Regulation of K+ Conductance by a Hydrogen Bond in Kv2.1, Kv2.2, and Kv1.2 Channels

Figures

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  Figure 1. Effects of the mutation of the equivalent residues controlling the slow inactivation of Shaker channels on the rat Kv2.1, Kv2.2, and Kv1.2 channels. (A) Sequence alignment of the selectivity filter and pore helix of various K+ channels: Kv2.1 (GI: 6981120), Kv2.2 (GI: 164663795), Kv1.2 (GI: 25742772), Shaker (GI: 288442), KcsA (GI: 61226909), and the Kv1.2/Kv2.1 chimeric channel (PDB: 2R9R). (B) Representative currents for the Kv2.1 wild-type, W365A, and T370A mutant channels (−100 to +40 mV, 200 ms, in 10-mV increments; n = 8~14). (C) Representative currents for the Kv2.2 wild-type, W373A, and T378A mutant channels (n = 7~12). (D) Representative currents for the Kv1.2 wild-type, W366A, and S371A mutant channels (n = 6–9). (E) G-V curves of the wild-type and mutant Kv channels.

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  Figure 2. Effects of the mutation of the time constants of the inactivation of the Kv2.1, Kv2.2, and Kv1.2 channels. The inactivation time constants for the wild-type and mutant Kv channels were determined by fitting a 5-s depolarization at +20 mV with single exponentials. Red lines show the representative fitting trajectories of the current traces. (A). Representative currents for the Kv2.1 wild-type, W365A, and T370A mutant channels (5-s pulses from −100 mV to 20 mV) (B). Representative currents for the Kv2.2 wild-type, W373A, and T378A mutant channels. (C) Representative currents for the Kv1.2 wild-type, W366A, and S371A mutant channels. (D) Comparison of the time constants of the inactivation of the wild-type and mutant Kv channels, n = 6~12, p > 0.05 (wild type versus mutants).

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  Figure 3. Selectivity filter structure of the Kv1.2/Kv2.1 chimeric channel (PDB: 2R9R) with the ion-binding sites S0–S4 labeled. The relevant pore helix residues and the three putative hydrogen bonds (blue lines) are labeled as well.

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  Figure 4. Effects of the mutations of the conserved tryptophan residue on the Kv2.1, Kv2.2, and Kv1.2 channel currents in HEK293 cells. (A) Representative currents for the Kv2.1 wild-type, W366A, and W366F mutant channels. (B) Representative currents for the Kv2.2 wild-type, W374A, and W374F mutant channels. (C) Representative currents for the Kv1.2 wild-type, W367A, and W367F mutant channels. (D) I-V plots of the outward currents of the Kv2.1, Kv2.2, and Kv1.2 wild-type and mutant channels (n = 7–14).

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  Figure 5. Cell surface expression of the various Kv channels in HEK293 cells. The Kv channel surface expression was detected using the cell surface biotinylation of HEK293 cells transfected with the wild-type/mutant Kv channels. The left panels were the biotinylated membrane protein samples, and the right panels were total protein samples. (A) Sample images showing endogenous or transfected wild-type/mutant Kv2.1 channel expression. (B) Sample images showing endogenous or transfected wild-type/mutant Kv2.2 channel expression. (C) Sample images showing endogenous or transfected wild-type/mutant Kv1.2 channel expression. The signals of the intracellular protein beta-tubulin were detected only in the total protein samples (right panels) and not in the biotinylated surface protein samples (left panels). Endogenous cell surface protein expression of the Kv2.1, Kv2.2, and Kv1.2 channels was undetectable. (D) The mutations did not alter the cell surface expression of the Kv channels in HEK293 cells (n = 3, p > 0.05, WT versus mutants).

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  Figure 6. Effects of the mutation of the conserved tyrosine residue on the Kv2.1, Kv2.2, and Kv1.2 channels in HEK293 cells. (A) Y376F (Kv2.1) mutant channels present no obvious outward currents. HEK293 cells present no obvious endogenous outward currents. (B,C) Y384F (Kv2.2) and Y377F (Kv1.2) mutant channels present no obvious outward currents. Currents are elicited by a step protocol (holding at −100 mV and depolarizing in 10-mV steps from −100 to +40 mV at 10-s intervals). (D) I-V plots of the outward currents of Kv2.1, Kv2.2, and Kv1.2 wild-type and mutant channels (n = 5~13).