期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 110, 期 26, 页码 10842-10847出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1304714110
关键词
membrane channels; protein reconstitution; knock-on mechanism; aquaporin; brain water homeostasis
资金
- Austrian Science Fund (Fonds zur Forderung der Wissenschaftlichen Forschung) [P19716]
- Stiftung Rheinland-Pfalz fur Innovation [961-386261/969]
- Austrian Science Fund (FWF) [P19716] Funding Source: Austrian Science Fund (FWF)
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 p(f) of (6.9 +/- 0.6) x 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.
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