Induction of endogenous channels by high levels of heterologous membrane proteins in Xenopus oocytes.
Xenopus oocytes are widely employed for heterologous expression of cloned proteins, particularly electrogenic molecules such as ion channels and transporters. The high levels of expression readily obtained permit detailed investigations without interference from endogenous conductances. Injection of min K mRNA into Xenopus oocytes results in expression of voltage-dependent potassium-selective channels. Recent data show that injections of high concentrations of min K mRNA also induce a chloride current with very different biophysical, pharmacological, and regulatory properties from the min K potassium current. This led to the suggestion that the min K protein acts as an inducer of endogenous, normally silent oocyte ion channels. We now report that high levels of heterologous expression of many membrane proteins in Xenopus oocytes specifically induce this chloride current and a hyperpolarization-activated cation-selective current. The current is blocked by 4,4'-diisothiocyanostilbene-2-2'-disulphonic acid and tetraethylammonium, enhanced by clofilium, and is pH-sensitive. Criteria are presented that distinguish this endogenous current from those due to heterologous expression of electrogenic proteins in Xenopus oocytes. Together with structure-function studies, these results support the hypothesis that the min K protein comprises a potassium-selective channel.
Vollum Institute, Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland 97201, USA.
T
T
Tzounopoulos
J
J
Maylie
J P
JP
Adelman
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000096:biosynthesis*; D009865:Oocytes / Q000502:physiology*; D015221:Potassium Channels / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014981:Xenopus
D018118:Chloride Channels; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D015221:Potassium Channels; D011994:Recombinant Proteins; C018564:dihydro-DIDS; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium