Water transport through potassium channels

Water was found to move through the bacterial potassium channel from Streptomyces lividans (KcsA) 20 times faster than predicted by diffusion (Saparov and Pohl. 2004. Proc.Natl.Acad.Sci.U.S.A 101:4805-4809). To resolve the molecular mechanism of this fast transport mode, the current proposal addresses the following questions: Do other members of the protein family exhibit similar transport characteristics? Does the water transport rate depend on the conformational state? As a starting point, we will investigate the single channel water permeability pf of Kv AP, a bacterial voltage sensitive K+ channel. A pf similar to KcsA would increase the possibility that excitable channels may contribute to water homeostasis in the brain. pf is derived from the water permeability Pf of reconstituted planar membranes and the number of open channels. Therefore the reconstituted membrane is exposed to an osmotic gradient and the solute concentration changes are measured by scanning microelectrodes adjacent to the membrane. A conformational state of the channel that is permeable to water but closed to ions would result in an underestimation of channel number, and thus, in an overestimation of pf . The recently discovered C-type inactivated state of KcsA may represent such a conformation. Whether pf dependends on the conformational state of KcsA will be investigated: (i) by suppressing the inactivation with an E71A mutation in the pore helix; (ii) by increasing the probability to be in the open state with mutations in the bundle cross, and (iii) by increasing the rate of entry into the inactivated state by membrane potential. Assignment of the substantial volume flow to the inactivated state would have important implications for other K + channels in mammalian excitable tissues since the amino acid sequence encoding the KcsA selectivity filter is conserved in K + channels throughout nature.

Potassium channels are essential for the propagation of excitation. They open in response to transmembrane voltage during an action potential. While still in the open state, the selectivity filter of potassium channels may inactivate, i.e. the channels become impermeable to ions. Using the bacterial potassium channel KcsA, we have shown that the inactivated state functions as a selective water channel. Therefore, we have reconstituted inactivating and non- inactivating variants of the channel into liposomes and monitored their shrinking in response to an osmotic gradient. Water flow through the inactivated filter should be important for excitable tissues as potassium extrusion may otherwise lead to an elevated osmotic pressure in synapses. So far the importance of water channels (aquaporin-4) has only been recognized for the efficient K+ clearance during high neuronal activity. Little attention has been paid to the pathway water takes when K + is extruded into the synaptic cleft. We propose that the K + efflux during high neuronal activity is facilitated by water flux through inactivated K+ channels.