Effects of Tryptophan Substitutions for Highly-Conserved Phenylalanines in the Skeletal Muscle L-Type Calcium Channel

Abstract

The motif FxxExxxK/R is highly conserved in S2 segments of voltage-gated cation channels, including the four repeats of the α1s subunit of the skeletal muscle L-type Ca2+ channel, and appears to represent an important structure for voltage-sensing. Moreover, mutating the conserved S2 phenylalanine within the drk1 K+ channel monomer to alanine causes a ∼50 mV depolarizing shift in activation (Li-Smerin et al., JGP 115:33-49). Here, we made homologous F to A substitutions in Repeat I (F97A), Repeat III (F843A) and Repeat IV (F1161A) of α1S N-terminally tagged with yellow fluorescent protein (YFP-α1S), and tested the ability of the mutants to conduct L-type Ca2+ current and serve as the voltage sensor for excitation-contraction (EC) coupling after expression in dysgenic (α1S null) myotubes. Confocal imaging of the YFP tag indicated that each of these constructs was targeted to plasma membrane junctions with the sarcoplasmic reticulum (SR). Measurement of intramembrane charge movements showed no significant difference in mutant expression relative to YFP-α1S (p > 0.05, ANOVA). The F843A and F1161A mutants both supported L-type currents and myoplasmic Ca2+ transients with similar amplitude and voltage-dependence to those of YFP-α1S. In contrast, the F97A mutant displayed substantial depolarizing shifts in activation of L-type current (24 mV) and SR Ca2+ release (13 mV). Together, these results indicate that: i) voltage-induced conformational changes in repeat I are directly important for activation of both L-type current and EC coupling, and ii) either repeats III and IV are less directly important for these functions or the F-A mutations have little influence on voltage-dependent conformational changes of these repeats. We are currently assessing the effects of the F475A mutation in Repeat II. Supported by NIH AR055104 and MDA4319 to K.G.B. and MDA4155 to R.A.B.