Filter gate closure inhibits ion but not water transport
Sprache des Vortragstitels:
Englisch
Original Tagungtitel:
541. WE-Heraeus-Seminar: Transport through Nanopores: From Understanding to Engineering
Sprache des Tagungstitel:
Englisch
Original Kurzfassung:
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
Sprache der Kurzfassung:
Englisch
Vortragstyp:
Hauptvortrag / Eingeladener Vortrag auf einer Tagung
Forschungs-