that all the mutants were correctly targeted to membrane-SR junctions. In striking contrast to the virtual loss of channel function observed with R174W, the K537W and K1245W mutants produced enormous L-type currents with peak amplitudes nearly 8-fold greater than wild-type CaV1.1 and greatly impaired deactivation. Even though these two gain of channel function mutants activated at substantially more hyperpolarized potentials (∼20 mV shift), the voltage-dependence of charge movement and SR Ca2+ release were largely unaffected. On the other hand, R906W produced currents of similar amplitude to wild-type CaV1.1 but with a consistent ∼15 mV depolarizing shift in activation. We are currently investigating the effects of the R906W mutation on the voltage dependence of charge movement and SR Ca2+ release in order to determine whether RIII plays a more critical role in engaging EC coupling than the other conserved Repeats of CaV1.1. Supported by NIH AR055104 (KGB) and AG038778 (RAB), and MDA176448 (KGB).