Human Neuronal Beta Subunits of Voltage-Operated Calcium Channels and the Use Thereof

The present invention relates to human neuronal beta subunits of voltage-operated calcium channels and the use thereof in screening methods for finding pharmaceuticals.

Calcium ions have a wide variety of functions in every biological system. Cellular calcium homeostasis plays an essential part specifically in the physiology of nerve cells. The intracellular calcium concentration is about 0.1 .mu.M, compared with 1 mM outside the nerve cell. This steep concentration gradient (.times.10,000) is regulated primarily by voltage-operated calcium channels (VOCC) which can be blocked by certain calcium antagonists. During a cerebral ischaemia (stroke) there is a considerable change in the calcium homeostasis in neurons in the area affected by the infarct. The voltage-operated calcium channels are kept in the open state by prolonged membrane depolarisations, the consequence of which is a massive influx of calcium ions. The intracellular calcium concentration increases 1000-fold during this. The large excess of calcium activates, owing to the binding to calmodulin, various calcium/calmodulin-dependent cellular enzyme systems, such as kinases, proteases and phospholipases, which together lead, when activation is prolonged, to irreversible damage to nerve cells.

One therapeutic approach to neuroprotection in cerebral ischemia is reversible blockade of the massive influx of calcium into the nerve cell. The voltage-operated neuronal calcium channels are a suitable pharmacological target in this case. The VOCCs exist in various muscle cells (vascular, cardiac and skeletal muscle), neurons and secretory cells with tissue-specific physio-logical properties.

Electrophysiological investigations (Tsien et al., 1988, Trends in Neurol. Sci 11: 431-438) indicate that there are at least three different types of VOCCs (L, N and T channels). The 1,4-dihydropyridines (DHPs) are potent blockers of L type calcium channels which are found both in muscle cells and in nerve cells. The rabbit skeletal muscle dihydropyridine receptor has been biochemically characterised and cloned (Tanabe et al., 1987, Nature 328: 313-318). The primary sequence of this .alpha.l SU of the VOCC has been derived from the cDNA data and is consistent with a 212 kD transmembrane protein with five N-glycosylation sites and seven possible phosphorylation sites. The protein contains four mutually similar trans-membrane domains, each of which has six--presumably .alpha.-helical--transmembrane segments (S1-S6). The fourth transmembrane segment (S4) of each domain contains an ordered pattern of positive charges (Lys, Arg) which may form the voltage sensor of the calcium channel. The structure of this cloned .alpha.l SU is consistent with an ion-conducting, voltage-controlled unit of the DHP-sensitive calcium channel.

Besides the alpha-1 subunit, which forms the actual calcium channel, four other proteins are involved in the structure of the complete channel complex, which are called the alpha-2, beta, gamma and delta subunits.

Recent investigations have shown, that, in particular, the beta subunit influences important parameters of channel function: when the .alpha..sub.1 and .beta. SUs are expressed together in vitro there is a change in the calcium flux, the activation and inactivation kinetics of the channel complex, and the binding affinity for dihydropyridines (Singer et al., 1991, Science 253: 1553; Lacerda et al., 1991, Nature 352: 527; Varadi et al., 1991, Nature 352: 159).

Thus, the availability of complete cDNA clones of the beta subunit is of great importance for the reconstitution of a physiologically relevant channel structure by gene expression in eukaryotic cells.

It was possible with the aid of an oligonucleotide which was derived from the DNA sequence of the beta subunit from rabbit skeletal muscle (Ruth et al., 1989, Science 245: 1115) to isolate 3 different cDNA types which encode human neuronal beta subunits. The cloned beta subunits are to be expressed together with subtypes, which are already present, of neuronal alpha-1 clones in transformed animal cells (for example cos cells, mouse L cells, CHO cells etc) (Gluzman, 1981, Cell 23: 175 and Chen et al., 1987, Mol. Cell. Biol. 7: 2745-2752). These constructs are employed in binding assays and/or functional assay systems which are used to find novel subtype-specific ligands of neuronal calcium channels.

