Large conductance voltage- and calcium-activated potassium (BKCa) channels are phosphorylated and inhibited by AMP-activated protein kinase (AMPK), which mediates, in part, the response to hypoxia of the oxygen-sensing carotid body type I cells 1. However, hypoxia-response coupling is tissue specific, despite the fact that BKCa channels are widely expressed across a variety of cell types that do not respond to hypoxia. We therefore investigated the possibility that AMPK-dependent regulation of BKCa may be conferred by the selective expression of splice variants of the pore-forming α-subunits of BKCa channels 2. All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986; carotid bodies were isolated as previously described 1. Bacterially-derived recombinant AMPK phosphorylated and inhibited BKCa channels (transiently expressed in HEK-293 cells) in a manner that was splice variant-specific. Current inhibition was observed in the ZERO variant during intracellular dialysis with thiophosphorylated AMPK which peaked at 36 ± 7 % inhibition (P<0.01) after 5 ± 0.4 min (n = 8). The inclusion of the stress-regulated exon (STREX) increased the stoichiometry of AMPK-dependent phosphorylation of the BKCa α-subunit from 1 to 2 moles/mole when compared with the ZERO variant. This increased phosphorylation of STREX prevented AMPK-dependent inhibition of the STREX variant BKCa current. Point mutation of a single serine (Ser-657) to alanine within the STREX insert reversed the increase in phosphorylation stoichiometry to 1 and recovered channel inhibition by AMPK, which peaked at 30 ± 9 % (P<0.01) after 6 ± 0.8 min (n=3). RT-PCR showed that carotid body type I cells express only the ZERO, but not STREX, variant, and intracellular dialysis of recombinant AMPK attenuated BKCa currents in these cells (35% ± 2; n = 6). Such conditional regulation of BKCa channel splice variants by AMPK may therefore contribute to the cell-specific nature of hypoxia-response coupling observed in carotid body type I cells and other oxygen-sensing cells.
University of Birmingham (2010) Proc Physiol Soc 20, PC21
Poster Communications: AMPK-dependent regulation of BKCa channels is splice variant specific
J. Rafferty1, M. L. Dallas2, F. A. Ross3, C. N. Wyatt4, O. Ogunbayo1, N. Ikematsu3, H. McClafferty1, L. Tian1, H. Widmer5, I. C. Rowe1, M. J. Shipston1, D. Hardie3, C. Peers2, A. Evans1
1. Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom. 2. School of Medicine, University of Leeds, Leeds, United Kingdom. 3. Division of Molecular Physiology, University of Dundee, Dundee, United Kingdom. 4. Department of Neuroscience, Cell Biology and Physiology, Wright State University, Dayton, Ohio, United States. 5. Govan Mbeki Health Building, Glasgow Caledonian University, Glasgow, United Kingdom.
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Where applicable, experiments conform with Society ethical requirements.