O2-sensitive K+ channels: role of the Kv1.2 -subunit in mediating the hypoxic response.
One of the early events in O2 chemoreception is inhibition of O2-sensitive K+ (KO2) channels. Characterization of the molecular composition of the native KO2 channels in chemosensitive cells is important to understand the mechanism(s) that couple O2 to the KO2 channels. The rat phaeochromocytoma PC12 clonal cell line expresses an O2-sensitive voltage-dependent K+ channel similar to that recorded in other chemosensitive cells. Here we examine the possibility that the Kv1.2 alpha-subunit comprises the KO2 channel in PC12 cells. Whole-cell voltage-clamp experiments showed that the KO2 current in PC12 cells is inhibited by charybdotoxin, a blocker of Kv1.2 channels. PC12 cells express the Kv1.2 alpha-subunit of K+ channels: Western blot analysis with affinity-purified anti-Kv1.2 antibody revealed a band at approximately 80 kDa. Specificity of this antibody was established in Western blot and immunohystochemical studies. Anti-Kv1.2 antibody selectively blocked Kv1.2 current expressed in the Xenopus oocyte, but had no effect on Kv2.1 current. Anti-Kv1.2 antibody dialysed through the patch pipette completely blocked the KO2 current, while the anti-Kv2.1 and irrelevant antibodies had no effect. The O2 sensitivity of recombinant Kv1.2 and Kv2.1 channels was studied in Xenopus oocytes. Hypoxia inhibited the Kv1.2 current only. These findings show that the KO2 channel in PC12 cells belongs to the Kv1 subfamily of K+ channels and that the Kv1.2 alpha-subunit is important in conferring O2 sensitivity to this channel.
Conforti
L
L
Department of Molecular Physiology, Institute of Molecular Pharmacology and Biophysics, College of Medicine, University of Cincinnati, Ohio, USA. laura.conforti@uc.edu
Bodi
I
I
Nisbet
J W
JW
Millhorn
D E
DE
D000818:Animals; D000918:Antibody Specificity; D015687:Cell Hypoxia / Q000502:physiology*; D018999:Charybdotoxin / Q000494:pharmacology; D051663:Kv1.2 Potassium Channel; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology*; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D018075:RNA, Complementary / Q000494:pharmacology; D051381:Rats; D014981:Xenopus
C496972:Kcna2 protein, rat; D051663:Kv1.2 Potassium Channel; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D018075:RNA, Complementary; D018999:Charybdotoxin; D010100:Oxygen