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J W Hell

Publications and source records attributed to J W Hell.

30 records · Page 2Linked to original sources
PubMed1995

Biochemical properties and subcellular distribution of the neuronal class E calcium channel alpha 1 subunit.

Anti-peptide antibodies specific for the neuronal calcium channel alpha 1E subunit (anti-CNE1 and anti-CNE2) were produced to study the biochemical properties and subcellular distribution of the alpha 1E polypeptide from rat brain. Immunoblotting identified a single size form of 245-255 kDa which was a substrate for phosphorylation by cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II. Ligand-binding studies of alpha 1E indicate that it is not a high affinity receptor for the dihydropyridine isradipine or the peptide toxins omega-conotoxin GVIA or omega-conotoxin MVIIC at concentrations which elicit high affinity binding to other channel types in the same membrane preparation. The alpha 1E subunit is widely distributed in the brain with the most prominent immunocytochemical staining in deep midline structures such as caudate-putamen, thalamus, hypothalamus, amygdala, cerebellum, and a variety of nuclei in the ventral midbrain and brainstem. Staining is primarily in the cell soma but is also prominent in the dendritic field of a discrete subset of neurons including the mitral cells of the olfactory bulb and the distal dendritic branches of the cerebellar Purkinje cells. Our observations indicate that the 245-255 kDa alpha 1E subunit is localized in cell bodies, and in some cases in dendrites, of a broad range of central neurons and is potentially modulated by multiple second messenger-activated protein kinase.

Animals↗
PubMed1994

Differential phosphorylation of two size forms of the N-type calcium channel alpha 1 subunit which have different COOH termini.

Two size forms of the class B N-type calcium channel alpha 1 subunit were recently identified with CNB1, an antipeptide antibody directed against an intracellular loop of this channel (Westenbroek, R.E., Hell, J.W., Warner, C., Dubel, S.J., Snutch, T.P., and Catterall, W.A. (1992) Neuron 9, 1099-1115). To investigate the biochemical differences between these two size forms, the antibodies CNB3 and CNB4 were raised against peptides with sequences corresponding to the COOH-terminal end of the full-length form. Immunoblot experiments demonstrated that both antibodies specifically recognize the longer form of 250 kDa, indicating that the COOH-terminal regions of the two size forms of the class B N-type channel alpha 1 subunit are different. Phosphorylation experiments with immunopurified calcium channels and different second messenger-activated protein kinases revealed that both the 220- and 250-kDa forms of the class B N-type calcium channel alpha 1 subunit are substrates for cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein kinase C. These three kinases incorporated approximately 1 mol of phosphate/mol of binding sites for omega-conotoxin (omega-CgTx) GVIA, a ligand specific for the N-type calcium channel, and may regulate the activity of both forms in vivo. In contrast, calcium- and calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated only the long form of the class B N-type calcium channel alpha 1 subunit, with a stoichiometry of 0.5 mol of phosphate/mol of total omega-CgTx GVIA binding sites. Specific phosphorylation of the long form of the class B alpha 1 subunit by CaM kinase II may differentially regulate the function of N-type calcium channels containing different size forms of their alpha 1 subunits in vivo.

Amino Acid Sequence↗
PubMed1993

Differential phosphorylation of two size forms of the neuronal class C L-type calcium channel alpha 1 subunit.

L-type calcium channels mediate long-lasting calcium currents which are modulated by protein phosphorylation. Using site-directed anti-peptide antibodies, we show that the alpha 1 subunit of the neuronal class C L-type calcium channel from rat brain exists in two size forms. The longer form, LC2, with an apparent molecular mass of 210-235 kDa was phosphorylated in vitro by cAMP-dependent protein kinase (cA-PK), but the shorter form, LC1, with an apparent molecular mass of 190-195 kDa was not a substrate for cA-PK. In contrast, LC1 and LC2 are both substrates for protein kinase C (PKC), calcium- and calmodulin-dependent protein kinase II, and cGMP-dependent protein kinase (cG-PK). The site-directed anti-peptide antibody CNC2 was produced against the COOH-terminal end of the class C L-type alpha 1 subunit as predicted by molecular cloning and sequencing of cDNA. CNC2 recognized LC2 but not LC1 by immunoblotting and immunoprecipitated only LC2 phosphorylated by either cA-PK or PKC. These results indicate that LC1 is truncated at its COOH-terminal end with respect to LC2 and that cA-PK preferentially phosphorylates sites in the COOH-terminal region of the alpha 1 subunit that are present in LC2 but not LC1. The selectivity of cA-PK for phosphorylation of the COOH-terminal region of LC2 suggests that the channel activities of the two alpha 1 subunit size forms may be differentially regulated by neurotransmitters and hormones which act through cAMP-dependent mechanisms, while both alpha 1 subunit isoforms may be modulated by PKC, cG-PK, and calcium- and calmodulin-dependent protein kinase II.

