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
1999-09-01
2002-04-02
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C536S023500, C530S350000, C435S071100, C435S071200, C435S325000, C435S471000, C435S320100, C435S252300, C435S254110
Reexamination Certificate
active
06365370
ABSTRACT:
BACKGROUND OF THE INVENTION
Serotonin (5-hydroxytryptamine, 5-HT) is a multifunctional chemical transmitter that signals though cell surface receptors. At least fourteen subtypes of serotonin receptors have been defined pharmacologically (Julius, 1996). Thirteen of the fourteen known receptors are G-protein coupled receptors and the only known ionotropic 5-HT receptor, the type 3 5-HT3 receptor, is a fast activating, ligand gated non-selective cation channel unique among known monoamine receptors (Derkach et al., 1989). The 5-HT3 receptor is exclusively localized on neurons in the central (Waeber et al., 1989; Yakel et al., 1991) and peripheral (Fozard, 1984) nervous systems. Activation of the 5-HT3 receptor leads to membrane depolarization and an increase in intracellular Ca
2+
. The 5-HT3 receptor is the target of antagonists (granisetron and ondansetron) selective against the nausea induced by cytotoxic chemotherapy and general anesthesia (Gralla, 1998). There is some evidence that serotonin 5-HT3 receptors are important in pain reception, anxiety, cognition, cranial motor neuron activity, sensory processing, modulation of affect, and the behavioral consequences of drug abuse (Greenshaw and Silverstone, 1997; Lambert et al., 1995; Passani and Corradetti, 1996).
There are two known subunits for the human 5-HT3 receptor: 5-HT3-A (Belelli et al., 1995; Miyake et al., 1995) and 5-HT3-B (Davies et al., 1999). They have structural and functional similarities with nicotinic, GABA-ergic and other ligand gated ion channels (Barnard, 1996; Gurley and Lanthorn, 1998; Maricq et al., 1991). The modifier subunit 5-HT3-B for the serotonin 5-HT3 receptor can explain certain observations that 5-HT3 receptors from a variety of preparations have distinct pharmacological, kinetic, permeation and voltage-dependent properties (Peters et al., 1992) (Dubin et al., manuscript submitted to JBC). However, biochemical evidence suggests that 5-HT3 receptors may exist as heteromultimers composed of more than 2 subunits. Receptors purified from a variety of sources by affinity chromatography usually reveal at least 2 major protein bands with molecular masses in the order of 54 and 38 kDa (Lambert et al., 1995). The 5-HT3-A receptor corresponds to the former (Turton et al., 1993). Affinity purified 5-HT3 receptor solubilized from pig cerebral cortex is composed of at least 3 separable components, based on silver staining of proteins on denaturing gels (Fletcher and Barnes, 1997). A number of these protein bands are not recognized by specific antibodies directed against the recombinant 5-HT3-A subunit (Fletcher and Barnes, 1997), and their sizes are too large (52-71 kDa) to be considered as degraded 5-HT3-A fragments (Fletcher and Barnes, 1998).
Heteromeric assembly of subunits often produces channels with altered function compared to homomeric channels. An increasing number of subunits have been reported to be electrically silent when expressed alone but profoundly inhibit the function of specific classes of channels in co-expression studies (Kv8, Kv9, Kir1.3) (Hugnot et al., 1996; Salinas et al., 1997; Salinas et al., 1997; Shuck et al., 1997; Stocker et al., 1999). Like 5-HT3 receptors, voltage-gated potassium channels are heteromers of similar subunits (Jan and Jan, 1997). Kv8.1 and members of the Kv9 family have the capability to abolish the functional expression of specific potassium channel family members when co-expressed at high levels (Hugnot et al., 1996; Salinas et al., 1997; Salinas et al., 1997; Stocker et al., 1999). Immunoprecipitation experiments reveal co-assembly of Kv8.1 with Kv2 (Hugnot et al., 1996). Kv8.1 does not appear to reach the plasma membrane when expressed alone (Salinas et al., 1997) and homomeric assembly of Kv9 subunits does not occur (Stocker et al., 1999).
