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

Strial marginal cells (SMC) electrogemically 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 mu M 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 mu M 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 (grebil: by 62 +/- 10%; rat: by 72 +/- 6%) in perforated-patch whole-cell recordings while the inactive analog, 4 alpha PMA, had no effect. By contrast, elevation of cytosolic [Ca2+] 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. (C) 1998 Elsevier Science B.V.