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Identifying the Site/S of Modification on Human L-type Calcium Channel Protein Isoforms During Oxidative Stress

Muralidharan, P., Ingley, E.ORCID: 0000-0002-8112-9134 and Hool, L. (2013) Identifying the Site/S of Modification on Human L-type Calcium Channel Protein Isoforms During Oxidative Stress. Heart, Lung and Circulation, 22 . S56.

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Reactive oxygen species and calcium contribute to the development of cardiac hypertrophy. The L-type calcium channel (LTCC) is the major route for calcium influx into cardiac myocytes. Alterations in cellular redox state influence the activity of the LTCC. Exposing the native channel to the thiol-specific oxidising compound 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) increases peak inward current while the thiol reducing agent dithiothreitol (DTT) decreases basal channel activity. The purpose of this study was to identify the cysteines responsible for modulating channel function during oxidative stress. Human long and short N terminal (NT) isoforms of Cav1.2 (alpha subunit) were expressed in HEK cells. Cysteines present on the alpha interacting domain (AID) of the long NT isoform were mutated to a serine or an alanine. The channel protein was purified by histidine tag purification and incorporated in liposomes for functional analysis. Exposure of the long NT isoform to 200 μM DTNB increased open probability (Po) of the channel from 0.032 ± 0.003 to 0.075 ± 0.011 and subsequent exposure to 1 mM DTT decreased Po to 0.032 ± 0.014 (n = 12; p < 0.05). Similar results were obtained with the short NT isoform that lacks the first 46 amino acids of the N terminus (n = 5). This suggests that the NT domain is not the reactive region. In contrast, DTNB and DTT had little effect on Po of the LNT containing mutated cysteines in AID region (n = 9). The results of this study suggest that the cysteines on the alpha interacting domain of Cav1.2 are required for alterations in the function of the channel during oxidative stress.

Item Type: Journal Article
Publisher: Elsevier B.V.
Copyright: © 2013 Published by Elsevier Ltd.
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