P2U purinergic receptor inhibits apical IsK/KvLQT1 channel via protein kinase C in vestibular dark cells

• Published in: American Journal of Physiology: Cell Physiology 1997-12, Vol.273 (6), p.C2022
• Main Authors: Marcus, Daniel C, Sunose, Hiroshi, Liu, Jianzhong, Shen, Zhijun, Scofield, Margaret A
• Format: Article
• Language: English
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.1997.273.6.c2022

1  Biophysics Laboratory, Boys Town National Research Hospital, Omaha 68131; and 2  Molecular Pharmacology Laboratory, Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska 68178 Vestibular dark cells (VDC) are known to 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 (P2Y 2 ) receptor (J. Liu, K. Kozakura, and D. C. Marcus. Audit. Neurosci. 2: 331-340, 1995). 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 macropatch recordings from gerbil VDC showed a decrease of the I sK channel current ( I IsK ) by 83 ± 7% during pipette perfusion of 10 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 (PMA, 20 nM) decreased I IsK by 79 ± 3% in perforated-patch whole cell recordings, whereas the inactive analog, 4 -PMA, had no effect. In contrast, elevation of cytosolic Ca 2+ concentration by A-23187 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 VDC in response to activation of the apical P 2U receptor and predict that the secretion of endolymph in the human vestibular system may be controlled by PKC in the same way as in our animal model. P2Y 2 receptor; phospholipase C; perforated-patch whole cell voltage clamp; minK channel; gerbil; self-referencing probe; slowly activating potassium channel