Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-07-16
2001-03-27
Grimes, Eric (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06207410
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the voltage-dependent calcium channel multigene family. More particularly, the present invention relates to the characterization and isolation of an insect calcium channel &agr;
1
and &bgr; subunits particularly from
Drosophila melanogaster.
BACKGROUND OF THE INVENTION
Early electrophysiological studies on invertebrate preparations revealed the presence of calcium currents and suggested the presence of multiple types of voltage-dependent calcium channels (reviewed by Hille, B., (1992), In:
Ion Channels of Excitable Membranes
, 2nd Ed., Sinauer, Sunderland, Mass.). Continuing studies of calcium channels have shown that they are ubiquitous since they are found in excitable cells in species ranging from Paramecium to humans. Calcium channels are involved in many cell functions including: membrane excitability, synaptic transmission, and differentiation (Tsien et al., (1988),
Trends Neurosci
., vol. 11, pp. 431-438). Voltage-dependent calcium channels have been studied extensively in vertebrate neuronal tissue using electrophysiological and pharmacological approaches and as a result have been divided into four classes designated L, N, T, and P (Bean, B. P., (1989),
Annu. Rev., Physiol
., vol. 51, pp. 367-384; Hess, P., (1990),
Annu. Rev. Neurosci
., vol. 13, pp. 1337-1356).
Gene cloning studies, which up to this point have focused exclusively on vertebrate species, have helped to elucidate the molecular nature of calcium channel structure and have suggested a remarkable degree of channel heterogeneity beyond that predicted from physiological and pharmacological approaches. This molecular diversity of calcium channels arises from several mechanisms. Calcium channels are comprised of multiple subunits designated &agr;
1
, &agr;
2
, &bgr;, &ggr;, and &dgr; (Catterall, W. A., (1991a),
Cell
, vol. 64, pp. 871-874; Catterall, W. A., (1991b),
Science
, vol. 253, pp. 1499-1500). The &agr;
2
and &dgr; subunits are encoded by the same gene and are cleaved during posttranslational processing whereas each of the other subunits arise from different genes. One way that calcium channel diversity arises is through the presence of a family of genes each encoding genetic variants of a given subunit. For example, in rat brain the &agr;
1
subunit appears to be encoded by a family of at least five different genes (Snutch et al., (1990),
Proc. Natl. Acad. Sci. USA
, vol. 87, pp. 3391-3395; Snutch et al., (1991),
Neuron
, vol. 7, pp. 45-57; Hui et al., (1991),
Neuron
, vol. 7, pp. 35-44; Starr et al., (1991),
Proc. Natl. Acad. Sci. USA
, vol. 88, pp. 5621-5625; Dubel et al., (1992),
Proc. Natl. Acad. Sci. USA
, vol. 89, pp. 5058-5062; Soong et al., (1993),
Science
, vol. 260, pp. 1133-1136). For each member of the gene family further diversity is introduced by alternative splicing (Biel et al., (1990),
FEBS Lett
., vol. 269, pp. 409-412; Koch et al., (1990),
J. Biol. Chem
., 265, pp. 17786-17791; Perez-Reyes et al., (1990),
J. Biol. Chem
., 265, pp. 20430-20436; Snutch et al., (1991),
Neuron
, vol. 7, pp. 45-57). Recent studies point to the existence of similar molecular diversity for the other subunits as well (Williams et al., (1992a),
Science
, vol. 257, pp. 389-395; Williams et al., (1992b),
Neuron
, vol. 8, pp. 71-84). The &agr;
1
subunit is the essential portion of the calcium channel as it is involved in forming the pore in a membrane. The &bgr; subunit is of special interest since its coexpression with &agr;
1
in an artificial expression system often significantly increases the channel current and the drug binding ability and brings channel kinetics closer to the physiological norm [Lacerda et al. (1991),
Nature
, Vol. 352, pp. 527-530; Varadi et al. (1991),
Nature
, vol 352, pp. 159-162; Neely et al. (1993),
Science
, vol 262, pp. 575-578]. In some cases, no functional expression is detected without coexpression of the &bgr; subunit. &bgr; subunits have also been implicated in the regulation of calcium channels by protein phosphorylation [Singer et al. (1992),
FEBS Lett
., vol 306, pp. 113-118].
