α1-adrenoceptors regulate only the caveolae-located subpopulation of cardiac K(V)4 channels.
In ventricular myocytes, α1-AR stimulates Gαs proteins and reduces the transient outward K(+) current (I(to)) via a cAMP/PKA-mediated pathway and thus regulates cardiac contraction and excitability. This I(to) reduction is compartmentalized and limited to discrete membrane regions since PKA-dependent phosphorylation of the I(to) 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 I(to) in response to α1-AR activation. I(to) 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 I(to) 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 Akinase anchoring protein AKAP100, exist in caveolae in living myocytes. Density gradients and immunoprecipitation experiments show that the components of the α1-AR/I(to) 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, Gαs 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 I(to) channel population localizes in non-caveolar membrane rafts and is not sensitive to α1-adrenergic regulation.
Department of Physiology, School of Pharmacy, University of the Basque Country, Bilbao, Spain.
Aintzane
A
Alday
Janire
J
Urrutia
Mónica
M
Gallego
Oscar
O
Casis
D000818:Animals; D021941:Caveolae; D051244:Caveolin 3 / Q000378:metabolism*; D002451:Cell Compartmentation; D006352:Heart Ventricles / Q000166:cytology; D021962:Membrane Microdomains; D046912:Multiprotein Complexes / Q000378:metabolism*; D032342:Muscle Cells / Q000737:chemistry / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D011941:Receptors, Adrenergic / Q000502:physiology*; D018340:Receptors, Adrenergic, alpha-1 / Q000502:physiology*; D051672:Shal Potassium Channels / Q000378:metabolism*
C547715:Adra1a protein, rat; D051244:Caveolin 3; D046912:Multiprotein Complexes; D011941:Receptors, Adrenergic; D018340:Receptors, Adrenergic, alpha-1; D051672:Shal Potassium Channels