Effect of an N-terminus deletion on voltage-dependent gating of the ClC-2 chloride channel.
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-2Delta16-61 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-2Delta16-61 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-2Delta16-61 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.
Varela
Diego
D
Centro de Estudios Científicos (CECS), Avenida Arturo Prat 514, Casilia 1469, Valdivia, Chile.
Niemeyer
María Isabel
MI
Cid
L Pablo
LP
Sepúlveda
Francisco V
FV
D000818:Animals; D000090007:CLC-2 Chloride Channels; D002460:Cell Line; D018118:Chloride Channels / Q000737:chemistry* / Q000502:physiology*; D004594:Electrophysiology; D017353:Gene Deletion; D006168:Guinea Pigs; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics*; D007476:Ionophores; D007700:Kinetics; D009761:Nystatin; D018408:Patch-Clamp Techniques; D051381:Rats
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D007476:Ionophores; D009761:Nystatin