These recombinant cell systems are furthermore to be used to develop functional calcium flux assays with the aid of which it is possible to check the agonistic or antagonistic action of specific ligands. The difference and main advantage of these recombinant assays compared with conventional assays (brain membrane preparations, cell lines) is the purity of the receptor/channel preparation because only the recombinantly expressed neuronal calcium channel subtype is present in a suitable number on an animal cell surface. This is an essential precondition for the selection of specific neuronal ligands which ought if possible to have no effect on calcium channels of non-neuronal tissue types.

Some examples of the use of the recombinant screening assays described above are listed hereinafter.

1. Receptor binding assay

The animal cells transformed with human calcium channels (example: see above) can be cultivated and employed for the preparation of membranes. These membrane preparations can be employed in binding studies with various classes of radioactively labelled substances (Examples 1-5) for screening novel ligands (competitive assay). Examples of known calcium channel binding substances are:

1. Phenylalkylamines,

2. Benzothiazepines,

3. Dihydropyridines,

4. Bisphenylbutylpiperidines,

5. Omega conotoxins.

2. Calcium-45 flux assay

The cell membranes of cultured cells which have been transformed with human calcium channel subtypes can be depolarised with potassium ions or with alkaloids such as, for example, Veratridine. Membrane depolarisation leads to opening of calcium channels, which results in an influx (flux) of calcium ions into the cells. This voltage-dependent calcium influx can be measured using radioactively labelled calcium (.sup.45 Ca) (Example: Messing et al., 1985, J. Pharmacology and Exp. Therapeutics 235: 407-411) and employed for the functional testing/screening of calcium channel antagonists or agonists.

3. Fura 2 assay

Human calcium channel expressing animal cells (see above) can be employed in the presence of calcium-sensitive, fluorescent dyes (for example fura 2 or fluoro 3) for measurements of the intracellular calcium concentration after opening and blocking of the calcium channels (Example: Rosario et al., 1989, Neurosci. 29, 735-747). The change in the intracellular calcium concentration can in this case be measured by fluorimetry (spectrophotometry). This recombinant cell system can be employed as functional assay for finding subtype-specific calcium channel ligands (agonists and antagonists).

4. Electrophysiology

The calcium currents generated by membrane depolarisation can be measured electrophysiologically (Example; Carbone et al., 1990, Pflugers Arch., 416: 170-179). The effect of potential calcium channel antagonists or agonists can be physically measured and pharmacologically characterised directly on human calcium channels using the recombinant animal cell lines (see above).

5. Indirect methods of measurement

Many cellular processes are controlled by the intracellular calcium ion concentration (for example receptor-mediated signal transmission, various enzyme reactions, such as, for example, phosphorylation, dephosphorylations, neurotransmitter release, Ca-dependent gene regulation etc). Some of these biochemical reactions can be measured using a specific assay. It is thus possible in a recombinant calcium channel-expressing cell system to detect indirectly (physiologically) the effect of calcium channel modulators on calcium-dependent cellular processes (Exhale: Zernig et al., 1986, Eur. J. Pharmacol. 128., 221-229).

It is additionally possible by modifications introduced by targeted mutageneses, such as, for example, point mutations, insertions, deletions, replacement of DNA segments of various calcium channel subtypes, to detect direct effects on physiological processes (Example: Yool and Schwarz, 1991, Nature 349: 700-704).

Cloning strategy

1. Screening of the cDNA library

1.1. Plating of the cDNA library and processing of the nitrocellulose filters

The plating of the cDNA library (human hippocampus in Lambda ZAPII, supplied by Strategene Inc., La Jolla, Calif., USA; Cat. No. 936205) and of the nitrocellulose filters was carried out as stated by the manufacturers or described by Sambrook et al., 1989, Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, New York, N.Y., USA.

1.2 Hybridisation probes

The primary hybridisation probe used was a synthetic antisense oligonucleotide which is complementary to a 40 bases-long fragment (pos. 361-400) of the DNA sequence of the beta subunit from rabbit skeletal muscle:

The said DNA sequence can be obtained from the EMBL data bank under Access No. M25817.

The 1.9 kB-long cDNA fragment HB26 was isolated from the abovementioned cDNA library with the aid of this oligonucleotide. This fragment was employed as hybridisation probe in all subsequent screening experiments.

