Investigating the putative glycine hinge in Shaker potassium channel

Shinghua Ding; Lindsey Ingleby; Christopher A Ahern; Richard Horn

doi:10.1085/jgp.200509287

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Investigating the putative glycine hinge in Shaker potassium channel

Journal article

Open access

Peer reviewed

Investigating the putative glycine hinge in Shaker potassium channel

Shinghua Ding, Lindsey Ingleby, Christopher A Ahern and Richard Horn

The Journal of general physiology, Vol.126(3), pp.213-226

09/2005

DOI: 10.1085/jgp.200509287

PMCID: PMC2266578

PMID: 16103276

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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.

Potassium - metabolism

Epitopes - analysis

Oligopeptides

Humans

Potassium Channels - metabolism

Ion Channel Gating - physiology

Transfection

Glycine - chemistry

Binding Sites

Potassium Channel Blockers - pharmacology

Glycine - genetics

Membrane Potentials - drug effects

Cell Line

Protein Structure, Secondary

Oocytes - metabolism

Xenopus laevis

Glycosylation

Potassium Channels - genetics

Potassium Channels - analysis

Point Mutation

Animals

Potassium Channels - chemistry

Mutation

Shaker Superfamily of Potassium Channels

Peptides - analysis

Quaternary Ammonium Compounds - pharmacology

Details

Title: Subtitle: Investigating the putative glycine hinge in Shaker potassium channel
Creators: Shinghua Ding - Department of Physiology, Institute of Hyperexcitability, Jefferson Medical College, Philadelphia, PA 19107, USA
Lindsey Ingleby
Christopher A Ahern
Richard Horn
Resource Type: Journal article
Publication Details: The Journal of general physiology, Vol.126(3), pp.213-226
DOI: 10.1085/jgp.200509287
PMID: 16103276
PMCID: PMC2266578
NLM abbreviation: J Gen Physiol
ISSN: 0022-1295
eISSN: 1540-7748
Publisher: United States
Grant note: R01 AR041691 / NIAMS NIH HHS R01 AR41691 / NIAMS NIH HHS
Language: English
Date published: 09/2005
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute
Record Identifier: 9984070278102771

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