Electro-Steric Mechanism of CLC-2 Chloride Channel Activation

Two-pore voltage-gated CLC chloride channels control neuronal and muscle excitability. They share a dimeric structure but their activation mechanism remains unresolved. Here we determine the step-by-step activation mechanism of the broadly expressed CLC-2 channel using homology modelling, molecular dynamic simulations and functional studies. We establish that a two-leaf gate formed by Tyr561-HO-Glu213 flanked by Lys568/Glu174 and Lys212 closes the canonical pore. Activation begins when a hyperpolarization-propelled intracellular chloride occupies the pore and splits Tyr561-HO-Glu213 by electrostatic/steric repulsion. Unrestrained Glu213 rotates outwardly to bind Lys212 but the pore remains closed. Protonation breaks the Glu213-Lys212 interaction while another chloride occupies the pore thus catalysing chloride exit via Lys212. Also, we found that the canonical pore is uncoupled from a cytosolic cavity by a Tyr561-containing hydrophobic gate that prevents Glu213 protonation by intracellular protons. Our data provide atomistic details about CLC-2 activation but this mechanism might be common to other CLC channels.