Injection of a K+ channel (Kv1.3) cRNA in fertilized eggs leads to functional expression in cultured myotomal muscle cells from Xenopus embryos.
The synthetic cRNA encoding for the major T lymphocyte K+ channel (Kv1.3) was injected into Xenopus fertilized eggs. Somites from embryos of stage 20-22 (about 40 h post-fertilization at 19 degrees C) were dissociated and myotomal muscle cells were cultured in vitro for 2 days. The whole cell configuration of the tight seal patch-clamp technique was used to record K+ channel activity in cultured myocytes. These myocytes have two endogenous delayed-rectifiers (sustained and transient) and an inward-rectifier K+ currents, all of which are insensitive to the scorpion toxin charybdotoxin. Cultured myocytes dissociated from embryos injected with the Kv1.3 cRNA expressed the exogenous Kv1.3 channel. The Kv1.3 channel was identified by its physiological (a very low recovery from inactivation) and its pharmacological properties (a high sensitivity to charybdotoxin). This work demonstrates that Xenopus cultured myotomal muscle cells represent a very efficient and practical assay system for the functional expression of cloned ion channels.
Honoré
E
E
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UPS 411, Valbonne, France.
Guillemare
E
E
Lesage
F
F
Barhanin
J
J
Lazdunski
M
M
D000818:Animals; D002478:Cells, Cultured; D018999:Charybdotoxin; D004553:Electric Conductivity; D015870:Gene Expression; D018014:Gene Transfer Techniques; D008845:Microinjections; D009132:Muscles / Q000196:embryology* / Q000378:metabolism*; D015221:Potassium Channels / Q000235:genetics* / Q000502:physiology; D018075:RNA, Complementary / Q000235:genetics*; D012604:Scorpion Venoms / Q000494:pharmacology; D014982:Xenopus laevis / Q000196:embryology
D015221:Potassium Channels; D018075:RNA, Complementary; D012604:Scorpion Venoms; D018999:Charybdotoxin