A dual-pipette technique that permits rapid internal dialysis and membrane potential measurement in voltage-clamped cardiomyocytes.
Guinea pig ventricular myocytes were voltage-clamped and dialysed using two glass patch pipettes (P1, P2) with tip openings of around 2 microns. A substantial improvement in the efficacy of dialysis from P2 was achieved by the application of positive pressure (15-30 cm H2O) to P2, and similar negative pressure to P1. Evidence of enhanced dialysis was obtained by measuring the effect on Ca channel current of P2 dialysates containing Ca, cAMP, GTP [gamma-S], trypsin, or the catalytic subunit of protein kinase A. Times to maximum response were 3-5 times shorter than those calculated or observed by others using a single-pipette method. The speeding-up was verified in comparative experiments with 100 microM GTP [gamma-S] dialysates; maximum stimulation of ICa occurred after 1.3-1.8 min with the dual-pipette method, versus 8.2 min with a single pipette. Other advantages of the dual-pipette method include the option of following a control dialysis from P1 with a test dialysis from P2, and the measurement of actual membrane potential. The disadvantages are that the rate of success is lower than with single-pipette experiments, and that smaller cardiomyocytes are difficult subjects.
Shuba
Y M
YM
II. Physiologisches Institut, Medizinische Fakultät, Universität des Saarlandes, Homburg/Saar, Federal Republic of Germany.
Hesslinger
B
B
Trautwein
W
W
McDonald
T F
TF
Pelzer
D
D
D000818:Animals; D003956:Dialysis / Q000295:instrumentation / Q000379:methods*; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods*; D006352:Heart Ventricles; D008564:Membrane Potentials; D009206:Myocardium / Q000166:cytology*