ABSTRACT
Activation of KCNQ potassium channels by stimulation of co-expressed dopamine D(2) receptors was studied electrophysiologically in Xenopus laevis oocytes and in mammalian cells. To address the specificity of the interaction between D(2)-like receptors and KCNQ channels, combinations of KCNQ1-5 channels and D(2)-like receptors (D(2L), D(3), and D(4)) were investigated in Xenopus oocytes. Activation of either receptor with the selective D(2)-like receptor agonist quinpirole (100 nM) stimulated all the KCNQ currents, independently of the subunit combination, indicating a common pathway of receptor-channel interaction. The KCNQ4 current was investigated in further detail and was increased by 19.9+/-1.6% ( n=20) by D(2L) receptor stimulation. The effect could be mimicked by injection of GTPgammaS and prevented by injection of Bordetella pertussis toxin, indicating that channel stimulation was mediated via a G protein of the G(alphai/o) subtype. Cells of the human neuroblastoma line SH-SY5Y were co-transfected transiently with KCNQ4 and D(2L) receptors. Stimulation of D(2L) receptors increased the KCNQ4 current ( n=6) as determined in whole-cell patch-clamp recordings. The specificity of the dopaminergic activation of the KCNQ channels was confirmed by co-expression of other neuronal K(+) channels (BK, K(V)1.1, and K(V)4.3) with the D(2L) receptor in Xenopus oocytes. None of these K(+) channels responded to stimulation of the D(2L) receptor. In the mammalian brain, dopamine D(2) receptors and KCNQ channels co-localise postsynaptically in several brain regions, so modulation of neuronal excitability by dopamine release could in part be mediated via an effect on KCNQ channels.
Subject(s)
Potassium Channels/metabolism , Receptors, Dopamine D2/metabolism , Animals , Biotransformation/drug effects , Brain Neoplasms/metabolism , Cell Line, Tumor , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Dopamine Agonists/pharmacology , Electrophysiology , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Humans , In Vitro Techniques , Membrane Potentials/physiology , Neuroblastoma/metabolism , Neurons/drug effects , Neurons/metabolism , Oocytes/metabolism , Patch-Clamp Techniques , Pertussis Toxin/pharmacology , Potassium Channels/genetics , Receptors, Dopamine D2/genetics , Transfection , Xenopus laevisABSTRACT
Planar silicon chips with 1-2-microm etched holes (average resistance: 2.04 +/- 0.02 MOmega in physiological buffer, n = 274) have been developed for patch-clamp recordings of whole-cell currents from cells in suspension. An automated 16-channel parallel screening system, QPatch 16, has been developed using this technology. A single-channel prototype of the QPatch system was used for validation of the patch-clamp chip technology. We present here data on the quality of patch-clamp recordings and from actual drug screening studies of human potassium channels expressed in cultured cell lines. Using Chinese hamster ovary (CHO) and human embryonic kidney cells (HEK), gigaseals of 4.1 +/- 0.4 GOmega (n = 146) and high-quality whole-cell current recordings were obtained from hERG and KCNQ4 potassium channels. Success rates for gigaseal recordings varied from 40 to 95%, and 67% of the whole-cell configurations lasted for >20 min. Cells were maintained in suspension up to 4 h in a cell storage facility that is integrated in the QPatch 16. No decline in patchability was observed during this time course. A series of screens was conducted with known inhibitors of the hERG and KCNQ4 potassium channels. Dose-response relationship characterizations of verapamil and rBeKm-1 blockage of hERG currents provided IC(50) values similar to values reported in the literature.
Subject(s)
Cell Culture Techniques/instrumentation , Drug Evaluation, Preclinical/instrumentation , Membrane Potentials/physiology , Patch-Clamp Techniques/instrumentation , Potassium Channels/physiology , Robotics/instrumentation , Animals , Biotechnology/instrumentation , Biotechnology/methods , CHO Cells , Cell Culture Techniques/methods , Cells, Cultured , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , Electrophysiology/instrumentation , Electrophysiology/methods , Equipment Design , Equipment Failure Analysis , Feasibility Studies , Humans , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Kidney/drug effects , Kidney/physiology , Membrane Potentials/drug effects , Microelectrodes , Patch-Clamp Techniques/methods , Potassium Channel Blockers/pharmacology , Potassium Channels/drug effects , Reproducibility of Results , Robotics/methods , Sensitivity and SpecificityABSTRACT
KCNE4 is a membrane protein belonging to a family of single transmembrane domain proteins known to have dramatic effect on the gating of certain potassium channels. However, no functional role of KCNE4 has been suggested so far. In the present paper we demonstrate that KCNE4 is an inhibitory subunit to KCNQ1 channels. Co-expression of KCNQ1 and KCNE4 in Xenopus oocytes completely inhibited the KCNQ1 current. This was reproduced in mammalian CHO-K1 cells. Experiments with delayed expression of mRNA coding for KCNE4 in KCNQ1-expressing oocytes suggested that KCNE4 exerts its effect on KCNQ1 channels already expressed in the plasma membrane. This notion was supported by immunocytochemical studies and Western blotting, showing no significant difference in plasma membrane expression of KCNQ1 channels in the presence or absence of KCNE4. The impact of KCNE4 on KCNQ1 was specific since no effect of KCNE4 could be detected if co-expressed with KCNQ2-5 channels or hERG1 channels. RT-PCR studies revealed high KCNE4 expression in embryos and adult uterus, where significant expression of KCNQ1 channels has also been demonstrated.
