Stephen G. Volsen

The I-II Loop of the Ca2+ Channel α1 Subunit Contains an Endoplasmic Reticulum Retention Signal Antagonized by the β Subunit

Abstract The auxiliary β subunit is essential for functional expression of high voltage-activated Ca 2+ channels. This effect is partly mediated by a facilitation of the intracellular trafficking of α 1 subunit toward the plasma membrane. Here, we demonstrate that the I-II loop of the α 1 subunit contains an endoplasmic reticulum (ER) retention signal that severely restricts the plasma membrane incorporation of α 1 subunit. Coimmunolabeling reveals that the I-II loop restricts expression of a chimera CD8-I-II protein to the ER. The β subunit reverses the inhibition imposed by the retention signal. Extensive deletion of this retention signal in full-length α 1 subunit facilitates the cell surface expression of the channel in the absence of β subunit. Our data suggest that the β subunit favors Ca 2+ channel plasma membrane expression by inhibiting an expression brake contained in β-binding α 1 sequences.

Complete loss of P/Q calcium channel activity caused by a CACNA1A missense mutation carried by patients with episodic ataxia type 2

Familial hemiplegic migraine, episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 are allelic disorders of the CACNA1A gene (coding for the alpha(1A) subunit of P/Q calcium channels), usually associated with different types of mutations (missense, protein truncating, and expansion, respectively). However, the finding of expansion and missense mutations in patients with EA2 has blurred this genotype-phenotype correlation. We report the first functional analysis of a new missense mutation, associated with an EA2 phenotype-that is, T-->C transition of nt 4747 in exon 28, predicted to change a highly conserved phenylalanine residue to a serine at codon 1491, located in the putative transmembrane segment S6 of domain III. Patch-clamp recording in HEK 293 cells, coexpressing the mutagenized human alpha(1A-2) subunit, together with human beta(4) and alpha(2)delta subunits, showed that channel activity was completely abolished, although the mutated protein is expressed in the cell. These results indicate that a complete loss of P/Q channel function is the mechanism underlying EA2, whether due to truncating or to missense mutations.

The expression of neuronal voltage-dependent calcium channels in human cerebellum.

Little is known about the comparative distribution of voltage-dependent calcium channel subtypes in normal human brain. Previous studies in experimental animals have predominantly focused on the regional expression of single alpha 1 genes. We describe the preparation of riboprobes and antisera specific for human alpha 1A, alpha 1B and alpha 1E subunits and their application in comprehensive mapping studies of the human cerebellum. Within the cerebellar cortex, these pore forming proteins were found to have differential localisations when examined in adjacent sections. The alpha 1A and alpha 1B subunits broadly colocalised and were both present, though at apparently different levels, in the molecular, Purkinje and granule cell layers whilst alpha 1E was predominantly expressed in Purkinje cells. In the dentate nucleus, an area which has received little attention in previous studies, alpha 1A was highly expressed in regions in which Purkinje cell nerve terminals form synapses with deep cerebellar neurones.

Identification of Pore-forming Subunit of P-type Calcium Channels: an Antisense Study on Rat Cerebellar Purkinje Cells in Culture

Treatment of cerebellar neurones in culture with an antisense oligonucleotide (ODN) against alpha1A, reduced the whole-cell P-type calcium channel current relative to mismatch ODN treated controls (p < 0.001). Therefore, AgaIVA (50 nM) reduced whole-cell calcium current in mismatch and antisense treated cells by 70 +/- 4 and 19 +/- 3%, respectively.

Expression and functional characterisation of a human chimeric nicotinic receptor with α6β4 properties

Abstract Despite being cloned several years ago, the expression of functional nicotinic acetylcholine receptors containing the human α6 subunit in recombinant mammalian cell lines has yet to be demonstrated. The resulting lack of selective ligands has hindered the evaluation of the role of α6-containing nicotinic receptors. We report that functional channels were recorded following co-transfection of human embryonic kidney (HEK-293) cells with a chimeric α6/α4 subunit and the β4 nicotinic receptor subunit. They displayed an agonist rank order potency of epibatidine≫1,1-dimethyl-4-phenylpiperazinium (DMPP)≥cytisine>acetylcholine>nicotine measured in a fluorescent imaging plate reader assay. Nicotine, cytisine, DMPP and epibatidine displayed partial agonist properties whilst α-conotoxin MII and methyllycaconitine blocked the functional responses elicited by acetylcholine stimulation. Co-transfection of the α6/α4 chimera with the β2 nicotinic receptor subunit did not result in functional receptors. The human α6/α4β4 chimeric nicotinic receptor expressed in HEK-293 cells may provide a valuable tool for the generation of subtype specific ligands.

