Activation of volume-regulated Cl- channels by ACh and ATP in Xenopus follicles

1. Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocyt es (follicles) were studied using electrophysiological techniques. Whole fo llicle currents were monitored using a two-electrode voltage clamp and sing le-channel activity was measured using the patch-clamp technique. 2. In follicles held at -60 mV two chloride currents were activated in exte rnal hyposmotic solutions. One was the habitual volume-regulated current el icited by external hyposmolarity (I-Cl,I-swell), and the second was a slow and smooth current (S-in) generated by BCh or ATP application. 3. In follicles, the permeability ratios for different anions with respect to Cl- were similar for both I-Cl,I-swell and S-in, with a sequence of: SCN - > I- > Br- greater than or equal to NO3- greater than or equal to Cl- > g luconate greater than or equal to cyclamate > acetate > SO42-. 4. Extracell ular ATP blocked the outward component of S-in. Also, extracellular pH modu lated the inactivation kinetics of S-in elicited by ACh; e.g. inactivation at +80 mV was similar to 100% slower at pH 8.0 compared with that at pH 6.0 . 5. Lanthanides inhibited I-Cl,I-swell and S-in. La3+ completely inhibited I-Cl,I-swell with a half-maximal inhibitory concentration (IC50) of 17 +/- 1.9 mu M, while S-in was blocked up to 55% with an apparent IC50 of 38 +/- 2.6 mu M. 6. Patch-clamp recordings in follicular cells showed that hyposmotic challe nge opened inward single-channel currents. The single channel conductance ( 4.7 +/- 0.4 pS) had a linear current-voltage relationship with a reversal m embrane potential close to -20 mV. This single-channel activity was increas ed by application of ACh or ATP. 7. The I-Cl,I-swell generation was not affected by pirenzepine or metoctram ine, and did not affect the purinergic activation of the chloride current n amed F-in. Thus; I-Cl,I-swell was not generated via neurotransmitters relea sed during cellular swelling. 8. All together, equal discrimination for different anions, similar modulat ory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity, strongly suggest that I-Cl,I-swell and S- in currents depend on the opening of the same type or a closely related cla ss of volume-regulated chloride channels.