Beyond the diffusion limit: Water flow through the empty bacterial potassium channel

Abstract

Water molecules are constrained to move with K+ ions through the narrow part of the Streptomyces lividans K+ channel because of the single-file nature of transport. In the presence of an osmotic gradient, a water molecule requires <10 ps to cross the purified protein reconstituted into planar bilayers. Rinsing K+ out of the channel, water may be 1,000 times faster than the fastest experimentally observed K+ ion and 20 times faster than the one-dimensional bulk diffusion of water. Both the anomalously high water mobility and its inhibition observed at high K+ concentrations are consistent with the view that liquid-vapor oscillations occur because of geometrical confinements of water in the selectivity filter. These oscillations, where the chain of molecules imbedded in the channel (the "liquid") cooperatively exits the channel, leaving behind a near vacuum (the "vapor"), thus far have only been discovered in hydrophobic nanopores by molecular dynamics simulations [Hummer, G., Rasaiah, J. C. & Noworyta, J. P. (2001) Nature 414, 188-190; and Beckstein, O. & Sansom, M. S. P. (2003) Proc. Natl. Acad. Sci. USA 100, 7063-7068].

Fig. 1.

Water and ion fluxes through planar bilayers reconstituted with purified KcsA. The K⁺ flux was equal to 1.5 pmol/s at a stationary potential of 50 mV, as revealed by current measurements. It was accompanied by a voltage-insensitive water flux of at least 250 pmol/s, as derived from the K⁺ dilution in the immediate vicinity of a membrane clamped to 0 mV. Cation polarization was measured by scanning microelectrodes at the indicated distances from planar lipid bilayers. In an open circuit, there is no ion transport across a membrane containing only cation-selective channels. Because of the single-file nature of transport, water flow is inhibited also. It is conducted only by the lipid bilayer, which enables a flux of 100 pmol/s. The buffer contained 50 mM KCl, 100 mM choline chloride, 100 μM MgCl₂, and 10 mM Hepes. Osmotic water flux was induced by 1 M urea.

Fig. 2.

Gating (A) and block (B) of water transport. (A) The osmotic water permeability (46 μm/s) was determined (13) from spatially resolved measurements of Mg²⁺ dilution in response to a 1 M urea gradient at pH 4. Augmenting pH to 7 closed the KcsA channels, as indicated by the increase of the electrical membrane resistance (measured at a frequency of 1 kHz), R, from 8 × 10⁵ to 10⁸ Ω. Simultaneously, channel closing decreased P_f to 25 μm/s. (B) Channel block by 15 mM Na⁺ changed R and P_f from 5 × 10⁶ to 5 × 10⁸ Ω and from 35 to 24 μm/s, respectively.

Fig. 3.

Single-channel water permeability. Membrane conductance, G, was measured in a four-electrode configuration. The black line denotes Mg²⁺ dilution near the unmodified bilayer by osmotic flow. Increase in the number of reconstituted KcsA channels (8,300, 17,000, 25,000, 36,000, and 45,000 channels with a unitary conductance of 40 pS) resulted in increasing concentration polarizations (from dark gray to light gray), which allowed calculation of the corresponding P_f values (27, 34, 43, 51, and 59μm/s, respectively). (Inset) Four additional runs of the experiment and three similar experiments, in which 50 mM K⁺ were substituted for 25 mM Rb⁺, were analyzed. From the slope of the plot P_f versus G and the known single-channel conductances of 40 and 10 pS for K⁺ and Rb⁺, respectively, p_f was calculated (9) to be 4.8 × 10^–12 cm³·s^–1.

Fig. 4.

Activation energy for water flux across KcsA channels. Osmotic volume flux-induced Mg²⁺ dilution was measured as a function of temperature. Reconstitution of KcsA channels increased G from 3 nS·cm^–2 (bare bilayer) to 20 mS·cm^–2. (Inset) Arrhenius plot for water flow across protein-free lipid bilayers and KcsA containing bilayers. From the slope, an activation energy of ≈14 kcal/mol was obtained for protein-free lipid bilayers (filled circles). Protein reconstitution decreased the activation energy (open squares). The incremental water permeability caused by KcsA corresponds to an activation energy of 5.1 kcal/mol (solid line).

Fig. 5.

p_f does not depend on the driving force. An increase of the osmotic gradient from 0.1 to 1.5 M urea (from dark to light gray) resulted in an increased Mg²⁺ dilution close to the membrane, allowing determination of P_f. (Inset) From G measured simultaneously, the number of open channels and thus the single-channel hydraulic conductivity were calculated. p_f was constant over the interval of applied osmotic pressure.

Fig. 6.

All-or-none mechanism of fast water conductance: inhibition of fast water transport by K⁺. G was equivalent to ≈20,000 open channels. At 10 and 100 mM KCl, the channels mediated a water permeability P_f,c of ≈30 μm/s (black solid and dashed lines). At 0.3 and 0.4 M K⁺, P_f,c was immeasurably small (Inset), indicating inhibition of the fast transport mode (gray solid and dashed lines). At the intermediate 0.2 M K⁺ concentration (dark gray), part of the channels showed high water permeability.