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Differences between the deep pores of K+ channels determined by an interacting pair of nonpolar amino acids
G E Kirsch1, J A Drewe, H A Hartmann
1Department of Anesthesiology, Baylor College of Medicine, Houston, Texas 77030.
Abstract:
The pore of a chimeric K+ channel, CHM, differed from its parental host channel, Kv2.1, by 9 amino acids. Four were located in a putative deep region and 5 in a nearby outer mouth. Point reversions were without restorative effects, and reversions V369I or L374V in the deep pore produced novel phenotypes. Among double mutations, only V369I and L374V were effective in restoring the Kv2.1 pore phenotype. Adding a change in charge at Q382K in the outer pore fully restored the parental phenotype. Thus, the pore appears to have an inner, deep region where ions such as K+ and TEA+ may be regulated by nonpolar residues and an outer region where ions may be regulated by charged residues.
Insights
Investigating a chimeric K+ channel (CHM) revealed distinct pore regions. Specific amino acid changes in the deep pore and outer mouth regions were crucial for restoring the parental Kv2.1 channel phenotype.
Area of Science:
- Molecular Biology
- Biophysics
- Ion Channel Research
Background:
- Chimeric potassium (K+) channels are engineered proteins used to study ion channel function.
- The Kv2.1 channel is a well-characterized voltage-gated potassium channel with a specific pore structure.
Purpose of the Study:
- To identify key amino acid residues within the pore of a chimeric K+ channel (CHM) responsible for its unique ion conduction properties.
- To elucidate the functional roles of different regions within the K+ channel pore, specifically the deep pore and outer mouth.
Main Methods:
- Site-directed mutagenesis was employed to introduce specific amino acid substitutions into the CHM and Kv2.1 channels.
- Phenotypic analysis of mutant channels was performed to assess ion permeation and selectivity, including the effects of K+ and tetraethylammonium (TEA+).
Main Results:
- Nine amino acid differences between CHM and Kv2.1 were identified in the pore region, with four in the deep pore and five in the outer mouth.
- Single point mutations in the deep pore (V369I, L374V) resulted in novel phenotypes, while double mutations V369I and L374V partially restored the Kv2.1 pore phenotype.
- A charge-altering mutation (Q382K) in the outer pore, combined with deep pore mutations, fully restored the parental Kv2.1 pore phenotype.
Conclusions:
- The K+ channel pore possesses distinct functional regions: a deep pore regulated by nonpolar residues and an outer mouth regulated by charged residues.
- Specific amino acid residues in both the deep and outer regions of the K+ channel pore are critical for determining ion selectivity and channel gating.
- Understanding these structure-function relationships is vital for designing K+ channels with specific properties.