Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants

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 AtHKT1/HKT1 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.     

Thylakoid potassium channel is required for efficient photosynthesis in cyanobacteria

A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b(6)f turnover and respiration pointed to a reduced generation of ΔpH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis.     

High Incidence of Morphological Myelodysplasia and Apoptotic Bone Marrow Cells in Behçet’s Disease

Beh?et's disease (BD) is a systemic inflammatory disorder of unknown etiology, and rarely complicated with myelodysplastic syndrome (MDS). In the present study, we investigated the morphological myelodysplasia and apoptotic rate of bone marrow cells in 15 patients with BD in comparison with MDS patients. Morphological myelodysplasia of bone marrow cells was detected in 53.3% of BD, but none showed chromosomal abnormalities. The apoptotic rate in BD patients (26.1 +/- 8.4%) was significantly higher in normal controls (11.3 +/- 2.4%; p < 0.005) and significantly lower in patients with MDS (50.8 +/- 14.0%; p < 0.0001). These findings suggest that myelodysplasia in patients with BD is more frequent than expected, and possibly due to excess induction of apoptosis of bone marrow cells in BD. However, the rate of apoptotic bone marrow cells is lower than MDS, which may explain the slight peripheral cytopenia in BD, distinct from that in MDS.     

Determination of transmembrane topology of an inward-rectifying potassium channel from Arabidopsis thaliana based on functional expression in Escherichia coli

We report here that the inward-rectifying potassium channels KAT1 and AKT2 were functionally expressed in K⁺ uptake-deficient Escherichia coli. Immunological assays showed that KAT1 was translocated into the cell membrane of E. coli. Functional assays suggested that KAT1 was inserted topologically correctly into the cell membrane. In control experiments, the inactive point mutation in KAT1, T256R, did not complement for K⁺ uptake in E. coli. The inward-rectifying K⁺ channels of plants share a common hydrophobic domain comprising at least six membrane-spanning segments (S1–S6). The finding that a K⁺ channel can be expressed in bacteria was further exploited to determine the KAT1 membrane topology by a gene fusion approach using the bacterial reporter enzymes, alkaline phosphatase, which is active only in the periplasm, and β-galactosidase. The enzyme activity from the alkaline phosphatase and β-galactosidase fusion plasmid showed that the widely predicted S1, S2, S5, and S6 segments were inserted into the membrane. Although the S3 segment in the alkaline phosphatase fusion protein could not function as an export signal, the replacement of a negatively charged residue inside S3 with a neutral amino acid resulted in an increase in alkaline phosphatase activity, which indicates that the alkaline phosphatase was translocated into the periplasm. For membrane translocation of S3, the neutralization of a negatively charged residue in S3 may be required presumably because of pairing with a positively charged residue of S4. These results revealed that KAT1 has the common six transmembrane-spanning membrane topology that has been predicted for the Shaker superfamily of voltage-dependent K⁺ channels. Furthermore, the functional complementation of a bacterial K⁺ uptake mutant in this study is shown to be an alternative expression system for plant K⁺ channel proteins and a potent tool for their topological analysis.     

Expression of the intestinal T-lymphocyte-associated-molecule recognized by the HML-1 antibody on mononuclear cells from HTLV-I-infected subjects

We investigated the expression of a monoclonal antibody (HML-1) defined antigen that appears on human intestinal T-lymphocytes in HTLV-I-related disease. We studied 25 ATL, and 24 healthy HTLV-I carriers. Patients with acute ATL showed a variety of the expression of the HML-1 antigen (range 0.4–74.8%). HML-1 expression on mononuclear cells (MNCs) in blood from patients with chronic ATL ranged from 1.7–43.6% (mean 13.5%). This level of expression was less than that of patients with acute ATL, but not significantly. In patients with smoldering ATL, the degree of patients with acute ATL, but not significantly. In patients with smoldering ATL, the degree of expression ranged from 1.6–13.3% (mean 8.0%). In contrast to patients wtih acute ATL, MNCs from patients with acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), and B-cell type chronic lymphocytic leukemia (B-CLL) did not express the HML-1 antigen, except for the 2 patients with ALL. Healthy HTLV-I carriers and healthy controls also were negative for HML-1 reactivity. In acute ATL, patients with gastrointestinal tract infiltration tended to have high expression of the HML-1 epitope. After stimulation with phytohemagglutinin (PHA), healthy HTLV-I carriers showed significantly increased expression of the HML-1 epitope (P < 0.05). Recently, the β7 integrin family has been found to play a specific role in mucosal localization or adhesion, and HML-1 protein was found to match the deduced β7 N-terminal sequence. We propose that the cellular gene responsible for HML-1 epitope expression may, like IL-2, IL-2R, etc., be transactivated by infection with HTLV-I, and that HML-1 antigen gene expression by HTLV-I infection may lead to infiltration of ATL cells with highly expressed HML-1 epitope into the gut mucosa.     

Muscular Metastasis of Hepatocellular Carcinoma: Case Report and Literature Review

There are few case reports of hepatocellular carcinoma (HCC) metastasis to the skeletal muscle. A 78-year-old man developed a mass in the right shoulder. Washout of contrast medium during contrast-enhanced ultrasonography (CEUS) in both the primary HCC and the metastatic site was detected. Several nodules were scattered throughout the liver on an autopsy. In addition, the moderately differentiated HCC had metastasized to the right teres major muscle. Rare muscular metastasis should be considered if a hepatic tumor is moderately or poorly differentiated HCC. Early washout during CEUS is consistent with a pathological diagnosis of moderately or poorly differentiated HCC.