Evidence for redox sensing by a human cardiac calcium channel.

Padmapriya Muralidharan,Evan Ingley,Livia Hool,Henrietta Cserne Szappanos

doi:10.1038/srep19067

Abstract

Ion channels are critical to life and respond rapidly to stimuli to evoke physiological responses. Calcium influx into heart muscle occurs through the ion conducting α1C subunit (Cav1.2) of the L-type Ca2+ channel. Glutathionylation of Cav1.2 results in increased calcium influx and is evident in ischemic human heart. However controversy exists as to whether direct modification of Cav1.2 is responsible for altered function. We directly assessed the function of purified human Cav1.2 in proteoliposomes. Truncation of the C terminus and mutation of cysteines in the N terminal region and cytoplasmic loop III-IV linker did not alter the effects of thiol modifying agents on open probability of the channel. However mutation of cysteines in cytoplasmic loop I-II linker altered open probability and protein folding assessed by thermal shift assay. We find that C543 confers sensitivity of Cav1.2 to oxidative stress and is sufficient to modify channel function and posttranslational folding. Our data provide direct evidence for the calcium channel as a redox sensor that facilitates rapid physiological responses.

Highlights

Ion channels are critical to life and respond rapidly to stimuli to evoke physiological responses
Voltage stepping the long NT isoform protein to –200 mV elicited an inward current approximately –4 pA in magnitude that decreased as voltage approached 0 mV (Fig. 1b) reversed at 0 mV with a slope conductance of approximately 21.3 pS, similar to that previously reported in bovine calcium channels[27]
The I–V relationship and the mean ± SEM channel open probability (Po) for currents recorded in control solution and currents recorded in the presence of DTT, dithio bis-nitrobenzoic acid (DTNB) and nisoldipine (Nisol) are shown below right. #,*p < 0.05 vs Con ANOVA and Tukey’s Posthoc test

Summary

Introduction

Ion channels are critical to life and respond rapidly to stimuli to evoke physiological responses. The channel protein contains many cysteines that are available to be covalently modified during changes in redox state It is not clear how modification of thiols alters channel function because contradictory responses have been reported, with some groups arguing that thiol reducing agents increase channel activity while others argue the opposite response[26]. The variation in functional responses could be a result of the influence of a signalling protein in the cellular environment, or an accessory protein or subunit that modifies channel function It is not clear whether direct reduction or oxidation of thiol groups on the channel protein is sufficient to alter function. Our results indicate that direct modification of the Cav1.2 channel protein is responsible and sufficient for alterations in channel function during oxidative stress

Methods

Results

Conclusion