30Two--pore voltage--gated CLC chloride channels control neuronal and muscle excitability. They 31 share a dimeric structure but their activation mechanism remains unresolved. Here we 32 determine the step--by--step activation mechanism of the broadly expressed CLC--2 channel using 33 homology modelling, molecular dynamic simulations and functional studies. We establish that a 34 two--leaf gate formed by Tyr561--H 2 O--Glu213 flanked by Lys568/Glu174 and Lys212 closes the 35 canonical pore. Activation begins when a hyperpolarization--propelled intracellular chloride 36 occupies the pore and splits Tyr561--H 2 O--Glu213 by electrostatic/steric repulsion. Unrestrained 37Glu213 rotates outwardly to bind Lys212 but the pore remains closed. Protonation breaks the 38 Glu213--Lys212 interaction while another chloride occupies the pore thus catalysing chloride exit 39 via Lys212. Also, we found that the canonical pore is uncoupled from a cytosolic cavity by a 40 Tyr561--containing hydrophobic gate that prevents Glu213 protonation by intracellular protons. 41Our data provide atomistic details about CLC--2 activation but this mechanism might be common 42 to other CLC channels. 43
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