Schwarzer, S; Nobles, M; Tinker, A; (2010) Do Caveolae Have a Role in the Fidelity and Dynamics of Receptor Activation of G-protein-gated Inwardly Rectifying Potassium Channels? J BIOL CHEM , 285 (36) 27817 - 27826. 10.1074/jbc.M110.103598.
In atrial and nodal cardiac myocytes, M2 muscarinic receptors activate inhibitory G-proteins (G(i/o)), which in turn stimulate G-protein-gated inwardly rectifying K+ channels through direct binding of the G beta gamma subunit. Despite also releasing G beta gamma, G(s)-coupled receptors such as the beta-adrenergic receptor are not able to prominently activate this current. An appealing hypothesis would be if components were sequestered in membrane domains such as caveolae/rafts. Using biochemical fractionation followed by Western blotting and/or radioligand binding experiments, we examined the distribution of the components in stable HEK293 and HL-1 cells, which natively express the transduction cascade. The channel, M2 muscarinic, and A1 adenosine receptors were located in noncaveolar/nonraft fractions. G(i)alpha(1/2) was enriched in both caveolar/raft and noncaveolar/nonraft fractions. In contrast, G(s)alpha was only enriched in caveolar/raft fractions. We constructed YFP-tagged caveolin-2 (YFP-Cav2) and chimeras with the M2 (M2-YFP-Cav2) and A1 (A1-YFP-Cav2) receptors. Analysis of gradient fractions showed that these receptor chimeras were now localized to caveolae-enriched fractions. Microscopy showed that M2-YFP and A1-YFP had a diffuse homogenous membrane signal. YFP-Cav2, M2-YFP-Cav2, and A1-YFP-Cav2 revealed a more punctuate pattern. Finally, we looked at the consequences for signaling. Activation via M2-YFP-Cav2 or A1-YFP-Cav2 revealed substantially slower kinetics compared with M2-YFP or A1-YFP and was reversed by the addition of methyl-beta-cyclodextrin. Thus the localization of the channel signal transduction cascade in non-cholesterol rich domains substantially enhances the speed of signaling. The presence of G(s)alpha solely in caveolae may account for signaling selectivity between G(i/o) and G(s)-coupled receptors.
|Title:||Do Caveolae Have a Role in the Fidelity and Dynamics of Receptor Activation of G-protein-gated Inwardly Rectifying Potassium Channels?|
|Open access status:||An open access publication|
|Keywords:||HETEROTRIMERIC G-PROTEINS, RAT ATRIAL MYOCYTES, GIRK CHANNELS, K+-CHANNEL, CARDIAC MYOCYTES, MUSCARINIC RECEPTOR, ADENYLYL-CYCLASE, PLASMA-MEMBRANE, XENOPUS-OOCYTES, LIVING CELLS|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Medicine (Division of) > Cardiovascular Medicine|
UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Medicine (Division of) > Metabolism and Experimental Therapeutics
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