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 pf 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 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.

Keywords: aquaporin; brain water homeostasis; knock-on mechanism; membrane channels; protein reconstitution.

Fig. 1.

Comparison of the inactivated (PDB ID code 3F7V, ref. 6), collapsed (low-K⁺; PDB ID code 1K4D, ref. 5), and noninactivating (E71A; PDB code 3OGC, ref. 24) crystal structures of KcsA. In its open noninactivated state, KcsA is known to conduct both water and ions. In its collapsed or inactivated state, the channel is closed to ions. Assuming a low binding affinity of K⁺ for these two states (31), we tested the hypothesis that, with an open cytoplasmic gate, both the inactivated and the collapsed states are permeable to water.

Fig. 2.

FCS for counting the number of KcsA channels per vesicle. Autocorrelation curves obtained for the proteoliposome suspension were used to extract both the residence times τ_D and the numbers w_P and w_L of fluorescently labeled protein and lipid particles in the confocal volumes of green (488-nm) and red (633-nm) lasers, respectively, by fitting the standard model (dotted gray lines) for two-component free 3D diffusion (47) to G(τ). The residence time of the free dye was fixed at 25 μs. The difference in particle numbers between the KcsA and lipid channels was a result of different confocal volumes as well as the presence of both protein-containing and empty vesicles. Vesicle dissolution by OG at a final detergent concentration of 2% resulted in a decrease in τ_D from ∼4.3 ms to ∼200 µs. Assuming that each micelle contained one KcsA tetramer at most, we determined the number w_M of micelles in the confocal volume. After correction for dilution, the ratio w_MV_L/w_LV_P = 2.84 gives the number n of tetramers per vesicle and the ratio w_M/w_P = 2.91 the number m of tetramers per proteoliposome. V_L and V_P are the confocal volumes of the lipid and the proton channels, respectively. The buffer contained 150 mM KCl, 25 mM Tris, and 25 mM Hepes (pH 7.50).

Fig. 3.

Fusion of a single proteoliposome to a preformed planar bilayer. Ten microliters of a vesicle suspension (5 mg/mL) with the inactivating protein (A and B) or noninactivating mutant (C and D) was added to the hyperosmotic compartment (260 mM KCl). The hypoosmotic compartment contained 150 mM KCl. Solutions were buffered with 25 mM Tris and 25 mM Hepes (pH 7.50). Vesicle fusion resulted in the insertion of KcsA channels into the bilayer, as indicated by a sudden increase in current (A and C). Subsequently, the current decreased in a stepwise manner upon channel inactivation. The constant voltage of +100 mV was turned off (↓) and switched on again after a total of about 12 s (↑). At that time, all channels had recovered from inactivation, as indicated by the current amplitude. Note the 10-fold expanded time scale in B and D compared with A and C.

Fig. 4.

Osmotic water permeability of proteoliposomes. Stopped-flow experiments were performed in the presence of 8 µM valinomycin and 5 µM CCCP. Two populations of vesicles were used: with 0 (cyan lines) and 9 mM K⁺ (black and red lines) on both the inside and outside. The average number of KcsA tetramers per vesicle is indicated. Dilution (1:1 vol/vol) of the vesicle suspension in buffer (150 mM choline chloride, 25 mM Tris, 25 mM Hepes, pH 7.50) containing 600 mM sucrose resulted in vesicle shrinkage. Partial inhibition of KcsA-mediated water flow was achieved by increasing the K⁺ concentration to 200 mM (green lines). To this end, the 9 mM K⁺ vesicle population (black lines) first was incubated in 200 mM K⁺ buffer and then subjected to a stopped-flow experiment. Measurements were done at 7 °C. Experimental traces were fitted by a single-exponential function to determine the characteristic time τ_SF.

Fig. 5.

Single-channel water permeability. (A) τ_SF^-1 is plotted as a function of n. τ_SF^-1 and n were determined in experiments such as those shown in Figs. 4 and 2, respectively. The color of the data points corresponds to the color of the bars in B. Using Eqs. 2 and 3, a linear fit (black line) through all data points allowed calculation of p_f ∼ (4.1 ± 0.4) × 10⁻¹³ cm³⋅s⁻¹. (B) Individual p_f values (with SE) calculated similarly for the inactivating mutant (Inact.; H25R/E118A) and the noninactivating mutant (Non-inact.; H25R/E71A/E118A) at 0, 9, and 200 mM KCl in both the vesicle interior and the buffer solutions. p_f of an empty lipid vesicle was defined as the water permeability of a lipid bilayer P_f,l multiplied by the surface area of one vesicle. The decrease in K⁺ concentration from 9 to 0 mM had no significant effect on water flow, whereas the increase in K⁺ concentration from 9 to 200 mM led to partial inhibition.