Glass-funnel technique for the recording of membrane currents and intracellular perfusion of Xenopus oocytes.
In this report we present a description of a modified version of the "glass-funnel" technique for the recording of membrane currents and intracellular perfusion of Xenopus laevis oocytes. The technique is based on the ability of the devitellinated oocyte to form a high-resistance seal with the glass, permitting separation of the oocyte into two, i.e., extra- and intracellular, compartments. The technique is fairly simple to use, provides a much higher clamp speed compared to the double-microelectrode voltage-clamp technique, and allows effective control of the composition of the intracellular milieu. To elucidate the performance of the technique with respect to various membrane currents we present data relating to the recording of Ca-channel currents expressed in X. laevis oocytes by means of mRNA extracted from the rat cerebellum and heart, as well as currents induced by cRNA for the skeletal muscle micro1 Na+ channel and the dog heart NCX1 Na+-Ca2+ exchanger. Due to effective elimination of intra- and extracellular Cl- it became possible to measure not only Ba2+ but also Ca2+ current through the expressed Ca channels, and to record the activity of the Na+-Ca2+ exchanger following dialysis of the oocyte with high-Ca2+ intracellular solutions. Corresponding currents showed properties identical to those obtained with other techniques, suggesting the adequacy of the glass-funnel technique for critical analysis of membrane ionic currents in Xenopus oocytes.
Shuba
Y M
YM
Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington DC 20007, USA.
Naidenov
V G
VG
Morad
M
M
D000818:Animals; D017920:Antiporters / Q000096:biosynthesis; D015220:Calcium Channels / Q000378:metabolism / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D002531:Cerebellum / Q000378:metabolism; D004285:Dogs; D008564:Membrane Potentials / Q000502:physiology; D017551:Microdialysis; D008839:Microelectrodes; D009132:Muscles / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000295:instrumentation*; D018075:RNA, Complementary / Q000096:biosynthesis / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D015222:Sodium Channels / Q000378:metabolism / Q000502:physiology; D019831:Sodium-Calcium Exchanger; D014982:Xenopus laevis
D017920:Antiporters; D015220:Calcium Channels; D018075:RNA, Complementary; D012333:RNA, Messenger; D015222:Sodium Channels; D019831:Sodium-Calcium Exchanger