Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, PC180

Poster Communications

Voltage-dependent gating currents from the CLC-5 Cl-/H+ exchanger

J. D. Lippiat1, A. J. Smith1

1. Institute of Membrane & Systems Biology, University of Leeds, Leeds, United Kingdom.

  • Figure 1: Whole-cell currents recorded from HEK293 cells expressing wild-type (A) and E268A (B) CLC-5.

Much of our understanding of the CLC family has come from functional studies of the Torpedo CLC-0, a voltage-gated Cl- channel, and from structural studies of prokaryotic CLC-ec1, a voltage-independent Cl-/H+ exchanger. Mammalian CLC-5, however, exhibits voltage-dependent activation and Cl-/H+ exchange. We carried out high resolution whole-cell patch clamp recordings of wild-type and mutant CLC-5 expressed in HEK293 cells, as described in Smith et al. (2009). Outward currents, corresponding to Cl- into and H+ out from the cytosol, were activated by positive potentials (Fig. 1A). Upon repolarisation, brief (< 1ms) inward tail currents were observed, which according to the apparent reversal potential did not relate to the movement of permeating ions. As seen previously (Zdebik et al., 2008), neutralisation of an internal proton acceptor (E268A) resulted in ablated ionic flux across the membrane. With this mutant we observed non-linear capacity transient currents lasting approximately 1 ms (Fig. 1B), which had charge-movement kinetics (V½ = 94.0 ± 0.8 mV, ze = 1.3 ± 0.03; mean ± s.e.m. of recordings from 5 cells) that correlated with the voltage-dependent activation of wild-type CLC-5 (V½ = 108.6 ± 2.0 mV, ze = 1.4 ± 0.05, n = 5). The brief tail current that were observed with wild-type CLC-5 appeared to relate to gating rather than permeation and had similar charge-movement kinetics. We found that the properties of the E268A transient currents were similar when extracellular Cl- was replaced by Br- (ze = 1.3 ± 0.10, n = 3), an anion that also permeates wild-type CLC-5. In contrast, the voltage-dependence was reduced in the presence of the impermeant anions aspartate (ze = 0.9 ± 0.05, n = 3) and methanesulphonate (ze = 0.9 ± 0.08, n = 3). Furthermore, similar gating transients were recorded from cells expressing wild-type CLC-5 in the presence of the impermeant anions. The higher gating charge (ze) observed with permeant anions suggest that these interact with or modify an intrinsic voltage-sensor with a ze of 0.9 and may provide the additional charge which moves through a fraction of the voltage field (δ ≈ 0.4). The impermeant anions are unlikely to contribute to the gating charge since it was similar with extracellular divalent SO42- ions (ze = 0.9 ± 0.04, n = 3) and persisted in near-complete removal of ions. Our data suggest that the voltage-sensor in CLC-5 may be formed by an intrinsic protein domain in combination with permeating anions. As demonstrated by the lack of sustained Cl-/H+ current with the E268A mutant, “gating” of the pore also requires H+ likely to be delivered from the intracellular E268 proton acceptor.

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