Side-chain accessibilities in the pore of a K+ channel probed by sulfhydryl-specific reagents after cysteine-scanning mutagenesis.
To gain insight into the secondary structure of the ion conduction pathway of a voltage-gated K+ channel, we used sulfhydryl-specific reagents of different diameters to probe amino acid side-chain accessibilities in the pore of the channel after cysteine-substitution mutagenesis. We identified five positions at which modified amino acid side chains are accessible from the aqueous lumen of the external channel vestibule. Covalent coupling of the 2-trimethylammonium-thioethyl group to cysteine thiols leads to position-dependent current reduction, suggesting a gradual narrowing of the pore. The fact that the modified side chains of two adjacent amino acids are reactive is not compatible with the ion conduction pathway forming a regular beta-pleated sheet at these positions. The smaller thiol reagent Cd2+ reacts with modified side chains that are also accessible to the larger (2-trimethylammoniumethyl)methanethiosulfate (MTSET) [corrected]. Our results imply that the outer vestibule of a potassium-selective ion channel narrows over a short distance of three amino acids near a position where a regular beta-structure is unlikely.
Kürz
L L
LL
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235-9111, USA.
Zühlke
R D
RD
Zhang
H J
HJ
Joho
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D005260:Female; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D013439:Sulfhydryl Reagents; D014981:Xenopus
D015221:Potassium Channels; D013439:Sulfhydryl Reagents; D003545:Cysteine