Amino Acid Sequence↗
PubMed1993

A gamma-aminobutyric acid transporter driven by a proton pump is present in synaptic-like microvesicles of pancreatic beta cells.

A variety of peptide-secreting endocrine cells contain a population of recycling microvesicles that share several major membrane polypeptides with neuronal synaptic vesicles (SVs). The function of these synaptic-like microvesicles (SLMVs) remains to be elucidated. It was previously suggested that SLMVs of pancreatic beta cells may store and secrete gamma-aminobutyric acid (GABA). GABA, the major nonpeptide inhibitory neurotransmitter of the central nervous system, is stored in and secreted from SVs. GABA uptake into SVs is mediated by a transporter that is driven by a vacuolar proton ATPase. GABA is also present at high concentration in the endocrine pancreas where it is selectively localized in insulin-secreting beta cells, the core cells of pancreatic islets. GABA is not present in peripheral islet cells (mantle cells), represented primarily by glucagon-secreting alpha cells. In this study, an immunoisolation procedure was used to purify SLMVs from cell lines derived from mouse beta cells and alpha cells. SLMVs obtained from the beta-cell line, but not those obtained from the alpha-cell line, displayed a GABA-transport activity dependent upon a proton electrochemical gradient generated by a vacuolar proton ATPase. These data support the hypotheses that (i) SLMVs have a secretory function similar to that of SVs and (ii) beta-cell SLMVs are involved in the secretion of GABA, which in turn may have a paracrine function on mantle cells of the islet.

Adenosine Triphosphate↗
PubMed1993

Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits.

To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D alpha 1 subunits were produced. Anti-CNC1 directed against class C immunoprecipitated 75% of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type alpha 1 subunit, LC1 and LC2, and two size forms of the class D L-type alpha 1 subunit, LD1 and LD2. The larger isoforms had apparent molecular masses of approximately 200-210 kD while the smaller isoforms were 180-190 kD, as estimated from electrophoresis in gels polymerized from 5% acrylamide. Immunocytochemical studies using CNC1 and CND1 antibodies revealed that the alpha 1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNC1, particularly in the dentate gyrus and the CA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calcium-dependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.

Amino Acid Sequence↗
PubMed1992

Biochemical properties and subcellular distribution of an N-type calcium channel alpha 1 subunit.

A site-directed anti-peptide antibody, CNB-1, that recognizes the alpha 1 subunit of rat brain class B calcium channels (rbB) immunoprecipitated 43% of the N-type calcium channels labeled by [125I]omega-conotoxin. CNB-1 recognized proteins of 240 and 210 kd, suggesting the presence of two size forms of this alpha 1 subunit. Calcium channels recognized by CNB-1 were localized predominantly in dendrites; both dendritic shafts and punctate synaptic structures upon the dendrites were labeled. The large terminals of the mossy fibers of the dentate gyrus granule neurons were heavily labeled, suggesting that the punctate labeling pattern represents calcium channels in nerve terminals. The pattern of immunostaining was cell specific. The cell bodies of some pyramidal cells in layers II, III, and V of the dorsal cortex, Purkinje cells, and scattered cell bodies elsewhere in the brain were also labeled at a low level. The results define complementary distributions of N- and L-type calcium channels in dendrites, nerve terminals, and cell bodies of most central neurons and support distinct functional roles in calcium-dependent electrical activity, intracellular calcium regulation, and neurotransmitter release for these two channel types.

Amino Acid Sequence↗
PubMed1991

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.

Animals↗
PubMed1990

Energy dependence and functional reconstitution of the gamma-aminobutyric acid carrier from synaptic vesicles.

