Proton Sensing of CLC-0 Mutant E166D

Sonia Traverso,Rita Aiello,Giovanni Zifarelli,Michael Pusch

doi:10.1085/jgp.200509340

Abstract

CLC Cl− channels are homodimers in which each subunit has a proper pore and a (fast) gate. An additional slow gate acts on both pores. A conserved glutamate (E166 in CLC-0) is a major determinant of gating in CLC-0 and is crucially involved in Cl−/H+ antiport of CLC-ec1, a CLC of known structure. We constructed tandem dimers with one wild-type (WT) and one mutant subunit (E166A or E166D) to show that these mutations of E166 specifically alter the fast gate of the pore to which they belong without effect on the fast gate of the neighboring pore. In addition both mutations activate the common slow gate. E166A pores have a large, voltage-independent open probability of the fast gate (popen), whereas popen of E166D pores is dramatically reduced. Similar to WT, popen of E166D was increased by lowering pHint. At negative voltages, E166D presents a persistent inward current that is blocked by p-chlorophenoxy-acetic acid (CPA) and increased at low pHext. The pHext dependence of the persistent current is analogous to a similar steady inward current in WT CLC-0. Surprisingly, however, the underlying unitary conductance of the persistent current in E166D is about an order of magnitude smaller than that of the transient deactivating inward Cl− current. Collectively, our data support the possibility that the mutated CLC-0 channel E166D can assume two distinct open states. Voltage-independent protonation of D166 from the outside favors a low conductance state, whereas protonation from the inside favors the high conductance state.

Highlights

CLC proteins are a multigene family present in all phyla (Jentsch et al, 2005)
Despite the small difference between these two amino acids the kinetics of the WT and of the mutant are very different (Traverso et al, 2003). We show that both mutants, E166A and E166D, have a drastically altered protopore gate
Mutating E166 in CLC-0 to alanine leads to an almost constitutively open channel (Dutzler et al, 2003; Traverso et al, 2003), whereas E166D slows down opening at positive potentials and increases the rate of closure at negative potentials (Traverso et al, 2003)

Summary

Introduction

CLC proteins are a multigene family present in all phyla (Jentsch et al, 2005). The founding member, called CLC-0, was identified as a voltage-dependent ClϪ channel in the electric organ of Torpedo californica (White and Miller, 1979; Miller and White, 1980). From the recent X-ray structure of bacterial CLCs it is clear that CLC proteins contain two identical subunits in which each subunit presents an independent pathway for ClϪ ions (Dutzler et al, 2002, 2003). Such a “double-barreled” architecture had been conjectured by Miller and colleagues early on from the single channel behavior of the Torpedo channel (Miller and White, 1984) and it was predicted from later mutagenesis work and a lower resolution structure (Ludewig et al, 1996; Middleton et al, 1996; Mindell et al, 2001).

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Conclusion