Protein kinase C mediates P2U purinergic receptor inhibition of K+ channel in apical membrane of strial marginal cells

Abstract Strial marginal cells (SMC) electrogenically secrete K + via slowly activating K + (I sK ) channels, consisting of I sK regulatory and KvLQT1 channel subunits, and the associated short circuit current ( I sc ) is inhibited by agonists of the apical P 2U receptor [Liu et al., Audit. Neurosci. 2 (1995) 331–340]. Measurements of relative K + flux (J K ) with a self-referencing K + -selective probe demonstrated a decrease in J K after apical perfusion of 100 μM ATP. On-cell macro patch recordings from the apical membrane of gerbil SMC showed a decrease of the I sK channel current ( I IsK ) by 88±8% during pipette perfusion of 100 μM ATP. The magnitude of the decrease of I sc by ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (20 nM) decreased I IsK (gerbil: by 62±10%; rat: by 72±6%) in perforated-patch whole-cell recordings while the inactive analog, 4αPMA, had no effect. By contrast, elevation of cytosolic [Ca 2+ ] by A23187 increased the whole-cell I IsK . The expression of the isk gene transcript was confirmed and the serine responsible for the species-specific response to PKC was found to be present in the gerbil I sK sequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the I sK channel of SMC in response to activation of the apical P 2U receptor and predict that the secretion of endolymph in the human cochlea may be controlled by PKC in the same way as in our animal model.