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 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

Ion permeation in K + channels occurs by direct Coulomb knock-on

“…However, these experiments were based on the application of high osmotic gradients. Water permeation as a result of an applied osmotic pressure is likely to lead to ion-depleted SF states in which individual ions are only occasionally dragged along by permeating water molecules, whereas bound ions are reported to completely block water flux (33,34). Such iondepleted, and water-permeable, filter states are therefore likely markedly different from the ion-conductive states at higher ion occupancy considered by crystallography and in our MD simulations.…”

“…This is seemingly in conflict with the ion/water cotranslocation ratio derived from measurements of water translocation through KcsA (32)(33)(34)(35). However, these experiments were based on the application of high osmotic gradients.…”

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

“…The presented PAP channel contains more hydrophobic regions (30) compared with its predecessor channel (23), which improves both its water permeability and its ability to insert into membranes. To determine single-channel permeability of PAPs, we combined stopped-flow light-scattering measurements of lipid vesicles containing PAPs with fluorescence correlation spectroscopy (FCS) (35,36). Stopped-flow experiments allow the kinetics of vesicle swelling or shrinking to be followed with millisecond resolution and water permeability to be calculated, whereas FCS makes it possible to count the number of channels per vesicle (36,37).…”

“…To determine single-channel permeability of PAPs, we combined stopped-flow light-scattering measurements of lipid vesicles containing PAPs with fluorescence correlation spectroscopy (FCS) (35,36). Stopped-flow experiments allow the kinetics of vesicle swelling or shrinking to be followed with millisecond resolution and water permeability to be calculated, whereas FCS makes it possible to count the number of channels per vesicle (36,37). The combination of the two techniques allows molecular characterization of channel properties with high resolution and demonstrates that PAP channels have a water permeability close to those of AQPs and CNTs.…”

“…To calculate the average single-channel permeability of PAP channels, an FCS technique was used to count the number of channels per vesicle (36,37). The channels were tagged with a fluorophore, tetramethylrhodamine cadaverine, using dicyclohexylcarbodiimide as the crosslinking reagent (Fig.…”