New mammalian chloride channel identified by expression cloning.

Ion channels selectively permeable to chloride ions regulate cell functions as diverse as excitability and control of cell volume. Using expression cloning techniques, a complementary DNA from an epithelial cell line has been isolated, sequenced and its putative structure examined by site-directed mutagenesis. This cDNA, encoding a 235-amino-acid protein, gave rise to a chloride-selective outward current when expressed in Xenopus oocytes. The expressed, outwardly rectifying chloride current was calcium-insensitive and was blocked by nucleotides applied to the cell surface. Mutation of a putative nucleotide-binding site resulted in loss of nucleotide block but incurred dependence on extracellular calcium concentration. The unusual sequence of this putative channel protein suggests a new class of ion channels not related to other previously cloned chloride channels.     

Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel

Voltage-gated sodium (Na(v)) channels are essential for the rapid depolarization of nerve and muscle, and are important drug targets. Determination of the structures of Na(v) channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Na(v) channels, exemplified by the Na(+)-selective channel of bacteria (NaChBac), provides a useful model system for structure-function analysis. Here we report the crystal structure of Na(v)Rh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 (Rickettsiales sp. HIMB114; denoted Rh), at 3.05?? resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr?178 and Leu?179 constitute an inner site within the selectivity filter where a hydrated Ca(2+) resides in the crystal structure. The outer mouth of the Na(+) selectivity filter, defined by Ser?181 and Glu?183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that Na(v)Rh is in an 'inactivated' conformation. Comparison of Na(v)Rh with Na(v)Ab reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.     

Soluble CD4 blocks the infectivity of diverse strains of HIV and SIV for T cells and monocytes but not for brain and muscle cells

The CD4 antigen has been subverted as a receptor by the human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV). Several groups have reported that recombinant, soluble forms of the CD4 molecule (sCD4) block the infection of T lymphocytes by HIV-1, as CD4 binds the HIV envelope glycoprotein, gp120, with high affinity. We now report that sCD4 blocks diverse strains of HIV-1, HIV-2 and SIV, but is less effective for HIV-2. The blocking effect is apparent even after adsorption of virions to CD4 cells. Soluble CD4 prevents HIV infection of T-lymphocytic and myelomonocytic cell lines, but neither sCD4 nor anti-CD4 antibodies inhibit infection of glioma and rhabdomyosarcoma cell lines.     

The mitochondrial calcium uniporter is a highly selective ion channel

During intracellular Ca2+ signalling mitochondria accumulate significant amounts of Ca2+ from the cytosol. Mitochondrial Ca2+ uptake controls the rate of energy production, shapes the amplitude and spatio-temporal patterns of intracellular Ca2+ signals, and is instrumental to cell death. This Ca2+ uptake is undertaken by the mitochondrial Ca2+ uniporter (MCU) located in the organelle's inner membrane. The uniporter passes Ca2+ down the electrochemical gradient maintained across this membrane without direct coupling to ATP hydrolysis or transport of other ions. Carriers are characterized by turnover numbers that are typically 1,000-fold lower than ion channels, and until now it has been unclear whether the MCU is a carrier or a channel. By patch-clamping the inner mitochondrial membrane, we identified a previously unknown Ca2+-selective ion channel sensitive to inhibitors of mitochondrial Ca2+ uptake. Our data indicate that this unique channel binds Ca2+ with extremely high affinity (dissociation constant < or =2 nM), enabling high Ca2+ selectivity despite relatively low cytoplasmic Ca2+ concentrations. The channel is inwardly rectifying, making it especially effective for Ca2+ uptake into energized mitochondria. Thus, we conclude that the properties of the current mediated by this novel channel are those of the MCU.     

Evolutionary genetics: Ambiguous role of CCR5 in Y. pestis infection

Mecsas and colleagues suggest that a deficiency in the chemokine receptor CCR5 in humans is unlikely to confer protection against plague, based on their study of Yersinia pestis infection in Ccr5-deficient mice. They were testing the hypothesis that a mutation in the CCR5 gene, frequently found in Caucasians, may have been selected for in the past because it provided protection against (bubonic) plague; the mutation, called CCR5Delta32, is characterized by a 32-base-pair deletion. We have also tested this hypothesis by using Y. pestis infection in mice and, in addition, we have done phagocytosis experiments with macrophages from wild-type and Ccr5-deficient mice. Although, like Mecsas et al., we did not see any difference in the survival of the two groups of mice, we did find that there was a significantly reduced uptake of Y. pestis by Ccr5-deficient macrophages in vitro. Our results indicate that the role of Ccr5 in Y. pestis infection may therefore be more complex than previously thought.     

Variable and conserved neutralization antigens of human immunodeficiency virus

Human immunodeficiency virus type 1 (HIV-1, HTLV-III/LAV), the retrovirus responsible for acquired immune deficiency syndrome (AIDS), shows a high degree of genetic polymorphism, particularly in the env gene. We have examined sera from rabbits and guinea pigs immunized with gp130, a recombinant env glycoprotein, and sera from HIV-1-infected subjects, to test their capacity to neutralize a panel of genetically divergent HIV-1 isolates. The sera raised against recombinant antigen specifically neutralized the virus strain from which the env gene was cloned (HTLV-IIIB), but not an independent isolate (HTLV-IIIRF). One rabbit serum tested on seven isolates cross-neutralized two at lower titres. In contrast, human sera from Britain and Uganda, chosen for ability to neutralize HTLV-IIIRF, cross-neutralized six other HIV-1 isolates. When serum and isolate were derived from the same subject, the serum was in some cases effective at slightly lower concentrations (higher titres). Human complement did not affect neutralization titres. These findings indicate that genetically diverse HIV-1 isolates carry both variable and widely conserved antigenic epitopes for neutralizing antibodies. The identification of shared epitopes may help the development of protective vaccines.