1.3 Labelling of hybridisation probes with radioactive DNA precursors

Oligonucleotides were enzymatically labelled with .sup.32 P-dCTP ("DNA Tailing Kit", Boehringer Mannheim GmbH, Postfach 310120, D-6800 Mannheim; Cat. No. 1028707).

cDNA fragments are labelled with .sup.32 P-dCTP using the "Random Primed Labeling Kits" (Boehringer Mannheim GmbH, Cat. No. 10004760).

1.4 Hybridisation and washing conditions

1.4.1. Oligonucleotides

The nitrocellulose filters were hybridised with the radioactively labelled hybridisation probe in the following solution at 42.degree. C. overnight:

5.times. Denhardt's solution

5.times. SSC

50 .mu.g/ml herring sperm DNA

50 mmol/l Na phosphate

1 mmol/l Na pyrophosphate

60 .mu.g/ml ATP

The filters were washed with 2.times.SSC, 0.1% SDS at 55.degree. C.

1.4.2. cDNA fragments

The nitrocellulose filters are hybridised with a radio-actively labelled probe in the solution mentioned in 1.4.1., which was, however, made up with 50% formamide, at 42.degree. C. overnight.

The filters were washed with 0.2.times.SSC, 0.1% SDS at 55.degree. C.

Then Kodak X-Omat AR X-ray film was exposed with intensifying screens to the filters at -80.degree. C. for various times.

2. Isolation of the lambda phages, subcloning and sequencing of the cDNA inserts

2.1 Conversion of the cDNA inserts into plasmids

The cDNA inserts from positive Lambda ZAPII phages were removed and converted into the plasmid form according to a protocol of the manufacturer (Stratagene) using an fl-derived helper phage.

2.2. Determination of size and analysis of sequence of the cDNA inserts

Plasmid DNA was prepared from XL1-blue cells which harboured a recombinant pBluescript plasmid (Sambrook, J., et al., (1989) in: Molecular cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, New York, N.Y.) and 0.5 .mu.g samples of this DNA were treated with the restriction enzyme EcoRI. It was possible to deduce the total length of the inserted cDNA from the number and size of the resulting DNA fragments. The nucleotide sequence of the cDNA present was determined with SEQUENASE (USB, Cleveland, Ohio, USA) by the Sanger method on double-stranded DNA.

3. Description of the cDNA clones isolated to date for human neuronal beta subunits

3.1. HB26: length 1.9 kB

This cDNA clone was isolated using the oligonucleotide described under 1.2. It contains a large portion of the coding region and an intron of about 450 base pairs.

HB26 was employed as cDNA probe for the isolation of other specific cDNAs from the human hippocampal Lambda ZAPII library:

3.2. HBB1: length 1.6 kB

This clone contains the complete coding region of a human neuronal beta subunit with 92% homology to the beta subunit from rabbit muscle. HBB1 is the sequenced portion of HB26-3 which is a total of 1.9 kB long.

3.3. HBB2: length 1.8 kB

This clone represents another subtype of human neuronal beta subunits with 74% homology to the beta subunit from rabbit muscle. HB28t7 is a partial sequence of the complete HBB2 clone. HB28t7 has a sequence that corresponds to bases 124-1299 of sequence HBB2. Since HB28t7 contains several reading errors which were corrected in sequence HBB2, there is no perfect identity of sequence HB28t7 and bases 124-1299 of sequence HBB2.

3.4. HBB3: length 1.8 kB

This clone represents a third subtype of human neuronal beta subunits. Up to position 1288, the cDNA shows 92% homology to the beta subunit from rabbit muscle. From position 1289 on, no homology to the rabbit muscle type is detectable.

All four listed clones show by comparison with the beta subunit of rabbit muscle a deletion of 45 amino acids which is characteristic of neuronal beta subunits.

The HBB1 sequence listing comprises 1612 bp and contains the complete coding region.

The HBB2 sequence listing comprises 1830 bp and contains the complete coding region.

The HBB3 sequence listing comprises 1805 bp and likewise contains the complete coding region.