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The vacuole-type ATPases (V-ATPases) are proton pumps in various intracellular compartments of eukaryotic cells. Prokaryotic V-ATPase of Enterococcus hirae, closely related to the eukaryotic enzymes, provides a unique opportunity to study ion translocation by V-ATPases because it transports [Na.sup.+] ions, which are easier to detect by x-ray crystallography and radioisotope experiments. The purified rotor ring (K-ring) of the E. hirae V-ATPase binds one [Na.sup.+] ion per K-monomer with high affinity, which is competitively inhibited by [Li.sup.] or [H.sup.+], suggesting that the K-ring can also bind these ions. This finding is also supported by the K-ring structure at 2.8 [Angstrom] in the presence of [Li.sup.+]. Association and dissociation rates of the [Na.sup.+] to and from the purified K-ring were extremely slow compared with the [Na.sup.+] translocation rate estimated from the enzymatic activity, strongly suggesting that interaction with the stator subunit (I-subunit) is essential for [Na.sup.+] binding to/release from the K-ring. membrane protein | rotary motor | x-ray crystallography | lithium ion Enterococcus hirae

Ion selectivity is critical for the biological functions of voltage-dependent cation channels and is achieved by specific ion binding to a pore region called the selectivity filter. In voltage-gated [K.sup.+], [Na.sup.+] and [Ca.sup.2+] channels, the selectivity filter is formed by a short polypeptide loop (called the H5 or P region) between the fifth and sixth transmembrane segments, donated by each of the four subunits or internal homologous domains forming the channel. While mutagenesis studies on voltage-gated [K.sup.+] channels have begun to shed light on the structural organization of this pore region, little is known about the physical and chemical interactions that maintain the structural stability of the selectivity filter. Here we show that in an inwardly rectifying [K.sup.+] (IRK) channel, IRK1, short range interactions of an ion pair in the H5 pore loop are crucial for pore structure and ion permeation. The two residues, a glutamate and an arginine, appear to form exposed salt bridges in the tetrameric channel. Alteration or disruption of such ion pair interactions dramatically alters ion selectivity and permeation. Since this ion pair is conserved in all IRK channels, it may constitute a general mechanism for maintaining the stability of the pore structure in this channel superfamily.

Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in [10.sup.9] transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins.