TEMPERATURE-DEPENDENCE OF FAST AND SLOW GATING RELAXATIONS OF CLC-O CHLORIDE CHANNELS

The chloride channel from the Torpedo electric organ, ClC-0, is the be st studied member of a large gene-family (Jentsch, T.J. 1996. Gum: Opi n. Neurobiol. 6:303-310.). We investigate the temperature dependence o f both the voltage- and chloride-dependent fast gate and of the slow g ate of the ''double-barreled'' ClC-0 expressed in Xenopus oocytes. Kin etics of the fast gate exhibit only a moderate temperature dependence with a Q(10) of 2.2. Steady-state p(open) of the fast gate is relative ly independent of temperature. The slow gate, in contrast, is highly t emperature sensitive. Deactivation kinetics at positive voltages are a ssociated with a Q(10) of similar to 40. Steady-state open probability of the slow gate (p(open)(slow) (V)) can be described by a Boltzmann distribution with an apparent gating valence of approximate to 2 and a variable ''offset'' at positive voltages. We note a positive correlat ion of this offset (i.e., the fraction of channels that are not closed by the slow gate) with the amount of expression. This offset is also highly temperature sensitive, being drastically decreased at high temp eratures. Paradoxically, the maximum degree of activation of the slow gate also decreases at higher temperatures. The strong temperature dep endence of the slow gate was also observed at the single channel level in inside-out patches. The results imply that within a Markovian-type description at least two open and two closed states are needed to des cribe slow gating. The strong temperature dependence of the slow gate explains the phenotype of several ClC-0 point-mutants described recent ly by Ludewig et al. (Ludewig, U., T.J. Jentsch, and M. Pusch. 1996. J . Physiol. (Lend.). In press). The large Q(10) of slow gating kinetics points to a complex rearrangement. This, together with the correlatio n of the fraction of noninactivating channels with the amount of expre ssion and the fact that the slow gate closes both protochannels simult aneously suggests that the slow gate is coupled to subunit interaction of the multimeric ClC-0 channel.