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Mechanism of ion permeation in skeletal muscle chloride channels
1Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2372, USA. cfahlke@mbio.mc.vanderbilt.edu
Abstract:
Voltage-gated Cl- channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl- channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I- and related anions. Extracellular and intracellular I- exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br-) and polyatomic (SCN-, NO3-, CH3SO3-) anions. In addition, I- block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I- causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I- slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined KD values for I- block in three distinct kinetic states and found that binding of I- to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I- binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.
Insights
Iodide (I-) ions block human skeletal muscle chloride channels (hClC-1) by interacting with two distinct sites within the pore. This block modulates channel gating, revealing a close link between permeation and channel opening/closing dynamics.
Area of Science:
- Molecular Biology
- Biophysics
- Ion Channel Physiology
Background:
- Voltage-gated chloride (Cl-) channels, particularly the ClC family, possess unique ion permeation and gating characteristics.
- Understanding the functional properties of human skeletal muscle Cl- channels (hClC-1) is crucial for comprehending muscle function.
Purpose of the Study:
- To functionally investigate the conduction pathway of hClC-1 using iodide (I-) and related anions as blockers.
- To elucidate the relationship between ion permeation and channel gating in hClC-1.
Main Methods:
- Recombinant hClC-1 was expressed in Xenopus oocytes and a mammalian cell line.
- Extracellular and intracellular iodide (I-) and other anions were used to probe the channel pore.
- Voltage-clamp electrophysiology was employed to measure channel currents and analyze gating kinetics.
Main Results:
- Extracellular and intracellular I- ions block hClC-1 currents in a concentration- and voltage-dependent manner.
- I- block affects channel gating differently depending on the blocker's location, altering kinetic states and deactivation rates.
- Two distinct functional ion binding sites within the hClC-1 pore were identified, with I- binding modulated by channel gating state.
Conclusions:
- The study reveals distinct ion binding sites within the hClC-1 pore, influencing both ion permeation and gating.
- Conformational changes during gating transitions affect these binding sites, highlighting the interplay between permeation and gating in ClC channels.
- These findings provide insights into ion selectivity mechanisms and the gating-permeation coupling in hClC-1.