title: Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K+ channels.
abstract: K(+) channels distinguish K(+) from Na(+) in the selectivity filter, which consists of four ion-binding sites (S1-S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of C-type inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K(+) binding at the S2 site. We also show that an ester substitution at the S2 site in the KvAP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K(+) channels in K(+) compared with Rb(+) are due to different ion occupancies at the S2 site.
authors.affiliation: Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Matulef
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Komarov
authors.forename: Corey A
authors.initials: CA
authors.lastname: Costantino
authors.forename: Francis I
authors.initials: FI
authors.lastname: Valiyaveetil
mesh_terms: D000577:Amides / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D003461:Crystallography; D004952:Esters / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology*; D016296:Mutagenesis / Q000235:genetics; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000577:Amides; D002412:Cations; D004952:Esters; D015221:Potassium Channels; D011994:Recombinant Proteins
keywords: K channels; crystallography; unnatural amino acids