P2U purinergic receptor inhibits apical IsK/KvLQT1 channel via protein kinase C in vestibular dark cells.
Vestibular dark cells (VDC) are known to electrogenically secrete K+ via slowly activating K+ (IsK) channels, consisting of IsK regulatory and KvLQT1 channel subunits, and the associated short-circuit current (Isc) is inhibited by agonists of the apical P2U (P2Y2) receptor (J. Liu, K. Kozakura, and D. C. Marcus. Audit. Neurosci. 2: 331-340, 1995). Measurements of relative K+ flux (JK) with a self-referencing K(+)-selective probe demonstrated a decrease in JK after apical perfusion of 100 microM ATP. On-cell macropatch recordings from gerbil VDC showed a decrease of the IsK channel current (IIsK) by 83 +/- 7% during pipette perfusion of 10 microM ATP. The magnitude of the decrease of Isc 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 IIsK by 79 +/- 3% in perforated-patch whole cell recordings, whereas the inactive analog, 4 alpha-PMA, had no effect. In contrast, elevation of cytosolic Ca2+ concentration by A-23187 increased the whole cell IIsK. 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 IsK sequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the IsK channel of VDC in response to activation of the apical P2U 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.
Marcus
D C
DC
Biophysics Laboratory, Boys Town National Research Hospital, Omaha, Nebraska 68131, USA.
Sunose
H
H
Liu
J
J
Shen
Z
Z
Scofield
M A
MA
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000502:physiology; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004963:Estrenes / Q000494:pharmacology; D005849:Gerbillinae; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; D008301:Maleimides / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000502:physiology*; D058473:Receptors, Purinergic P2Y2; D016415:Sequence Alignment; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014722:Vestibule, Labyrinth / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*
D015220:Calcium Channels; D017931:DNA Primers; D004791:Enzyme Inhibitors; D004963:Estrenes; D007211:Indoles; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; C496911:Kcnq1 protein, rat; D008301:Maleimides; C546385:P2RY2 protein, human; C546387:P2ry2 protein, rat; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011760:Pyrrolidinones; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C059983:potassium channel protein I(sk); C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate