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L-type calcium channel -subunit and protein kinase inhibitors modulate Rem-mediated regulation of current

Departments of ¹Physiology and ²Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky

Submitted 7 September 2005 ; accepted in final form 20 April 2006

Cardiac voltage-gated L-type Ca channels (Ca_V) are multiprotein complexes, including accessory subunits such as Ca_V2 that increase current expression. Recently, members of the Rad and Gem/Kir-related family of small GTPases have been shown to decrease current, although the mechanism remains poorly defined. In this study, we evaluated the contribution of the L-type Ca channel -subunit (Ca_V1.2) to Ca_V2-Rem inhibition of Ca channel current. Specifically, we addressed whether protein kinase A (PKA) modulation of the Ca channel modifies Ca_V2-Rem inhibition of Ca channel current. We first tested the effect of Rem on Ca_V1.2 in human embryonic kidney 293 (HEK-293) cells using the whole cell patch-clamp configuration. Rem coexpression with Ca_V1.2 reduces Ba current expression under basal conditions, and Ca_V2a coexpression enhances Rem block of Ca_V1.2 current. Surprisingly, PKA inhibition by 133 nM H-89 or 50 µM Rp-cAMP-S partially relieved the Rem-mediated inhibition of current activity both with and without Ca_V2a. To test whether the H-89 action was a consequence of the phosphorylation status of Ca_V1.2, we examined Rem regulation of the PKA-insensitive Ca_V1.2 serine 1928 (S1928) to alanine mutation (Ca_V1.2-S1928A). Ca_V1.2-S1928A current was not inhibited by Rem and when coexpression with Ca_V2a was not completely blocked by Rem coexpression, suggesting that the phosphorylation of S1928 contributes to Rem-mediated Ca channel modulation. As a model for native Ca channel complexes, we tested the ability of Rem overexpression in HIT-T15 cells and embryonic ventricular myocytes to interfere with native current. We find that native current is also sensitive to Rem block and that H-89 pretreatment relieves the ability of Rem to regulate Ca current. We conclude that Rem is capable of regulating L-type current, that release of Rem block is modulated by cellular kinase pathways, and that the Ca_V1.2 COOH terminus contributes to Rem-dependent channel inhibition.

cardiac myocyte; monomeric GTPase; Rad and Gem/Kir-related family; insulin-secreting cell; calcium imaging; heart development

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