Acceleration of activation and inactivation by the beta subunit of the skeletal muscle calcium channel.

The L-type voltage-dependent calcium channel is an important link in excitation-contraction coupling of muscle cells (reviewed in refs 2 and 3). The channel has two functional characteristics: calcium permeation and receptor sites for calcium antagonists. In skeletal muscle the channel is a complex of five subunits, alpha 1, alpha 2, beta, gamma and delta. Complementary DNAs to these subunits have been cloned and their amino-acid sequences deduced. The skeletal muscle alpha 1 subunit cDNA expressed in L cells manifests as specific calcium-ion permeation, as well as sensitivity to the three classes of organic calcium-channel blockers. We report here that coexpression of the alpha 1 subunit with other subunits results in significant changes in dihydropyridine binding and gating properties. The available number of drug receptor sites increases 10-fold with an alpha 1 beta combination, whereas the affinity of the dihydropyridine binding site remains unchanged. Also, the presence of the beta subunit accelerates activation and inactivation kinetics of the calcium-channel current.     

Cloning of cDNA for the glutamate-binding subunit of an NMDA receptor complex.

The amino acids L-glutamic and L-aspartic acids form the most widespread excitatory transmitter network in mammalian brain. The excitation produced by L-glutamic acid is important in the early development of the nervous system, synaptic plasticity and memory formation, seizures and neuronal degeneration. The receptors activated by L-glutamic acid are a target for therapeutic intervention in neurodegenerative diseases, brain ischaemia and epilepsy. There are two types of receptors for the excitatory amino acids, those that lead to the opening of cation-selective channels and those that activate phospholipase C (ref. 11). The receptors activating ion channels are NMDA (N-methyl-D-aspartate) and kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive receptors. The complementary DNAs for the kainate/AMPA receptor and for the metabotropic receptor have been cloned. We report here on the isolation and characterization of a protein complex of four major proteins that represents an intact complex of the NMDA receptor ion channel and on the cloning of the cDNA for one of the subunits of this receptor complex, the glutamate-binding protein.     

Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation.

Cystic fibrosis is associated with a defect in epithelial chloride ion transport which is caused by mutations in a membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator). Heterologous expression of CFTR produces cyclicAMP-sensitive Cl(-)-channel activity. Deletion of phenylalanine at amino-acid position 508 in CFTR (delta F508 CFTR) is the most common mutation in cystic fibrosis. It has been proposed that this mutation prevents glycoprotein maturation and its transport to its normal cellular location. We have expressed both CFTR and delta F508 CFTR in Vero cells using recombinant vaccinia virus. Although far less delta F508 CFTR reached the plasma membrane than normal CFTR, sufficient delta F508 CFTR was expressed at the plasma membrane to permit functional analysis. delta F508 CFTR expression induced a reduced activity of the cAMP-activated Cl- channel, with conductance, anion selectivity and open-time kinetics similar to those of CFTR, but with much greater closed times, resulting in a large decrease of open probability. The delta F508 mutation thus seems to have two major consequences, an abnormal translocation of the CFTR protein which limits membrane insertion, and an abnormal function in mediating Cl- transport.     

Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.     

A three-base-pair deletion in the peripherin-RDS gene in one form of retinitis pigmentosa.

The group of retinopathies termed retinitis pigmentosa (RP) greatly contribute to visual dysfunction in man with a frequency of roughly 1 in 4,000. We mapped the first autosomal dominant RP (adRP) gene to chromosome 3q, close to the gene encoding rhodopsin, a rod photoreceptor pigment protein. Subsequently, mutations in this gene have been implicated as responsible for some forms of adRP. Another adRP gene has been mapped to chromosome 8p. A third adRP gene in a large Irish pedigree has been mapped to chromosome 6p, showing tight linkage with the gene for peripherin, a photoreceptor cell-specific glycoprotein, which is thus a strong candidate for the defective gene. We have now identified a three-base-pair deletion which results in the loss of one of a pair of highly conserved cysteine residues in the predicted third transmembrane domain of peripherin. This deletion segregates with the disease phenotype but is not present in unaffected controls, and suggests that mutant peripherin gives rise to retinitis pigmentosa.     

Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA.

