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

Alberto L. Pérez-Samartín, Ricardo Miledi*† and Rogelio O. Arellano†

1. Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique.

2. In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (I_Cl,swell), and the second was a slow and smooth current (S_in) generated by ACh or ATP application.

3. In follicles, the permeability ratios for different anions with respect to Cl^- were similar for both I_Cl,swell and S_in, with a sequence of: SCN^- > I^- > Br^- NO₃^- Cl^- > gluconate cyclamate > acetate > SO₄^2-.

4. Extracellular ATP blocked the outward component of S_in. Also, extracellular pH modulated the inactivation kinetics of S_in elicited by ACh; e.g. inactivation at +80 mV was ~100 % slower at pH 8·0 compared with that at pH 6·0.

5. Lanthanides inhibited I_Cl,swell and S_in. La³⁺ completely inhibited I_Cl,swell with a half-maximal inhibitory concentration (IC₅₀) of 17 ± 1·9 µM, while S_in was blocked up to 55 % with an apparent IC₅₀ of 36 ± 2·6 µM.

6. Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4·7 ± 0·4 pS) had a linear current-voltage relationship with a reversal membrane potential close to -20 mV. This single-channel activity was increased by application of ACh or ATP.

7. The I_Cl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named F_in. Thus, I_Cl,swell was not generated via neurotransmitters released during cellular swelling.

8. All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La³⁺, and the same single-channel activity, strongly suggest that I_Cl,swell and S_in currents depend on the opening of the same type or a closely related class of volume-regulated chloride channels.