Effect of an N-terminus deletion on voltage-dependent gating of the ClC-2 chloride channel

• Published in: The Journal of physiology 2002-10, Vol.544 (2), p.363-372
• Main Authors: Varela, Diego, Niemeyer, María Isabel, Cid, L. Pablo, Sepúlveda, Francisco V.
• Format: Article
• Language: English
• Subjects: Animals; Cell Line; Chloride Channels - chemistry; Chloride Channels - physiology; Electrophysiology; Gene Deletion; Guinea Pigs; Humans; Ion Channel Gating - genetics; Ionophores; Kinetics; Nystatin; Original; Patch-Clamp Techniques; Rats
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2002.026096

ClC-2, a chloride channel widely expressed in mammalian tissues, is activated by hyperpolarisation and extracellular acidification. Deletion of amino acids 16–61 in rat ClC-2 abolishes voltage and pH dependence in two-electrode voltage-clamp experiments in amphibian oocytes. These results have been interpreted in terms of a ball-and-chain type of mechanism in which the N-terminus would behave as a ball that is removed from an inactivating site upon hyperpolarisation. We now report whole-cell patch-clamp measurements in mammalian cells showing hyperpolarization-activation of rClC-2Δ16–61 differing only in presenting faster opening and closing kinetics than rClC-2. The lack of time and voltage dependence observed previously was reproduced, however, in nystatin-perforated patch experiments. The behaviour of wild-type rClC-2 did not differ between conventional and nystatin-perforated patches. Similar results were obtained with ClC-2 from guinea-pig. One possible explanation of the results is that some diffusible component is able to lock the channel in an open state but does so only to the mutated channel. Alternative explanations involving the osmotic state of the cell and cytoskeleton structure are also considered. Low extracellular pH activates the wild-type channel but not rClC-2Δ16–61 when expressed in oocytes, a result that had been interpreted to suggest that protons affect the ball-and-chain mechanism. In our experiments no difference was seen in the effect of extracellular pH upon rClC-2 and rClC-2Δ16–61 in either recording configuration, suggesting that protons act independently from possible effects of the N-terminus on gating. Our observations of voltage-dependent gating of the N-terminal deleted ClC-2 are an argument against a ball-and-chain mechanism for this channel.