SummaryIt is thought that Na + and K + homeostasis is crucial for salt-tolerance in plants. To better understand the Na + and K + homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K + -Na + symporter was isolated from japonica rice, cv Nipponbare (Ni-OsHKT1). We also isolated two cDNAs homologous to Ni-OsHKT1 from salt-tolerant indica rice, cv Pokkali (Po-OsHKT1, Po-OsHKT2). The predicted amino acid sequence of Ni-OsHKT1 shares 100% identity with Po-OsHKT1 and 91% identity with Po-OsHKT2, and they are 66±67% identical to wheat HKT1. Low-K + conditions (less than 3 mM) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mM Na + . We further characterized the ion-transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K + -uptake de®ciency mutation in yeast, whereas OsHKT1 was not under K + -limiting conditions. When OsHKTs were expressed in Na + -sensitive yeast, OsHKT1 rendered the cells more Na + -sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na + , but not K + , shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na + or K + in the external solution shifted the reversal potential toward depolarization under the mixed Na + and K + containing solutions. These results suggest that two isoforms of HKT transporters, a Na + transporter (OsHKT1) and a Na + -and K + -coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice.

Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH, and KdpA transporter subunits and fungal Trk proteins. These transporters contain four loop domains in one polypeptide with a proposed distant homology to K ؉ channel selectivity filters. Functional expression in yeast and Xenopus oocytes revealed that wheat HKT1 mediates Na ؉ -coupled K ؉ transport. Arabidopsis AtHKT1, however, transports only Na ؉ in eukaryotic expression systems. To understand the molecular basis of this difference we constructed a series of AtHKT1͞HKT1 chimeras and introduced point mutations to AtHKT1 and wheat HKT1 at positions predicted to be critical for K ؉ selectivity. A single-point mutation, Ser-68 to glycine, was sufficient to restore K ؉ permeability to AtHKT1. The reverse mutation in HKT1, Gly-91 to serine, abrogated K ؉ permeability. This glycine in P-loop A of AtHKT1 and HKT1 can be modeled as the first glycine of the K ؉ channel selectivity filter GYG motif. The importance of such filter glycines for K ؉ selectivity was confirmed by interconversion of Ser-88 and Gly-88 in the rice paralogues OsHKT1 and OsHKT2. Surprisingly, all HKT homologues known from dicots have a serine at the filter position in P-loop A, suggesting that these proteins function mainly as Na ؉ transporters in plants and that Na ؉ ͞K ؉ symport in HKT proteins is associated with a glycine in the filter residue. These data provide experimental evidence that the glycine residues in selectivity filters of HKT proteins are structurally related to those of K ؉ channels.

We report a method for the successful reconstitution of the KcsA potassium channel with either an outside-out or inside-out orientation in giant unilamellar vesicles, using the droplet-transfer technique. The procedure is rather simple. First, we prepared water-in-oil droplets lined with a lipid monolayer. When solubilized KcsA was encapsulated in the droplet, it accumulated at monolayers of phosphatidylglycerol (PG) and phosphoethanolamine (PE) but not at a monolayer of phosphatidylcholine (PC). The droplet was then transferred through an oil/water interface having a preformed monolayer. The interface monolayer covered the droplet so as to generate a bilayer vesicle. By creating chemically different lipid monolayers at the droplet and oil/water interface, we obtained vesicles with asymmetric lipid compositions in the outer and inner leaflets. KcsA was spontaneously inserted into vesicles from the inside or outside, and this was accelerated in vesicles that contained PE or PG. Integrated insertion into the vesicle membrane and the KcsA orientation were examined by functional assay, exploiting the pH sensitivity of the opening of the KcsA when the pH-sensitive cytoplasmic domain (CPD) faces toward acidic media. KcsA loaded from the inside of the PG-containing vesicles becomes permeable only when the intravesicular pH is acidic, and the KcsA loaded from the outside becomes permeable when the extravesicular pH is acidic. Therefore, the internal or external insertion of KcsA leads to an outside-out or inside-out configuration so as to retain its hydrophilic CPD in the added aqueous side. The CPD-truncated KcsA exhibited a random orientation, supporting the idea that the CPD determines the orientation. Further application of the droplet-transfer method is promising for the reconstitution of other types of membrane proteins with a desired orientation into cell-sized vesicles with a targeted lipid composition of the outer and inner leaflets.

Membrane lipids modulate the function of membrane proteins. In the case of ion channels, they bias the gating equilibrium, although the underlying mechanism has remained elusive. Here we demonstrate that the N-terminal segment (M0) of the KcsA potassium channel mediates the effect of changes in the lipid milieu on channel gating. The M0 segment is a membrane-anchored amphipathic helix, bearing positively charged residues. In asymmetric membranes, the M0 helix senses the presence of negatively charged phospholipids on the inner leaflet. Upon gating, the M0 helix revolves around the axis of the helix on the membrane surface, inducing the positively charged residues to interact with the negative head groups of the lipids so as to stabilize the open conformation (i.e., the "rolland-stabilize model"). The M0 helix is thus a charge-sensitive "antenna," capturing temporary changes in lipid composition in the fluidic membrane. This unique type of sensory device may be shared by various types of membrane proteins.fluorescence measurements | single-channel current | gating kinetics | pH-dependence | activation gate T he cell membrane bears distinctly different kinds of membrane proteins within the matrix of membrane lipids (1), and the lipids are not merely structural building blocks, but substantially modulate the function of membrane proteins (2). The membrane matrix deforms and readily changes its curvature in the manner of an elastic material, and its membrane-embedded proteins are subjected to a variety of physical stresses (3, 4). At the boundary of the matrix, physical effects, such as lateral pressure and tension, modulate the functional properties of the membrane proteins (5-7). In addition to these nonspecific modulating effects, membrane lipids have been suggested to react with specific parts of the membrane proteins. In the fluidic membrane, the lateral diffusion of lipid molecules facilitates the exchange of lipids at the boundary of the membrane proteins, and the membrane matrix is the reaction platform for signal transduction (8).Data have been reported on the functionally modifying effects of membrane lipids on channel proteins that result in the gating equilibrium being altered (6, 9-13). In voltage-gated channels, the voltage-sensor domain (VSD) primarily senses changes in the membrane electric field, but this sensing is modulated by the lipid composition (9, 11, 13). For the non-voltage-gated (the twotransmembrane-helix inward-rectifier type) channels, such as the KcsA potassium channel from Streptomyces lividans, the underlying mechanism of the effect exerted by lipids is still undergoing extensive investigation, even though the structural information on the channel proteins cocrystallized with lipid molecules has already been reported (14-16).Here we demonstrate that there is a specialized structural interface in the KcsA potassium channel that senses the membrane milieu and mediates the effect of changes in the lipid composition on channel gating. The N-terminal M0 segment of the KcsA channel is not ...