Movement of hCIC-1 C-termini during common gating and limits on their cytoplasmic location
Date
2011
Authors
Ma, L.
Rychkov, G.
Bykova, E.
Zheng, J.
Bretag, A.
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Biochemical Journal, 2011; 436(2):415-428
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Linlin Ma, Grigori Y. Rychkov, Katerina A. Bykova, Jie Zheng and Allan H. Bretag
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Abstract
Functionally, the dimeric human skeletal muscle chloride channel hClC-1 is characterized by two distinctive gating processes, fast (protopore) gating and slow (common) gating. Of these, common gating is poorly understood, but extensive conformational rearrangement is suspected. To examine this possibility, we used FRET (fluorescence resonance energy transfer) and assessed the effects of manipulating the common-gating process. Closure of the common gate was accompanied by a separation of the C-termini, whereas, with opening, the C-termini approached each other more closely. These movements were considerably smaller than those seen in ClC-0. To estimate the C-terminus depth within the cytoplasm we constructed a pair of split hClC-1 fragments tagged extracellularly and intracellularly respectively. These not only combined appropriately to rescue channel function, but we detected positive FRET between them. This restricts the C-termini of hClC-1 to a position close to its membrane-resident domain. From mutants in which fast or common gating were affected, FRET revealed a close linkage between the two gating processes with the carboxyl group of Glu²³² apparently acting as the final effector for both.
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© The Authors Journal compilation © 2011 Biochemical Society