Seiichiro Nishimura

Involvement of the brain type of ryanodine receptor in T-cell proliferation

Cloning and sequence analysis of cDNA showed that the brain type of ryanodine receptor (RYR) is expressed in human Jurkat T‐lymphocyte cells. Fura‐2 measurements revealed that the RYR in T‐cells functions as a ryanodine‐sensitive, caffeine‐insensitive Ca2+ release channel. Furthermore, ryanodine stimulated proliferation and altered the growth pattern of cultured human T‐cells when added together with FK506.

Requirement of the calcium channel β subunit for functional conformation

The cardiac dihydropyridine‐sensitive L‐type calcium channel was stably expressed in Chinese hamster ovary cells by transfecting the rabbit cardiac calcium channel α1 subunit cDNA with or without coexpression of the β subunit of skeletal muscle calcium channel. Whereas coexpression of the β subunit significantly increased DHP binding activity and calcium channel activity, it did not affect the amount of the α1 subunit expressed, as judged by RNA blot hybridization analysis and immunoblotting analysis. The results suggest that association with the β subunit is necessary for the α1 subunit protein to take a proper conformation suitable for a functional calcium channel.

A brain-specific transcript from the 3'-terminal region of the skeletal muscle ryanodine receptor gene.

We have shown previously that the skeletal muscle ryanodine receptor mRNA of ∼16,000 nucleotides codes 5,037 amino acid residues constituting the calcium release channel in skeletal muscle. In this study, RNA blot hybridization analysis shows that the brain contains an RNA species with an estimated size of ∼2,400 nucleotides hybridizable with the 3′‐terminal region of the skeletal muscle ryanodine receptor cDNA. cDNA cloning and genome analysis indicated that two transcripts differing in their start sites are produced from the skeletal muscle ryanodine receptor gene in a tissue‐specific fashion, and that the mRNA in brain may code the carboxyl‐terminal region of the ryanodine receptor molecule. cDNA expression experiments suggested that the ATG triplet encoding Met4382 of the skeletal muscle ryanodine receptor can function as a translation initiation codon, and that the expressed protein composed of the carboxy terminal 656 amino acid residues of the receptor is located on the endoplasmic reticulum membrane.

Molecular cloning and characterization of a human brain ryanodine receptor.

We have cloned and sequenced the cDNA of the human brain ryanodine receptor (RyR3), which is composed of 4866 amino acids and shares characteristic structural features with the rabbit RyR3. Northern blot analysis shows that the human RyR3 mRNA is abundantly expressed in hippocampus, caudate nucleus and amygdala as well as in skeletal muscle. The human RyR3 mRNA is also detected in several cell lines derived from human brain tumors. Functional expression of RyR3 and a chimeric RyR suggests that RyR3 forms a calcium‐release channel with a very low Ca2+ sensitivity.

Stable expression and characterization of human PN1 and PN3 sodium channels.

Nociceptive transduction in inflammatory and neuropathic pain involves peripherally expressed voltage-gated sodium channels, such as tetrodotoxin (TTX)-sensitive PN1 and TTX-resistant PN3. We generated recombinant cell lines stably expressing the human PN1 and PN3 sodium channels in Chinese hamster ovary (CHO) cells using inducible expression vectors. The PN1 and PN3 cDNAs were isolated from human adrenal gland and heart poly(A)+ RNAs, respectively. The recombinant human PN1 currents exhibited rapid activation and inactivation kinetics and were blocked by TTX with a half-maximal inhibitory concentration (IC50) of 32.6 nM. The human PN3 channel expressed in stable transfectants showed TTX-resistant inward currents with slow inactivation kinetics. The IC50 value for TTX was 73.3 microM. The voltage-dependence of activation of the PN3 channel was shifted to the depolarizing direction, compared to that of the PN1 channel. Lidocaine and mexiletine exhibited tonic and use-dependent block of PN1 and PN3 channels. The PN1 channel was more susceptible to inhibition by mexiletine than PN3. These results suggest that stable transfectants expressing the human PN1 and PN3 sodium channels will be useful tools to define subtype selectivity for sodium channel blockers.

The lethal expression of the GluR2flip/GluR4flip AMPA receptor in HEK293 cells

α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) ‐type glutamate receptors play a critical role in excitotoxicity associated with cerebral hypoxia, ischaemia and other acute brain insults. AMPA receptors are composed of GluR1–GluR4 subunits in homomeric and heteromeric assemblies, forming nonselective cation channels. In addition, each subunit has alternative splice variants, flip and flop forms. Heterologous expression studies showed that the AMPA receptor channels exhibit diverse properties depending on subunit/variant composition. For example, the absence of the GluR2 subunit makes AMPA receptor assemblies Ca2+‐permeable. Excitotoxicity induced by activating AMPA receptor channels has been linked to excessive Ca2+ influx through the GluR2‐lacking channels. Here we demonstrate that coexpression of the AMPA receptor GluR2flip and GluR4flip subunits exerts a lethal effect on HEK293 cells, whereas no lethal activity is observed in other homomeric or heteromeric combinations of AMPA receptor subunits. Patch clamp recordings and Ca2+ imaging analyses have revealed that this GluR2flip/GluR4flip receptor exhibits a low Ca2+ permeability. This subunit combination, however, showed prolonged Na+ influx following AMPA stimulation, even in the absence of cyclothiazide, which attenuates AMPA receptor desensitization. Furthermore, the GluR2flip/GluR4flip‐mediated lethality was potentiated by the interruption of cellular Na+ extrusion mechanisms using ouabain or benzamil. These observations suggest that the GluR2flip/GluR4flip receptor‐mediated excitotoxicity is attributed to Na+ overload, but not Ca2+ influx.

Cyclic AMP-dependent phosphorylation and regulation of the cardiac dihydropyridine-sensitive Ca channel.

A polyclonal antibody, CR2, prepared using the C-terminal peptide of the alpha 1 subunit of the rabbit cardiac DHP-sensitive Ca channel, specifically immunoprecipitated the [3H]PN200-110-labeled Ca channel solubilized from cardiac microsomes. The antibody recognized 250 and 200-kDa cardiac microsomal proteins as determined by immunoblotting, and cAMP-dependent protein kinase phosphorylated the 250-kDa, but not the 200-kDa protein in vitro. CHO cells, transfected with the cardiac alpha 1 subunit cDNA carried by an expression vector, synthesized a 250-kDa protein which was recognized by CR2. Adding db-cAMP or forskolin to the transformed CHO cells induced phosphorylation of the 250-kDa protein and stimulated the DHP-sensitive Ba current under patch-clamp conditions. These results suggested that the cardiac DHP-sensitive Ca channel was regulated by cAMP-dependent phosphorylation of the alpha 1 subunit.