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α1-Adrenoreceptors regulate only the caveolae-located subpopulation of cardiac KV4 channels
In ventricular myocytes, α1-AR stimulates Gas proteins and reduces the transient outward K + current (Ito) via a cAMP/PKA-mediated pathway and thus regulates cardiac contraction and excitability. This Ito reduction is compartmentalized and limited to discrete membrane regions since PKA-dependent pho...
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Published in: | Channels (Austin, Tex.) Tex.), 2010-05, Vol.4 (3), p.168-178 |
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Main Authors: | , , , |
Format: | Article |
Language: | English |
Subjects: | |
Online Access: | Get full text |
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Summary: | In ventricular myocytes, α1-AR stimulates Gas proteins and reduces the transient outward K
+
current (Ito) via a cAMP/PKA-mediated pathway and thus regulates cardiac contraction and excitability. This Ito reduction is compartmentalized and limited to discrete membrane regions since PKA-dependent phosphorylation of the Ito channels after α1-AR stimulation requires the integrity of both the sarcoplasmic membrane and the cytoskeleton. The aim of this work was to investigate the mechanisms involved in the compartmentalization of the PKA-dependent modulation of Ito in response to α1-AR activation. Ito current recordings were performed by the Patch-Clamp technique. Membrane rafts from isolated ventricular myocytes were extracted by centrifugation in a sucrose density gradient. The different proteins were visualized by western blot and protein-protein interactions determined by coimmunoprecipitation experiments. Localization of Ito channel in caveolae, particular subtypes of membrane rafts, was achieved by electron microscopy. Patch-Clamp recordings show that a functional supramolecular complex, kept together by the A kinase anchoring protein AKAP100, exist in caveolae in living myocytes. Density gradients and immunoprecipitation experiments show that the components of the a1-AR/Ito pathway localize in caveolae, forming two different groups of proteins. The K
V
4.2/K
V
4.3 channel forms a supramolecular complex with PKA through AKAP100 and is attached to caveolae by interacting with caveolin-3. On the other hand, α1-AR, Gas and adenylate cyclase gather in a second group also connected to caveolin-3. Therefore, both groups of preassembled proteins are maintained in close proximity by caveolin-3. A different Ito channel population localizes in non-caveolar membrane rafts and is not sensitive to a1-adrenergic regulation. |
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ISSN: | 1933-6950 1933-6969 |
DOI: | 10.4161/chan.4.3.11479 |