Removal of gating in voltage-dependent ClC-2 chloride channel by point mutations affecting the pore and C-terminus CBS-2 domain.
Functional and structural studies demonstrate that Cl(-) channels of the ClC family have a dimeric double-barrelled structure, with each monomer contributing an identical pore. Studies with ClC-0, the prototype ClC channel, show the presence of independent mechanisms gating the individual pores or both pores simultaneously. A single-point mutation in the CBS-2 domain of ClC-0 has been shown to abolish slow gating. We have taken advantage of the high conservation of CBS domains in ClC channels to test for the presence of a slow gate in ClC-2 by reproducing this mutation (H811A). ClC-2-H811A showed faster opening kinetics and opened at more positive potentials than ClC-2. There was no difference in [Cl(-)](i) dependence. Additional neutralization of a putative pore gate glutamate side chain (E207V) abolished all gating. Resolving slow and fast gating relaxations, however, revealed that the H811A mutation affected both fast and slow gating processes in ClC-2. This suggests that slow and fast gating in ClC-2 are coupled, perhaps with slow gating contributing to the operation of the pore E207 as a protopore gate.
Centro de Estudios Cientificos (CECS), Av. Arturo Prat 514, Valdivia, Chile.
Yamil R
YR
Yusef
Leandro
L
Zúñiga
Marcelo
M
Catalán
María Isabel
MI
Niemeyer
L Pablo
LP
Cid
Francisco V
FV
Sepúlveda
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000090007:CLC-2 Chloride Channels; D002460:Cell Line; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007668:Kidney / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016062:Porosity; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels