Johannes W. Hell

Biochemical properties and subcellular distribution of an N-type calcium hannel α1 subunit

Abstract A site-directed anti-peptide antibody, CNB-1, that recognizes the α1 subunit of rat brain class B calcium channels (rbB) immunoprecipitated 43% of the N-type calcium channels labeled by [ 125 I]ω-conotoxin. CNB-1 recognized proteins of 240 and 210 kd, suggesting the presence of two size forms of this α1 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 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.

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.

GABA and glycine in synaptic vesicles: storage and transport characteristics

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.

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.

Phosphorylation of presynaptic and postsynaptic calcium channels by cAMP-dependent protein kinase in hippocampal neurons.

Phosphorylation by cAMP‐dependent protein kinase (PKA) and other second messenger‐activated protein kinases modulates the activity of a variety of effector proteins including ion channels. Anti‐peptide antibodies specific for the alpha 1 subunits of the class B, C or E calcium channels from rat brain specifically recognize a pair of polypeptides of 220 and 240 kDa, 200 and 220 kDa, and 240 and 250 kDa, respectively, in hippocampal slices in vitro. These calcium channels are localized predominantly on presynaptic and dendritic, somatic and dendritic, and somatic sites, respectively, in hippocampal neurons. Both size forms of alpha 1B and alpha 1E and the full‐length form of alpha 1C are phosphorylated by PKA after solubilization and immunoprecipitation. Stimulation of PKA in intact hippocampal slices also induced phosphorylation of 25‐50% of the PKA sites on class B N‐type calcium channels, class C L‐type calcium channels and class E calcium channels, as assessed by a back‐phosphorylation method. Tetraethylammonium ion (TEA), which causes neuronal depolarization and promotes repetitive action potentials and neurotransmitter release by blocking potassium channels, also stimulated phosphorylation of class B, C and E alpha 1 subunits, suggesting that these three classes of channels are phosphorylated by PKA in response to endogenous electrical activity in the hippocampus. Regulation of calcium influx through these calcium channels by PKA may influence calcium‐dependent processes within hippocampal neurons, including neurotransmitter release, calcium‐activated enzymes and gene expression.

Immunochemical Identification and Differential Phosphorylation of Alternatively Spliced Forms of the α1A Subunit of Brain Calcium Channels

Biochemical properties of the α1 subunits of class A brain calcium channels (α1A) were examined in adult rat brain membrane fractions using a site-directed anti-peptide antibody (anti-CNA3) specific for α1A. Anti-CNA3 specifically immunoprecipitated high affinity receptor sites for ω-conotoxin MVIIC (Kd ∼100 pM), but not receptor sites for the dihydropyridine isradipine or for ω-conotoxin GVIA. In immunoblotting and immunoprecipitation experiments, anti-CNA3 recognized at least two distinct immunoreactive α1A polypeptides, a major form with an apparent molecular mass of 190 kDa and a minor, full-length form with an apparent molecular mass of 220 kDa. The 220- and 190-kDa α1A polypeptides were also specifically recognized by both anti-BI-Nt and anti-BI-1-Ct antibodies, which are directed against the NH2- and COOH-terminal ends of α1A predicted from cDNA sequence, respectively. These data indicate that the predicted NH2 and COOH termini are present in both size forms and therefore that these isoforms of α1A are created by alternative RNA splicing rather than post-translational proteolytic processing of the NH2 or COOH termini. The 220-kDa form was phosphorylated preferentially by cAMP-dependent protein kinase, whereas protein kinase C and cGMP-dependent protein kinase preferentially phosphorylated the 190-kDa form. Our results identify at least two distinct α1A subunits with different molecular mass, demonstrate that they may result from alternative mRNA splicing, and suggest that they may be differentially regulated by protein phosphorylation.

Bioenergetic characterization of γ-aminobutyric acid transporter of synaptic vesicles.

Publisher Summary This chapter describes the bioenergetic characterization of γ-aminobutyric acid transporter of synaptic vesicles. Neurotransmitters are stored in synaptic vesicles and, on depolarization and subsequent calcium influx, released from nerve terminals by exocytosis. The main inhibitory neurotransmitter in the mammalian brain is γ-aminobutyric acid (GABA), whereas glycine is the prevailing inhibitory neurotransmitter in the spinal cord. The GABA uptake into synaptic vesicles is mediated by the vesicular GABA transporter, which works as an electrogenic GABA proton antiporter in contrast to the sodium-driven GABA transporter in the plasma membrane. This chapter describes the technical details for measuring GABA uptake in synaptic vesicles, for the solubilization and functional reconstitution of the vesicular GABA transport system, and for the creation of ΔΨ and ΔpH by salt gradients and ionophores to energize the reconstituted GABA transporter independently from the vesicular ATPase.