L-type calcium channel alpha-subunit and protein kinase inhibitors modulate Rem-mediated regulation of current.
Cardiac voltage-gated L-type Ca channels (Ca(V)) are multiprotein complexes, including accessory subunits such as Ca(V)beta2 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 alpha-subunit (Ca(V)1.2) to Ca(V)beta2-Rem inhibition of Ca channel current. Specifically, we addressed whether protein kinase A (PKA) modulation of the Ca channel modifies Ca(V)beta2-Rem inhibition of Ca channel current. We first tested the effect of Rem on Ca(V)1.2 in human embryonic kidney 293 (HEK-293) cells using the whole cell patch-clamp configuration. Rem coexpression with Ca(V)1.2 reduces Ba current expression under basal conditions, and Ca(V)beta2a coexpression enhances Rem block of Ca(V)1.2 current. Surprisingly, PKA inhibition by 133 nM H-89 or 50 microM Rp-cAMP-S partially relieved the Rem-mediated inhibition of current activity both with and without Ca(V)beta2a. To test whether the H-89 action was a consequence of the phosphorylation status of Ca(V)1.2, we examined Rem regulation of the PKA-insensitive Ca(V)1.2 serine 1928 (S1928) to alanine mutation (Ca(V)1.2-S1928A). Ca(V)1.2-S1928A current was not inhibited by Rem and when coexpression with Ca(V)beta2a 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(V)1.2 COOH terminus contributes to Rem-dependent channel inhibition.
Crump
Shawn M
SM
Dept. of Physiology, MS-508, Univ. of Kentucky College of Medicine, 800 Rose St. Lexington, KY 40536-0298, USA.
Correll
Robert N
RN
Schroder
Elizabeth A
EA
Lester
William C
WC
Finlin
Brian S
BS
Andres
Douglas A
DA
Satin
Jonathan
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000187:drug effects* / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008647:Mesocricetus; D051379:Mice; D008813:Mice, Inbred ICR; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D007546:Isoquinolines; C419385:L-type calcium channel alpha(1C); D047428:Protein Kinase Inhibitors; C108135:Rem protein, mouse; D013449:Sulfonamides; D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D020559:Monomeric GTP-Binding Proteins; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium