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Investigating the putative glycine hinge in Shaker potassium channel.

Ding S, Ingleby L, Ahern CA, Horn R - J. Gen. Physiol. (2005)

Bottom Line: Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation.The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467.Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute of Hyperexcitability, Jefferson Medical College, Philadelphia, PA 19107, USA.

ABSTRACT
The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523-526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.

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Tests of steric clash. (A) Two opposing subunits of KcsA from a high-resolution crystal structure (1K4C; Zhou et al., 2001b), highlighting the position of the pore-lining residue homologous to Val467 in Shaker (red). Drawn with Swiss-PdbViewer (http://us.expasy.org) and POV-Ray (www.povray.org). (B) Single KcsA subunits showing the homologue to Shaker's Met440 (orange) and Gly466 (green) on the left, and the expected clash due to the G466A mutation on the right. K+ ions are depicted as gray spheres. (C) Western blot as in Fig. 2 showing poor glycosylation of all mutants containing G466A.
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fig10: Tests of steric clash. (A) Two opposing subunits of KcsA from a high-resolution crystal structure (1K4C; Zhou et al., 2001b), highlighting the position of the pore-lining residue homologous to Val467 in Shaker (red). Drawn with Swiss-PdbViewer (http://us.expasy.org) and POV-Ray (www.povray.org). (B) Single KcsA subunits showing the homologue to Shaker's Met440 (orange) and Gly466 (green) on the left, and the expected clash due to the G466A mutation on the right. K+ ions are depicted as gray spheres. (C) Western blot as in Fig. 2 showing poor glycosylation of all mutants containing G466A.

Mentions: What could account for the large increase of koff in the double mutant? A previous study showed that a mutation in Kv1.4 equivalent to V467A of Shaker caused an ∼1 kcal/mol destabilization of TBA block (Zhou et al., 2001a). Moreover, crystal structures of potassium channels show that the residues homologous to Val467 extend their side chains into the central permeation pathway (Fig. 10 A). These facts suggest that the decreased potency of TBA block in the G466A/V467G mutant is due more to the loss of the greasy side chain of Val467 than to the G466A mutation. This proposal was confirmed by measuring TBA block of the single mutant V467G, which increased Kd from 3.77 μM to 2.47 ± 0.20 mM (3.8 kcal/mol, n = 3), an effect even larger than observed for the double mutant in which Kd was 0.73 mM (Fig. 7). This suggests that the G466A mutation itself modestly stabilizes TBA block.


Investigating the putative glycine hinge in Shaker potassium channel.

Ding S, Ingleby L, Ahern CA, Horn R - J. Gen. Physiol. (2005)

Tests of steric clash. (A) Two opposing subunits of KcsA from a high-resolution crystal structure (1K4C; Zhou et al., 2001b), highlighting the position of the pore-lining residue homologous to Val467 in Shaker (red). Drawn with Swiss-PdbViewer (http://us.expasy.org) and POV-Ray (www.povray.org). (B) Single KcsA subunits showing the homologue to Shaker's Met440 (orange) and Gly466 (green) on the left, and the expected clash due to the G466A mutation on the right. K+ ions are depicted as gray spheres. (C) Western blot as in Fig. 2 showing poor glycosylation of all mutants containing G466A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266578&req=5

fig10: Tests of steric clash. (A) Two opposing subunits of KcsA from a high-resolution crystal structure (1K4C; Zhou et al., 2001b), highlighting the position of the pore-lining residue homologous to Val467 in Shaker (red). Drawn with Swiss-PdbViewer (http://us.expasy.org) and POV-Ray (www.povray.org). (B) Single KcsA subunits showing the homologue to Shaker's Met440 (orange) and Gly466 (green) on the left, and the expected clash due to the G466A mutation on the right. K+ ions are depicted as gray spheres. (C) Western blot as in Fig. 2 showing poor glycosylation of all mutants containing G466A.
Mentions: What could account for the large increase of koff in the double mutant? A previous study showed that a mutation in Kv1.4 equivalent to V467A of Shaker caused an ∼1 kcal/mol destabilization of TBA block (Zhou et al., 2001a). Moreover, crystal structures of potassium channels show that the residues homologous to Val467 extend their side chains into the central permeation pathway (Fig. 10 A). These facts suggest that the decreased potency of TBA block in the G466A/V467G mutant is due more to the loss of the greasy side chain of Val467 than to the G466A mutation. This proposal was confirmed by measuring TBA block of the single mutant V467G, which increased Kd from 3.77 μM to 2.47 ± 0.20 mM (3.8 kcal/mol, n = 3), an effect even larger than observed for the double mutant in which Kd was 0.73 mM (Fig. 7). This suggests that the G466A mutation itself modestly stabilizes TBA block.

Bottom Line: Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation.The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467.Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute of Hyperexcitability, Jefferson Medical College, Philadelphia, PA 19107, USA.

ABSTRACT
The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523-526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.

Show MeSH