Analysis of the CLC-1 ATP Binding Site by Structure-Guided Mutagenesis

Abstract

The CLC-1 chloride channel is abundantly expressed on the cell membrane of skeletal muscles, and thus plays an important role in controlling the muscle excitability. We previously showed that ATP can modulate the common gating of CLC-1, presumably by binding to a potential binding site at the C-terminal cytoplasmic region of the channel. The X-ray crystallographic study by others revealed the structure of the ATP-binding site, and two residues, Y617 and D727, were identified to be important for the ATP binding. Based on the CLC-5 C-terminal structure, we identify that besides V634 and E865 of CLC-1 (corresponding to Y617 and D727 of CLC-5), V613 and E860 are also important for the ATP modulation of the CLC-1 common gating. Mutating V634 to either alanine or aspartate abolishes the ATP modulation effect. Surprisingly, mutations of V634 to aromatic amino acids increase the ATP affinity: V634F and V634Y mutations decrease the apparent K1/2 by ∼30- and ∼13-fold, respectively. Mutations of E865 to alanine and serine abolish the ATP modulation, while a charge-conserved mutation, E865D, greatly decreases the efficacy of the ATP effect_the maximal shift of the V1/2 of the common gate P-V curve is ∼10-20 % of that in the WT channel. Mutations of V860 and V613 to various amino acids also greatly reduce the efficacy of the ATP modulation. Double mutant cycle analyses show that V634 and V613 are strongly energetically coupled, with a coupling coefficient (Ω) of ∼25, while V634 and E865 may be only weakly coupled with a Ω value of ∼1.6. These results support the idea that ATP likely binds directly to the C-terminus of CLC-1 to modulate the common gating of the channel.