Clustering of protein kinase A-dependent CFTR chloride channels in the sarcolemma of guinea-pig ventricular myocytes.
Cardiac myocytes express protein kinase A-dependent Cl(-) (Cl(PKA)) channels that are thought to represent cardiac expression of CFTR. In the present study, the 'Smart' patch clamp technique was used to investigate the distribution of Cl(PKA) channels at the cell surface of isolated guinea-pig ventricular myocytes. Imaging the cell surface using scanning ion conductance microscopy allowed the identification of the mouths to t-tubules and lateral z-grooves with a spacing of 1.86 microm. Cell-attached patch clamp recordings were made from specified locations within the imaged area. Perfusion of the cells with an activating cocktail of isoprenaline (5 microM), forskolin (10 microM) and isobutylmethylxanthine (50 microM) activated large, noisy anion-selective currents in which unitary channel currents could not be identified. Currents were recorded both from within z-grooves and in the inter-groove region but not at the mouths of t-tubules. Power spectral and noise analyses indicated the involvement of 13.5pS channels occurring in clusters of >50 channels. Channel activity was lost on excision of the patch from the cell but could be recovered in inside-out excised patches by application of the catalytic subunit of PKA. These results suggest that CFTR Cl(PKA) channels occur in clusters in the sarcolemma of guinea-pig ventricular myocytes; there was no evidence of a heterogeneous distribution of clusters between the z-grooves and the inter-groove region.
James
Andrew F
AF
Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan. a.james@bristol.ac.uk
Sabirov
Razshan Z
RZ
Okada
Yasunobu
Y
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000378:metabolism*; D008564:Membrane Potentials; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010477:Perfusion; D012508:Sarcolemma / Q000378:metabolism*
D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases