Facilitators of a Proton-Glutamate in a Cl -/H + Exchanger, CLC-EC1

Abstract

The CLC-ec1 is a bacterial homologue of the CLC Cl-/H+ exchanger that catalyzes 2:1 exchange of Cl- and H+. Functionally intact monomers form a dimer in bilayer membranes. Gating-glutamate (or Gluex, E148) is the pH-dependent external gate of both Cl- and H+ transport and proton-glutamate (or Gluin, E203) mediates proton-transport from intracellular solution to the protein interior. Mutations on either glutamate with non-protonable amino acids abolish H+ transport across the membrane. However, it is still puzzling how protons in the intracellular solution could be transferred to the proton-gluatamate forming a salt bridge with arginine (R28) in swapped N-terminal domain from neighboring subunit, which structurally isolates the proton-glutamate from the intracellular solution. In order to find functional facilitators for proton transport, we have been examining several residues near E203. Salt-bridge breaking mutants, R28L, Q and E do not alter the functional activity at all. Moreover, N-terminal truncation mutants (Delta16 and Delta29), which remove an apparent “lid” covering the proton-glutamate, preserve intact Cl-/H+ exchanger activity. Mutations on two proximate glutamates (E113L and E202L) decrease H+ transport rate without changing Cl- turnover rate. Currently, we are investigating functional and structural changes of multiple mutations.