Protein kinase C suppresses spontaneous, transient, outwards K+ currents through modulation of the Na/Ca exchanger in guinea-pig gastric myocytes

The effect of protein kinase C (PKC) on the Ca2+-activated K+ current (I-k,I-Ca) in guinea-pig gastric myocytes was studied using the whole-cell voltage-clamp technique. At a holding potential of 0 mV, I-K,I-Ca, recorded as spontaneous, transient, outwards currents (STOCs), was markedly inhibited, both in mean amplitude (54+/-54%) and frequency (60+/-8%) by 1 muM phorbol 12, 13 dibutyrate (PDBu, n=6). These effects were antagonized by pretreatment with 10 nM bisindolylmaleimide I (n=5), a selective inhibitor of PKC. The possibility that the inhibition of STOCs was due to direct channel inhibition by PKC was addressed using inside-out or open-cell-attached patch-clamp techniques, the latter established using beta -escin. PDBu did not alter the conductance or open probability of the K-Ca channel in any mode, suggesting that PKC does not inhibit the K-Ca channel directly. To study the involvement of the Na/Ca exchanger in the inhibition of STOCs by PDBu, its operation was prevented by replacing Na+ in the internal solution by tris(hydroxymethyl)ami nomethane (TRIS) and external Na+ by equimolar K+ and Ca2+-activated inwards K+ currents recorded at a holding potential of 0 mV. Neither the mean amplitude (96+/-8%) nor the frequency of these currents was inhibited significantly by 1 muM PDBu (n=5). Like PDBu, 5 muM 2-{2-[4-(4-nitrobenzyloxy)phenyl]ethyl}isothiourea methanesulphonate (KB-R7943), a selective inhibitor of the reverse mode Na/Ca exchanger, also inhibited the mean amplitude (45+/-64%) and frequency (26+/-2%) of STOCs at the holding potential of 0 mV (n=6). The results suggest that the suppression of STOCs by PKC is mediated by inhibition of the Na/Ca exchanger.