P-2U PURINERGIC RECEPTOR INHIBITS APICAL I-SK KVLQT1 CHANNEL VIA PROTEIN-KINASE-C IN VESTIBULAR DARK CELLS/

Vestibular dark cells (VDC) are known to electrogenically secrete K+ v ia 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 (P2Y(2)) receptor (J. Liu, K. Kozakura, and D. C. Marcus. Audit. Neurosci. 2: 331-340, 1 995). Measurements of relative K+ flux (J(K)) with a self-referencing K+-selective probe demonstrated a decrease in J(K) after apical perfus ion of 100 mu M ATP. On-cell macropatch recordings from gerbil VDC sho wed a decrease of the I-sK channel current (I-IsK) by 83 +/- 7% during pipette perfusion of 10 mu M ATP. The magnitude of the decrease of I- sc by ATP was diminished in the presence of inhibitors of phospholipas e C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activatio n of PKC by phorbol 12-myristate 13-acetate (PMA, 20 nM) decreased I-I sK by 79 +/- 3% in perforated-patch whole cell recordings, whereas the inactive analog, 4 alpha-PMA, had no effect. In contrast, elevation o f cytosolic Ca2+ concentration by A-23187 increased the whole cell I-I sK. The expression of the isk gene transcript was conformed, and the s erine responsible for the species-specific response to PKC was found t o 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 o n the I-sK channel of VDC in response to activation of the apical P-2U receptor and predict that the secretion of endolymph in the human ves tibular system may be controlled by PKC in the same way as in our anim al model.