Cloning and functional expression of rKCNQ2 K(+) channel from rat brain.
By homologue cloning, we have isolated a cDNA encoding a voltage-gated K(+) channel, rKCNQ2, from a rat brain cDNA library using RACE. The open reading frame of the translated protein comprises 852 amino acids with 6 transmembrane segments and a pore motif between S5 and S6. rKCNQ2 shares 96% amino acid identity with human KCNQ2 in which mutations cause a form of epilepsy known as benign familial neonatal convulsions (BFNC). Northern blotting with a rKCNQ2-specific probe revealed a robust single band of 8.6-kb transcript expressed in brain not in other tissues. Functional expression of rKCNQ2 in an HEK 293 cell line by whole-cell current recording and in Xenopus oocytes by two-electrode voltage clamp showed outward K(+) selective currents that displayed delayed rectifier-type kinetics. The G-V curve, fitted with a Boltzmann function, showed voltage dependence of activation with a threshold of activation approximately -60 mV. The rKCNQ2 currents were sensitive to TEA block with a Ki of 0.1 mM. In addition, rKCNQ2 currents were down-regulated upon exposure of cells to either a broad-spectrum tyrosine kinase inhibitor genistein or a Src-like tyrosine kinase inhibitor herbimycin A. Our findings add a rodent member to the KCNQ channel subfamily, providing new information of the channel modulation, and will facilitate generation of rodent models of epilepsy.
Jow
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F
Department of Neuroscience, Wyeth-Ayerst Research, CN 8000, 08543, Princeton, NJ, USA. wangk@war.wyeth.com
Wang
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K
D000818:Animals; D016227:Benzoquinones; D001923:Brain Chemistry / Q000502:physiology*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004827:Epilepsy / Q000235:genetics / Q000503:physiopathology; D015870:Gene Expression / Q000502:physiology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D051658:KCNQ2 Potassium Channel; D007668:Kidney / Q000166:cytology; D047029:Lactams, Macrocyclic; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000235:genetics; D024642:Potassium Channels, Voltage-Gated; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017828:Rifabutin / Q000031:analogs & derivatives; D017386:Sequence Homology, Amino Acid; D019789:Tetraethylammonium / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism; D014981:Xenopus
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D051658:KCNQ2 Potassium Channel; C496914:KCNQ2 protein, human; C496915:Kcnq2 protein, rat; D047029:Lactams, Macrocyclic; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011809:Quinones; D012333:RNA, Messenger; D017828:Rifabutin; D014443:Tyrosine; D019789:Tetraethylammonium; C020754:herbimycin; D019833:Genistein; D011505:Protein-Tyrosine Kinases