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Selectivity changes during activation of mutant Shaker potassium channels
1Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
The Journal of General Physiology
|August 1, 1997
Summary
Mutations in Shaker channel pore residue T442 significantly alter ion channel gating kinetics. T442S and T442G mutations dramatically slow deactivation and prolong channel opening, revealing kinetic intermediates.
Area of Science:
- Molecular biology
- Ion channel biophysics
- Neuroscience
Background:
- Shaker potassium channels are crucial for neuronal excitability.
- The pore region, particularly residue T442, is known to influence channel gating.
- Previous studies indicated T442 mutations impact channel kinetics.
Purpose of the Study:
- To investigate the effects of pore-region residue T442 mutations on Shaker channel kinetics.
- To characterize the functional expression and gating properties of T442 mutants.
- To explore the role of T442 in ion permeation and subconductance states.
Main Methods:
- Site-directed mutagenesis of the T442 residue in Shaker H4 and Shaker-NGK2 chimeric channels.
- Expression of mutant channels in Xenopus oocytes.
- Electrophysiological recordings, including macroscopic currents and single-channel analysis.
Main Results:
- Mutations T442C, D, H, V, Y resulted in undetectable channel expression.
- T442S and T442G mutants exhibited functional channels with 100-1000 fold longer deactivation time constants and open times.
- Mutant channels displayed two subconductance levels (37% and 70%), representing kinetic intermediates in gating, with distinct ion conductance sequences.
Conclusions:
- The T442 residue is critical for normal Shaker channel gating kinetics.
- Mutations at T442 can dramatically slow channel deactivation and prolong opening by introducing stable kinetic intermediates.
- Subconductance states are integral to the gating process and exhibit differential ion selectivity.