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) × 10 −13 cm 3 ·s −1 for both mutants. "Collapse" of the selectivity filter upon K + removal did not alter p f 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. membrane channels | protein reconstitution | knock-on mechanism | aquaporin | brain water homeostasis
Phase diagrams offer a wealth of
thermodynamic information on aqueous
mixtures of bilayer-forming lipids and micelle-forming detergents,
providing a straightforward means of monitoring and adjusting the
supramolecular state of such systems. However, equilibrium phase diagrams
are of very limited use for the reconstitution of membrane proteins
because of the occurrence of irreversible, unproductive processes
such as aggregation and precipitation that compete with productive
reconstitution. Here, we exemplify this by dissecting the effects
of the K+ channel KcsA on the process of bilayer self-assembly
in a mixture of Escherichia coli polar lipid extract
and the nonionic detergent octyl-β-d-glucopyranoside.
Even at starting concentrations in the low micromolar range, KcsA
has a tremendous impact on the supramolecular organization of the
system, shifting the critical lipid/detergent ratios at the onset
and completion of vesicle formation by more than 2-fold. Thus, equilibrium
phase diagrams obtained for protein-free lipid/detergent mixtures
would be misleading when used to guide the reconstitution process.
To address this issue, we demonstrate that, even under such nonequilibrium
conditions, high-sensitivity isothermal titration calorimetry can
be exploited to monitor the progress of membrane-protein reconstitution
in real time, in a noninvasive manner, and at high resolution to yield
functional proteoliposomes with a narrow size distribution for further
downstream applications.
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