The energy dependence of gamma-aminobutyric acid (GABA) uptake was characterized in rat brain synaptic vesicles and in proteoliposomes reconstituted with a new procedure from vesicular detergent extracts. The proteoliposomes displayed high ATP-dependent GABA uptake activity with properties virtually identical to those of intact vesicles. GABA uptake was similar at chloride concentrations of 0 and 150 mM, i.e. conditions under which either the membrane potential (delta psi) or the pH difference (delta pH) predominates. Delta psi was gradually dissipated by increasing the concentration of SCN-. GABA uptake was reduced by 10 mM SCN-, showing less sensitivity to delta psi reduction than glutamate uptake but more than dopamine uptake. Dissipation of delta pH with NH+4 abolished GABA uptake at pH 7.3, whereas no significant inhibition occurred at pH 6.5. In contrast, dopamine uptake was inhibited more strongly, even at pH 6.5, and glutamate uptake was not reduced in either condition. We conclude that GABA uptake is driven by both components of the proton electrochemical gradient, delta pH and delta psi, and that this is different from the uptake of both dopamine and glutamate, which is more strongly dependent on delta pH and delta psi, respectively. Thus, our data suggest that GABA uptake is electrogenic and occurs in exchange for protons.

Adenosine Triphosphate↗
PubMed1990

Amino acid neurotransmission: spotlight on synaptic vesicles.

The vesicle hypothesis describing quantal release of neurotransmitter at the cholinergic neuromuscular junction was introduced in 1956. Since then, the concept of vesicular storage and release of acetylcholine has become firmly established and extended to include other synapses and neurotransmitters. However, for the amino acids, which are the major class of neurotransmitters in the mammalian CNS, there was no direct experimental evidence of the participation of synaptic vesicles in neurotransmission. This area of research has now moved out of the shadows and this article discusses recent findings which indicate that amino acid neurotransmitters are accumulated and stored by synaptic vesicles in presynaptic nerve endings.

Amino Acids↗
PubMed1988

Glutamate uptake by brain synaptic vesicles. Energy dependence of transport and functional reconstitution in proteoliposomes.

The dependence of glutamate uptake on ATP-generated proton electrochemical potential was studied in a highly purified preparation of synaptic vesicles from rat brain. At low chloride concentration (4 mM), the proton pump present in synaptic vesicles generated a large membrane potential (inside-positive), associated with only minor acidification. Under these conditions, the rate of L-[3H]glutamate uptake was maximal. In addition, L-glutamate induced acidification of the vesicle interior. D-Glutamate produced only 40% of the effect, and L-aspartate or gamma-aminobutyric acid produced less than 5%. The initial rate of glutamate-induced acidification increased with increasing glutamate concentration. It was saturable and showed first-order kinetics (KM = 0.32 mM). Correspondingly, L-glutamate induced a small reduction in the membrane potential. The rate of ATP hydrolysis was unaffected. In comparison, glutamate had no effect on acidification or membrane potential in resealed membranes of chromaffin granules. At high chloride concentration (150 mM), the vesicular proton pump generated a large pH difference, associated with a small change in membrane potential. Under these conditions, uptake of L-[3H]glutamate by synaptic vesicles was low. For reconstitution, vesicle proteins were solubilized with the detergent sodium cholate, supplemented with brain phospholipids, and incorporated into liposomes. Proton pump and glutamate uptake activities of the proteoliposomes showed properties similar to those of intact vesicles indicating that the carrier was reconstituted in a functionally active form. It is concluded that glutamate uptake by synaptic vesicles is dependent on the membrane potential and that all components required for uptake are integral parts of the vesicle membrane.

Acridine Orange↗
PubMed1988

Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure.

Uptake of GABA was demonstrated in rat brain synaptic vesicles which were prepared by a new and efficient procedure. The uptake activity co-purified with the synaptic vesicles during the isolation procedure. The purity of the vesicle fraction was rigorously examined by analysis of marker enzymes and marker proteins and also by immunogold electron microscopy using antibodies against p38 (synaptophysin). Contamination by other cellular components was negligible, indicating that GABA uptake by the synaptic vesicle fraction is specific for synaptic vesicles and not due to the presence of other structure possessing GABA uptake or binding activities. GABA uptake was ATP dependent and similar to the uptake of glutamate, which was assayed for a comparison. Both uptake activities were independent of sodium. They were inhibited by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, indicating that the energy for the uptake is provided by an electrochemical proton gradient. This gradient is generated by a proton ATPase of the vacuolar type as suggested by the effects of various ATPase inhibitors on neurotransmitter uptake and proton pumping. Competition experiments revealed that the transporters for GABA and glutamate are selective for the respective neurotransmitters.

Adenosine Triphosphatases↗