Regulation of caveolar cardiac sodium current by a single Gsalpha histidine residue.
Cardiac sodium channels (voltage-gated Na(+) channel subunit 1.5) reside in both the plasmalemma and membrane invaginations called caveolae. Opening of the caveolar neck permits resident channels to become functional. In cardiac myocytes, caveolar opening can be stimulated by applying beta-receptor agonists, which initiates an interaction between the stimulatory G protein subunit-alpha (G(s)alpha) and caveolin-3. This study shows that, in adult rat ventricular myocytes, a functional G(s)alpha-caveolin-3 interaction occurs, even in the absence of the caveolin-binding sequence motif of G(s)alpha. Consistent with previous data, whole cell experiments conducted in the presence of intracellular PKA inhibitor stimulation with beta-receptor agonists increased the sodium current (I(Na)) by 35.9 +/- 8.6% (P < 0.05), and this increase was mimicked by application of G(s)alpha protein. Inclusion of anti-caveolin-3 antibody abolished this effect. These findings suggest that G(s)alpha and caveolin-3 are components of a PKA-independent pathway that leads to the enhancement of I(Na). In this study, alanine scanning mutagenesis of G(s)alpha (40THR42), in conjunction with voltage-clamp studies, demonstrated that the histidine residue at position 41 of G(s)alpha (H41) is a critical residue for the functional increase of I(Na). Protein interaction assays suggest that G(s)alphaFL (full length) binds to caveolin-3, but the enhancement of I(Na) is observed only in the presence of G(s)alpha H41. We conclude that G(s)alpha H41 is a critical residue in the regulation of the increase in I(Na) in ventricular myocytes.
Palygin
Oleg A
OA
Dept. of Molecular Physiology and Biophysics, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242-1109, USA.
Pettus
Janette M
JM
Shibata
Erwin F
EF
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D051244:Caveolin 3 / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D015640:Ion Channel Gating; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*
D000318:Adrenergic beta-Agonists; C495549:Cav3 protein, rat; D051244:Caveolin 3; D062554:NAV1.5 Voltage-Gated Sodium Channel; D047428:Protein Kinase Inhibitors; D011943:Receptors, Adrenergic, beta; C568322:Scn5a protein, rat; D015222:Sodium Channels; D006639:Histidine; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D019205:GTP-Binding Protein alpha Subunits, Gs; D007545:Isoproterenol