CA-2+ AND CA-2+-ACTIVATED CL- CURRENTS IN RABBIT ESOPHAGEAL SMOOTH-MUSCLE

1. An inward current carried by Ca2+ was recorded from single smooth muscle cells of rabbit oesophageal muscularis mucosae using a whole-cell gigaseal technique with physiological (2 mM) external calcium concentration ([Ca2+]o) in the presence of intracellular Cs+ ([Cs+]i 130 mm). Only one type of Ca2+ current could be identified. The threshold for its activation was approximately - 30 mV and maximum inward current (approximately 300 pA) was recorded at 0 mV. 2. This inward current was blocked by Co2+ (4 mm), Cd2+ (0.5 mm) and nifedipine (1 mum) and was enhanced by Bay K 8644 (5 mum). We therefore classify it as a L-type Ca2+ current and denote it I(Ca). 3. Steady-state inactivation data were well-fitted by a Boltzmann distribution, indicating that inactivation of the Ca2+ current is strongly modulated by membrane potential. However, the inactivation of I(Ca). slowed significantly and became less complete when BaCl2 replaced CaCl2 in the Tyrode solution suggesting that the inactivation of Ic. may also be dependent on [Ca2+]i. The steady-state activation and inactivation curves for I(Ca) overlap between - 40 and 0 mV indicating that there may be a Ca2+ window current in this range of potentials. 4. When EGTA was omitted from the pipette-filling solution, depolarizations positive to - 10 mV resulted in a transient as opposed to a maintained inward Ca2+ current which was followed by a relatively large outward current. Under these conditions, slowly decaying inward tail currents were also recorded upon repolarization to the holding potential, - 60 mV. However, when EGTA was omitted from the pipette, marked 'run-down' of the Ca2+ current occurred within 10 min after starting the whole-cell recording. 5. This run-down of I(Ca) was reduced significantly when the nystatin perforated patch technique was used. Under these conditions stable I(Ca) records could be obtained for over 1 h. Outward currents and slow decaying inward tail currents similar to those recorded with no EGTA in the pipette were also obtained consistently using the nystatin recording technique. 6. In nystatin perforated patch recordings, CoCl2 (2 mm) completely abolished the Ca2+ current, the outward currents and the slow inward tails. These findings suggest that the outward currents and slow inward tails are activated by a transmembrane influx of Ca2+. 7. Ion replacement and pharmacological tests provided evidence that both the outward current's and the slow inward tails are due to Ca2+-activated Cl- current (I(Cl(Ca))). Thus, (i) the reversal potential of the tail currents was dependent on the electrochemical gradient for Cl-, and (ii) the current onsets and tails were significantly reduced by niflumic acid (10 muM) and anthracene-9-carboxylic acid (9-AC; 300 muM). 4.4'-Diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamindo-4-iso-thiocyanostilbene-2,2'-disulphonic acid (SITS) also partially blocked these current changes. 8. Our results provide the first information on the biophysical properties of I(Ca) and I(Cl(Ca)) in oesophageal smooth muscle. The data obtained using the nystatin technique demonstrate that I(Ca) is physiologically important both as a source of electrogenic current and as an activator of other Ca2+-dependent current systems, e.g, I(Cl(Ca)).