Generally the cloned vertebrate calcium channel &bgr; subunits are highly conserved, but there are three nonconserved regions within the molecule. We report here a PCR strategy that showed that invertebrate calcium channels also contain a &bgr; subunit. We report here the deduced amino acid structure, expression pattern, and chromosome mapping of a &bgr; subunit from the fruitfly
Drosophila melanogaster
. We also show that alternative splicing occurs in at least three regions to generate multiple isoforms. These studies set the stage for genetic analysis of &bgr; subunit function in the intact organism.
If each subunit variant can interact with more than one form of each of the other subunits to form functional channels, then there is a potential for even further molecular diversity.
Although studies of the molecular diversity of calcium channels in Drosophila are just beginning, there is evidence for structural and functional heterogeneity in this system. Binding of phenylalkylamines (calcium channel blocking agents) to Drosophila head extracts showed curvilinear Scatchard plots indicative of multiple classes differing in ligand affinity (Greenberg et al., (1989),
Insect Biochem
., vol. 19, 309-322). Pelzer et al., (1989),
EMBO J
., vol. 8, pp. 2365-2371, reported at least 8 distinct voltage-sensitive calcium channels in Drosophila head membranes following reconstitution into phospholipid bilayers. Patch clamp studies on cultured embryonic Drosophila myocytes and neurons also showed variability of channel properties, suggesting at least two types of calcium channels in Drosophila (Leung and Byerly, 1991). Further evidence for channel heterogeneity comes from differential sensitivity of Drosophila calcium channels to a purified toxin from the spider
Hololena curta
(Leung, H. T. and Byerly, L., (1991),
J. Neurosci
., vol. 11, pp. 3047-3059). This heterogeneity is further supported in another insect (Periplaneta americana) where radiotracer flux studies have demonstrated the presence of dihydropyridine-insensitive and -sensitive components of phenylalkylamine-sensitive calcium uptake in nervous system and skeletal muscle membranes, respectively (Skeer et al., (1992),
Insect Biochem. Molec. Biol
., vol. 22, pp. 539-545).
Given the heterogeneity of calcium channels in insects, Drosophila provides an ideal system for a molecular genetic approach to define the significance of channel diversity by mutating individual subunit genes and determining the physiological and behavioral consequences.
Other ion channels have also been reported to date. For example, electrophysiological studies of ligand-gated ion currents in insect nerve and muscle cells provide evidence for the existence of chloride channels gated by glutamate, histamine, and taurine, as well as those gated by &agr;-Aminobutyric acid (“GABA”) (Sattelle, D. B., (1990), Adv.
Insect Physiol
., vol. 22, pp. 41-56 and Lummis et al., (1990),
Annu. Rev. Entomol
., vol. 35, pp. 345-377). Although these findings imply the existence of a large and diverse gene family encoding ligand-gated chloride channels in insects, very little is known about homologous channels of invertebrates. In ffrench-Constant et al., (1991),
Proc Natl. Acad. Sci. USA
, vol. 88, pp. 7209-7213, a
Drosophila melanogaster
cDNA having significant predicted amino acid sequence identity to vertebrate ligand-gated chloride channel genes was isolated and mapped to a genetic locus (“Rdl1”) that confers resistance to cyclodiene insecticides and other blockers of GABA-gated chloride channels. Rdl was shown to encode a GABA subunit by the expression of functional homomultimeric GABA in
Xenopus oocytes
following injection with Rdl mRNA (ffrench-Constant et al., (1993),
Nature
, vol. 363, pp. 449-451).
One other example of a ligand-gated chloride channel gene from an invertebrate species is a GABA &bgr;-like subunit gene isolated from the pond snail,
Lymnaea stagnalis
(
Dubald Manuel Marcel Paul
Hall Linda M.
Ren Dejian
Zheng Wei
Burns Doane Swecker & Mathis L.L.P.
Grimes Eric
The Research Foundation of State University of New York
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