Functional expression of the inward potassium channel Kir1.3 in Xenopus oocytes was not detectable, however, co-expression of Kir1.3 with either Kir1.1 or Kir1.2 reduced the currents resulting from expression of these inward-rectifier subunits alone, consistent with a negative influence on Kir1.1 and Kir1.2 expression (Shuck et al., 1997).
A number of naturally occurring mutant channels exist that reduce channel function. In one form of episodic ataxia, mutations in the gene encoding Kv1.1 abolish the function of wild-type Kv1.1 subunits (Mathur et al., 1999; Zerr et al., 1998). In the LQT1 form of long QT syndrome KvLQT1 mutations A177P or T311I have a dominant negative effect on currents produced by KvLQT1 with or without minK when channel subunit combinations are expressed in Xenopus oocytes (Shalaby et al., 1997). Expression of these KvLQT1 mutants either individually or in combination yielded inactive channels when expressed individually and inhibit wild-type KvLQT1 currents in a dominant-negative fashion.
A naturally occurring HERG mutant G601S is a hypomorphic mutation (ie., a specific mutant form of a gene that exhibits function qualitatively similar to the normal state but with quantitatively less function), resulting in a reduced current amplitude and represents a novel mechanism underlying LQT2 (Furutani et al., 1999).
A wide range of single channel conductances has been reported for the 5-HT3-A receptor subunit and endogenous 5-HT3 receptors in native cells. This variation is attributable in part to heteromeric formation of 5-HT3-A with 5-HT3-B subunits (Davies et al., 1999). Homomeric receptors revealed a sub-pS conductance whereas heteromeric receptors displayed large single channel conductance (16 pS) (Davies et al., 1999). Modulation of the conductance of 5-HT3 receptors has been reported. Van Hooft and colleagues have shown that the conductance of 5-HT3 receptors in N1E-115 cells is dependent on phosphorylation conditions (Van Hooft and Vijverberg, 1995).
An unmet need in this field is the ability to reconstitute a recombinant expression system that fully mimics the 5-HT3 receptor complex in natural tissue, likely due to incomplete receptor complexes localized at the cell surface. By creating a more physiologically relevant model of the 5-HT3 receptor complex using in vitro systems, it will be easier to develop and test new therapeutic compounds for treatment of diseases associated with the 5-HT3 serotonin receptor. This unmet need has been met by the isolation and characterization of a novel 5-HT3 receptor subunit cDNA molecule, hereafter termed 5-HT3-C. Using a recombinant expression system, functional DNA molecules encoding the subunit have been isolated. The biological and structural properties of this protein are disclosed, as is the amino acid and nucleotide sequence. The recombinant protein is useful to identify modulators of the 5-HT3 serotonin receptor complex, and to reconstitute a more realistic physiological 5-HT3 receptor response in recombinant systems. Co-expression of the 5-HT3-A with the 5-HT3-C subunit reduces the biological function of the 5-HT3-A receptor for some known modulators of 5-HT3 receptors. Modulators identified in the assay disclosed herein are useful as therapeutic agents. The recombinant DNA molecules, and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules.
SUMMARY OF THE INVENTION
A DNA molecule encoding a human subunit with homology to the 5-HT3-A serotonin receptor has been cloned and when co-expressed with the short form of the human serotonin 5-HT3-A receptor modifies the function of the 5-HT3 receptor. Using a recombinant expression system, functional DNA molecules encoding the human serotonin 5-HT3 receptor modifier protein (5-HT3-C) have been isolated. The biological and structural properties of these proteins are disclosed, as is the amino acid and nucleotide sequence. The recombinant protein is useful to identify modulators of human 5-HT3 receptors composed of both 5-HT3 receptors and the modifier subunit (5-HT3-C). Modulators identified in the assay disclosed herein are useful as therapeut
Buehler Lukas K.
Dubin Adrienne E.
Erlander Mark G.
Huvar Arne
Huvar Rene
Mertz Prema
Ortho-McNeil Pharmaceutical , Inc.
Wallen III John W.
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