Splicing of pre-messenger RNA in eukaryotic cells occurs in a multicomponent complex termed the spliceosome, which contains small nuclear ribonucleoprotein particles (snRNPs), protein factors and substrate pre-mRNA. Assembly of the spliceosome involves the stepwise binding of snRNPs and protein factors to the pre-mRNA through a poorly understood mechanism which probably involves specific RNA-RNA, RNA-protein and protein-protein interactions. Of particular interest are the interactions between snRNPs, which are likely to be important not only for assembly of the spliceosome but also for catalysis. U1 snRNP interacts with the 5' splice site and U2 snRNP with the branch site of the pre-mRNA; both of these interactions involve Watson-Crick base pairing. But very little is known about how other factors such as the U4/U6 and U5 snRNPs reach the spliceosome and function in splicing. Here we report evidence that U6 snRNA interacts directly with U2 snRNA by a mechanism involving base-pairing, and that this interaction can be necessary for splicing of a mammalian pre-mRNA in vivo.     

CD4+ and CD8+ T cells acquire specific lymphokine secretion potentials during thymic maturation.

Peripheral CD4+ and CD8+ T lymphocytes carry out different functions during immune reactions, partly as a result of the distinct patterns of lymphokines that they secrete upon stimulation. Using thymic cells from adult and newborn mice as well as from fetal organ cultures, we show here that this functional differentiation occurs inside the thymus and is completed during the single positive stage by the time the T-cell receptor becomes fully coupled to the intracellular activation pathways leading to lymphokine secretion. Surprisingly, CD4+8- thymocytes differ from their immediate progeny, naive peripheral CD4+ cells, in that they secrete a broader range of lymphokines, including interleukins 4, 5 and 10 and gamma-interferon, and more closely resemble immunologically experienced (activated or memory) CD4+ lymphocytes.     

Type 1 diabetes in mice is linked to the interleukin-1 receptor and Lsh/Ity/Bcg genes on chromosome 1.

Human type 1 (insulin-dependent) diabetes is a common auto-immune disease of the insulin-producing beta cells of the pancreas which is caused by both genetic and environmental factors. Several features of the genetics and immunopathology of diabetes in nonobese diabetic (NOD) mice are shared with the human disease. Of the three diabetes-susceptibility genes, Idd-1 -3 and -4 that have been mapped in mice to date, only in the case of Idd-1 is there any evidence for the identity of the gene product: allelic variation within the murine immune response I-A beta gene and its human homologue HLA-DQB1 correlates with susceptibility, implying that I-A beta is a component of Idd-1. We report here the mapping of Idd-5 to the proximal region of mouse chromosome 1. This region contains at least two candidate susceptibility genes, the interleukin-1 receptor gene and Lsh/Ity/Bcg, which encodes resistance to bacterial and parasitic infections and affects the function of macrophages.     

Rapid spread of an inherited incompatibility factor in California Drosophila

In Drosophila simulans in California, an inherited cytoplasmic incompatibility factor reduces egg hatch when infected males mate with uninfected females. The infection is spreading at a rate of more than 100 km per year; populations in which the infection was rare have become almost completely infected within three years. Analyses of the spread using estimates of selection in the field suggest dispersal distances far higher than those found by direct observation of flies. Hence, occasional long-distance dispersal, possibly coupled with local extinction and recolonization, may be important to the dynamics. Incompatibility factors that can readily spread through natural populations may be useful for population manipulation and important as a post-mating isolating mechanism.     

Hsp104 is a highly conserved protein with two essential nucleotide-binding sites

Most eukaryotic cells produce proteins with relative molecular masses in the range of 100,000 to 110,000 after exposure to high temperatures. These proteins have been studied only in yeast and mammalian cells. In Saccharomyces cerevisiae, heat-shock protein hsp104 is vital for tolerance to heat, ethanol and other stresses. The mammalian hsp110 protein is nucleolar and redistributes with growth state, nutritional conditions and heat shock. The relationships between hsp110, hsp104 and the high molecular mass heat-shock proteins of other organisms were unknown. We report here that hsp104 is a member of the highly conserved ClpA/ClpB protein family first identified in Escherichia coli and that additional heat-inducible members of this family are present in Schizosaccharomyces pombe and in mammals. Mutagenesis of two putative nucleotide-binding sites in hsp104 indicates that both are essential for function in thermotolerance.