Filter gate closure inhibits ion but not water transport

Beschreibung

Filter gate closure inhibits ion but not water transport through potassium channels Torben Hoomann,1 Nadin Jahnke,2 Andreas Horner,1 Sandro Keller,2 Peter Pohl,1 1 Johannes Kepler University Linz, Institute of Biophysics, Linz, Austria 2 Molecular Biophysics, University of Kaiserslautern, Germany 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 non-inactivating. 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)x10-13cm3s-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

Zeitraum	30 Juli 2013
Ereignistitel	541. WE-Heraeus-Seminar: Transport through Nanopores: From Understanding to Engineering
Veranstaltungstyp	Konferenz
Ort	DeutschlandAuf Karte anzeigen