Dynamical model of the CLC-2 ion channel reveals conformational changes associated with selectivity-filter gating

Abstract

This work reports a dynamical Markov state model of CLC-2 "fast" (pore) gating, based on 600 microseconds of molecular dynamics (MD) simulation. In the starting conformation of our CLC-2 model, both outer and inner channel gates are closed. The first conformational change in our dataset involves rotation of the inner-gate backbone along residues S168-G169-I170. This change is strikingly similar to that observed in the cryo-EM structure of the bovine CLC-K channel, though the volume of the intracellular (inner) region of the ion conduction pathway is further expanded in our model. From this state (inner gate open and outer gate closed), two additional states are observed, each involving a unique rotameric flip of the outer-gate residue GLU ex. Both additional states involve conformational changes that orient GLU ex away from the extracellular (outer) region of the ion conduction pathway. In the first additional state, the rotameric flip of GLU ex results in an open, or near-open, channel pore. The equilibrium population of this state is low (*1%), consistent with the low open probability of CLC-2 observed experimentally in the absence of a membrane potential stimulus (0 mV). In the second additional state, GLU ex rotates to occlude the channel pore. This state, which has a low equilibrium population (*1%), is only accessible when GLU ex is protonated. Together, these pathways model the opening of both an inner and outer gate within the CLC-2 selectivity filter, as a function of GLU ex protonation. Collectively, our findings are consistent with published experimental analyses of CLC-2 gating and provide a high-resolution structural model to guide future investigations.

Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel

“…Computational docking was performed to generate binding site predictions for AK-42 and to gain insight into the molecular basis for CLC homolog selectivity. Absent a high-resolution structure of CLC-2, we capitalized on a CLC-2 structural homology model that we developed and refined with 600 μs of molecular dynamics simulations (60). Guided by prior mutagenesis and computational modeling studies of fenamate inhibitors (in the same structural class as AK-42) against CLC-Ka and CLC-Kb, AK-42 was docked to an extracellular site on the channel.…”

Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel

“…To gain insight into the molecular basis for the CLC homolog selectivity, we performed a computational docking study and experimentally tested the binding-site predictions. Absent a high-resolution structure of CLC-2, we took advantage of a CLC-2 structural homology model we had developed and refined with 600 µs of molecular dynamics simulations 44 . Based on the kinetics and reversibility of inhibition (Extended Data Figures 3, 4), we reasoned that AK-42 is likely binding to the extracellular side of channel pore.…”

“…The receptor surface and ligand were parameterized using the AMBER parm99 force field 72 . et al 44 , as well as CLC-2 homology models generated from the 5TQQ and 5TR1 cryo-EM structures of bovine CLC-K 45 . Alignment of the CLC-2 sequence with these structures for generation of the homology models was accomplished with the UCSF Chimera sequence alignment tool 71 .…”

Electro-Steric Mechanism of CLC-2 Chloride Channel Activation

“…1F). This conformation is 145 similar to that of Glu gate in CmCLC and in rCLC--2 models built using the CmCLC structure (Feng et 146 al., 2010;McKiernan et al, 2020). We propose that the Tyr561--H 2 O--Glu gate array forms the gate 147 that maintains CLC--2 in the closed state.…”

“…suggested by McKiernan et al,(McKiernan et al, 2020). These authors combined Markov state 559 modelling with MD simulations and a homology structure of rat CLC--2 built using the CmCLC Cl --560 /H + exchanger structure 3ORG as a template(Feng et al, 2010).…”