SK3 channels are activated by peroxynitrite

Matthew P. Burnham & Arthur H. Weston. Faculty of Life Sciences, G38 Stopford Building, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK.

The endothelium-derived hyperpolarizing factor (EDHF) pathway is critically dependent upon activation of endothelial small- and intermediate-conductance Ca2+-activated K+ channels (SKCa and IKCa, respectively). Previous studies have shown that endothelial SKCa are formed from SK3 subunits (Burnham et al., 2002).

We have investigated the effect of peroxynitrite (ONOO-) on SK3 channels expressed (calcium phosphate transfection method) in HEK293 cells using whole-cell patch-clamp techniques. Recorded under quasi-physiological K+ gradients, with 3 µM free Ca2+ internal solution, whole-cell currents displayed a reversal potential of -77 ± 1mV (n=3), close to the calculated K+ equilibrium potential of -82mV, and were inhibited (approximately 80%) by 200nM apamin, consistent with robust expression and function of SK3 under these conditions. Working dilutions of ONOO- (Calbiochem) were prepared immediately prior to use from frozen stocks and decomposed ONOO- (DC-ONOO-) negative controls were prepared by heating working solutions to 50 oC for 1h. Application of DC-ONOO- (control) was without effect in all cases (n=7). In the presence of 2mM extracellular Ca2+, exposure to 40 µM ONOO- increased outward current at +20mV from 942 ± 79pA to 1677 ± 42pA (mean ± sem; n=3) and this current was reduced to 713 ± 95pA (n=3) by 200nM apamin. The requirement for extracellular Ca2+ was determined by substituting 1mM EGTA for Ca2+ in the bathing solution. Under these conditions, exposure to 40 µM ONOO- increased outward current at +20mV from 1656 ± 146pA to 3217 ± 186pA (n=4) and 200nM apamin reduced this to 413 ± 103pA (n=4). Furthermore, application of 4 µM ONOO- under these conditions stimulated outward current at +20mV from 1258 ± 331pA to 2907 ± 44pA (n=3) and 200nM apamin reduced this to 366 ± 32pA (n=3).

Given that cells were dialysed with pipette solution for approximately 10min prior to application of ONOO-, it is unlikely that SK3 activation was secondary to release of intracellular Ca2+. These data are consistent with a direct activation of the SK3 channel complex by peroxynitrite independent of changes in intracellular Ca2+.

Burnham M.P. et al., (2002). Br. J. Pharmacol. 135, 1133-1143.

This work was supported by the British Heart Foundation.