| Popis: |
Members of the CLC family function as Cl- channels or as H+/Cl- exchangers. Despite this mechanistic divide the two CLC sub-classes share many functional and structural traits. CLC-ec1, a transporter, can be transformed into a Cl--selective pore by simultaneously removing the intra- and extra-cellular gates. This entailed mutating two residues, E148 and Y445, which are conserved in both CLC channels and transporters. While the extracellular gate regulates channel gating, the intracellular one does not. We hypothesized that in the channels the intracellular gate's position is shifted so that it does not regulate ion passage anymore.The linker connecting helices R and Q is 1-2 amino-acids shorter in the channels than in the transporters. We investigated whether shortening this linker disrupts the intracellular gate of CLC-ec1 while preserving Y445. To this end we deleted two residues, G441 and K442, in the Q-R linker. This deletion, ΔGK, functionally mirrors the gate-removed Y445A mutant: H+ transport is nearly abolished with slight effects on Cl- transport. We then tested the effects of single-residue deletions. The ΔG mutant is similar to the WT while the ΔK deletion impairs H+ transport like the ΔGK deletion. K442 however is not involved in H+ transport: Alanine or Methionine substitutions have no significant effects.Lastly, we incorporated in CLC-ec1 increasing portions of the CLC-0 channel. Introduction of the CLC-0 Q-R linker into CLC-ec1 recapitulates the functional effects of the ΔGK and ΔK mutations. Introduction into CLC-ec1 of up to the full helix R from CLC-0 led to no further effects on H+ transport.In conclusion we found that the length of the Q-R linker is critical for H+/Cl- coupling in CLC-ec1 while the chemical properties of the residues in the linker do not seem to matter. |
