title: Filter gate closure inhibits ion but not water transport through potassium channels.
abstract: The selectivity filter of K(+) channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K(+) hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K(+). Here, we show that K(+) channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K(+) channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability pf of (6.9 ± 0.6) × 10(-13) cm(3)⋅s(-1) for both mutants. "Collapse" of the selectivity filter upon K(+) removal did not alter pf and was fully reversible, as demonstrated by current measurements through planar bilayers in a K(+)-containing medium to which K(+)-free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K(+) in the aqueous solution, which indicates an effective K(+) dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K(+) ions in the selectivity filter act to mutually destabilize binding.
authors.affiliation: Institute of Biophysics, Johannes Kepler University of Linz, 4020 Linz, Austria.
authors.forename: Torben
authors.initials: T
authors.lastname: Hoomann
authors.forename: Nadin
authors.initials: N
authors.lastname: Jahnke
authors.forename: Andreas
authors.initials: A
authors.lastname: Horner
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
authors.forename: Peter
authors.initials: P
authors.lastname: Pohl
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability; D015640:Ion Channel Gating; D017136:Ion Transport; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048170:Streptomyces lividans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014867:Water / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D015221:Potassium Channels; D011510:Proteolipids; D011994:Recombinant Proteins; C107790:prokaryotic potassium channel; C020485:proteoliposomes; D014867:Water
keywords: aquaporin; brain water homeostasis; knock-on mechanism; membrane channels; protein reconstitution