The Ca²⁺-inactivated Cl⁻ Channel at Work: Selectivity, Blocker Kinetics and Transport Visualization

Abstract.

Removal of extracellular divalent cations activated a Cl⁻ channel in the plasma membrane of Xenopus laevis oocytes. This so-called Ca²⁺-inactivated Cl⁻ channel (CaIC) was present in every oocyte and was investigated using two-electrode whole-cell voltage clamp and single-channel patch-clamp techniques. Beside other Cl⁻ channel inhibitors, anthracene-9-carboxylic acid (9-AC) and 3′azido-3′deoxythymidine (AZT), a nucleoside analogue commonly used as an antiviral drug, blocked at least partly the CalC-mediated currents.

Using the Cl⁻-sensitive dye 6-methoxy-N-(sulfopropyl)quinolinium (SPQ) we could visualize the transport of Cl⁻ from the oocyte cytoplasm to the surrounding medium after activation of the CaIC by Ca²⁺ removal. In the absence of external Cl⁻ and Ca²⁺, the emission intensity of SPQ declined continuously, indicating a quenching of fluorescence by the efflux of Cl⁻ in the millimolar range. In the presence of external Ca²⁺, no emission changes could be observed during the same time period. Chelating external Ca²⁺ in absence of Cl⁻ immediately activated Ca²⁺-inactivated Cl⁻ channels leading to subsequent emission decrease of SPQ.

Investigations on the selectivity of the CaIC revealed only poor discrimination between different anions. With single-channel measurements, we found an anion selectivity sequence I⁻ > Br⁻ > Cl⁻≫ gluconate as it is also typical for maxi Cl⁻ channels.

Contrary to the majority of all other transport systems of the Xenopus oocyte, which show reduced activity due to membrane depolarization or endocytotic removal of the transport protein from the plasma membrane during oocyte maturation, the CaIC remained active in maturated oocytes. Single-channel measurements on maturated oocytes, also known as eggs, showed the presence of Ca²⁺-inactivated Cl⁻ channels. However, this egg CaIC revealed an altered sensitivity to external Ca²⁺ concentrations.

All these data confirm and extend our previous observations on the CaIC and give clear evidence that this channel is peculiar among all Cl⁻ channels described up to now.