A 63 residue peptide, p7, encoded by hepatitis C virus was synthesised and tested for ion channel activity in lipid bilayer membranes. Ion channels formed by p7 had a variable conductance: some channels had conductances as low as 14 pS. The reversal potential of currents £owing through the channels formed by p7 showed that they were permeable to potassium and sodium ions and less permeable to calcium ions. Addition of Ca + to solutions made channels formed by p7 less potassium-or sodium-selective. Hexamethylene amiloride, a drug previously shown to block ion channels formed by Vpu encoded by HIV-1, blocked channels formed by p7. In view of the increasing number of peptides encoded by viruses that have been shown to form ion channels, it is suggested that ion channels may play an important role in the life cycle of many viruses and that drugs that block these channels may prove to be useful antiviral agents. ß

A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix. Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential. The channels were more permeable to Na+ than to Cl ions and were inhibited when the trans potential was made positive. Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen. Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber. Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber. The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential. The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber. The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies.

The influenza B virus protein, NB, was expressed in Escherichia coli, either with a C-terminal polyhistidine tag or with NB fused to the C-terminus of glutathione S-transferase (GST), and purified by affinity chromatography. NB produced ion channel activity when added to artificial lipid bilayers separating NaCl solutions with unequal concentrations (150-500 mM cis, 50 mM trans). An antibody to a peptide mimicking the 25 residues at the C-terminal end of NB, and amantadine at high concentration (2-3 mM), both depressed ion channel activity. Ion channels had a variable conductance, the lowest conductance observed being approximately 10 picosiemens. At a pH of 5.5 to 6.5, currents reversed at positive potentials indicating that the channel was more permeable to sodium than to chloride ions (PNa/PCl approximately 9). In asymmetrical NaCl solutions at a pH of 2.5, currents reversed closer to the chloride than to the sodium equilibrium potential indicating that the channel had become more permeable to chloride than to sodium ions (PCl/PNa approximately 4). It was concluded that, at normal pHs, NB forms cation-selective channels.

A chemically synthesized peptide consisting of the C-terminus of the M protein of the Dengue virus type 1 strain Singapore S275/90 (DVM-C) produced ion channel activity in artificial lipid bilayers. The channels had a variable conductance and were more permeable to sodium and potassium ions than to chloride ions and more permeable to chloride ions than to calcium ions. Hexamethylene amiloride (100 microM) and amantadine (10 microM), blocked channels formed by DVM-C. Ion channels may play an important role in the life cycle of many viruses and drugs that block these channels may prove to be useful antiviral agents.

The secretory Na ؉ -K ؉ -2Cl ؊ cotransporter (NKCC1) is a member of a small gene family of electroneutral salt transporters that play essential roles in salt and water homeostasis in many mammalian tissues. We have identified a highly conserved residue (Ala-483) in the sixth membrane-spanning segment of rat NKCC1 that when mutated to cysteine renders the transporter sensitive to inhibition by the sulfhydryl reagents 2-aminoethyl methanethiosulfonate (MTSEA) and 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The mutation of Ala-483 to cysteine (A483C) results in little or no change in the affinities of NKCC1 for substrate ions but produces a 6-fold increase in sensitivity to the inhibitor bumetanide, suggesting a specific modification of the bumetanide binding site. When residues surrounding Ala-483 were mutated to cysteine, only I484C was sensitive to inhibition by MTSEA and MTSET. Surprisingly I484C showed increased transport activity in the presence of low concentrations of mercury (1-10 M), whereas A483C showed inhibition. The inhibition of A483C by MTSEA was unaffected by the presence or absence of sodium and potassium but required the presence of extracellular chloride. Taken together, our results indicate that Ala-483 lies at or near an important functional site of NKCC1 and that the exposure of this site to the extracellular medium is dependent on the conformation of the transporter. Specifically, our results indicate that the cysteine introduced at residue 483 is only available for interaction with MTSEA when chloride is bound to NKCC1 at the extracellular surface.Ϫ cotransporters (NKCCs) 1 mediate the electroneutral transport of Na ϩ , K ϩ , and Cl Ϫ across cell membranes with a stoichiometry of 1Na ϩ :1K ϩ :2Cl Ϫ (1, 2). By providing a concentrative chloride entry step in chloride secreting and absorbing epithelia, these transporters play a central role in trans-epithelial salt movements across these tissues (1, 2). The NKCCs belong to a small gene family with homologues in vertebrates, crustaceans, insects, worms, plants, and some microorganisms. Nine members of this family have been identified in vertebrates, and of these, seven have been shown to be electroneutral cation-chloride cotransporters (3). These include two Na ϩ -K ϩ -2Cl Ϫ cotransporter isoforms (NKCC1 and NKCC2), a Na ϩ -Cl Ϫ cotransporter (NCC), and four K ϩ -Cl Ϫ cotransporter isoforms (KCC1, KCC2, KCC3, and KCC4). The function of the remaining two vertebrate sequences as well as all of the homologues identified in lower organisms remains to be definitively established. NKCC1, the "secretory" isoform of the NaϪ cotransporters, is the most extensively studied member of the cationchloride cotransport family at the molecular level. This transporter is relatively widely expressed in both epithelial and nonepithelial tissues and has been of considerable interest because of its roles in cell volume regulation as well as trans-epithelial chloride secretion (1, 2). The activity of NKCC1 typically is highly regulated by physiological ...