Preparation and purification of antibodies specific to human neuronal voltage-dependent calcium channel subunits.

Neuronal voltage-dependent calcium channels (VDCCs) each comprising of alpha 1, alpha 2 delta, and beta subunits, are one mechanism by which excitable cells regulate the flux of calcium ions across the cell membrane following depolarisation Studies have shown the expression of several alpha 1 and beta subtypes within neuronal tissue. The comparative distribution of these in normal human brain is largely unknown. The aim of this work is to prepare antibodies directed specifically to selected subunits of human neuronal VDCCs for use in biochemical and mapping studies of calcium channel subtypes in the brain. Previous studies have defined DNA sequences specific for each subunit Comparison of these sequences allows the selection of unique amino acid sequences for use as immunogens which are prepared as glutathione-S-transferase (GST) fusion proteins in E. coli. Polyclonal antibodies raised against these fusion proteins are purified by Protein A chromatography, followed by immunoaffinity chromatography and extensive adsorptions using the appropriate fusion protein-GST Sepharose 4B columns. The resultant antibodies are analysed for specificity against the fusion proteins by ELISA, and by immunofluorescence and Western immunoblot analysis of recombinant HEK293 cells stably transfected with cDNAs encoding alpha 1, alpha 2 delta and beta subunits.

Immunohistochemical and in situ mRNA hybridisation techniques to determine the distribution of ion channels in human brain: a study of neuronal voltage-dependent calcium channels

The molecular, structural and functional characterisation of ion channels in the CNS forms an area of intense investigation in current brain research. For strategic and logistical reasons, rodents have historically been the species of choice for these studies. The examination of human CNS tissues generally presents the investigator with specific challenges that are often less problematic in animal studies, e.g. post-mortem delay/agonal status, and thus both the experimental design and techniques must be manipulated accordingly. Since much pharmaceutical interest is currently focused on neuronal ion channels, the examination of their expression in human brain material is of particular importance. We describe here the details of methods that we have developed and used successfully in the study of the expression of voltage-dependent calcium channels (VDCCs) in human CNS tissues. Presynaptic neuronal VDCCs control neurotransmitter release and are important new drug targets. They are composed of three subunits, alpha 1, beta and alpha 2/delta and multiple gene classes of each protein have been identified. Little is known, however, about the distribution of neuronal VDCCs in the human central nervous system, although initial studies have been performed in rat and rabbit.

[21] Application of antisense techniques to characterize neuronal ion channels in vitro

Publisher Summary Current gene cloning and genomic initiatives provide neuroscientists with an exponentially increasing bank of genetic sequence data that details the molecular identity of novel brain proteins. Often in the initial absence of selective ligands, the functional properties of the more recently cloned brain proteins remain unclear. The antisense approach conceptually offers a solution to this problem. Multiple mechanisms have been proposed that describe the molecular events that precipitate gene-specific knockdown by antisense oligonucleotides. These include inhibition of transcription, translational arrest, disruption of ribonucleic acid (RNA) processing, and RNase H–mediated transcript degradation. Despite the many questions that remain unanswered, antisense oligonucleotides have been applied successfully in the central nervous system (CNS) research. This chapter discusses their in vitro applications and describes both the strategies and methods that have been developed and applied successfully to the studies of neuronal voltage-dependent ion channels. Wherever possible, the effects of antisense treatment have been examined at multiple levels—that is, transcription transduction, messenger RNA (mRNA), protein and/or functionally by electrophysiological measurements. Each level of analysis affords complementary data that when taken together greatly facilitate the final interpretation of results.