Junichi Nakai

Primary structure and distribution of a novel ryanodine receptor/calcium release channel from rabbit brain

The complete amino acid sequence of a novel ryanodine receptor/calcium release channel from rabbit brain has been deduced by cloning and sequence analysis of the EDNA. This protein is composed of 4872 amino acids and shares characteristic structural features with the skeletal muscle and cardiac ryanodine receptors. RNA blot hybridization analysis shows that the brain ryanodine receptor is abundantly expressed in corpus striatum, thalamus and hippocampus, whereas the cardiac ryanodine receptor is more uniformly expressed in the brain. The brain ryanodine receptor gene is transcribed also in smooth muscle.

Primary structure and functional expression from cDN A of the cardiac ryanodine receptor/calcium release channel

The sequence of 4968 (or 4976 with an insertion) amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by cloning and sequencing the cDNA. This protein is homologous in amino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopus oocytes injected with mRNA derived from the cardiac ryanodine receptor cDNA exhibit Ca2+‐dependent Cl− current in response to caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor mRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac mRNA. This result, in conjunction with cloning and analysis of partial cDNA sequences, suggests that the brain contains a cardiac type of ryanodine receptor mRNA.

Primary structure and functional expression of the ω-conotoxin-sensitive N-type calcium channel from rabbit brain

The complete amino acid sequence of a rabbit brain calcium channel (BIII) has been deduced by cloning and sequencing the cDNA. The open reading frame encodes 2339 amino acids, which corresponds to an M(r) of 261,167. A phylogenetic tree representing evolutionary relationships indicates that BIII is grouped together with the other rabbit brain calcium channels, BI and BII, into a subfamily that is distinct from the dihydropyridine-sensitive L-type subfamily. Transient expression in cultured skeletal muscle myotubes derived from muscular dysgenic mice demonstrates that the BIII channel mediates an omega-conotoxin-sensitive calcium current with kinetics and voltage dependence like those previously reported for whole-cell N-type current. Cell-attached patch recordings, with isotonic barium as the charge carrier, revealed distinct single channels with an average slope conductance of 14.3 pS.

Two regions of the ryanodine receptor involved in coupling with L-type Ca2+ channels.

Ryanodine receptors (RyRs) are present in the endoplasmic reticulum of virtually every cell type and serve critical roles, including excitation-contraction (EC) coupling in muscle cells. In skeletal muscle the primary control of RyR-1 (the predominant skeletal RyR isoform) occurs via an interaction with plasmalemmal dihydropyridine receptors (DHPRs), which function as both voltage sensors for EC coupling and as l-type Ca2+ channels (Rios, E., and Brum, G. (1987) Nature325, 717–720). In addition to “receiving” the EC coupling signal from the DHPR, RyR-1 also “transmits” a retrograde signal that enhances the Ca2+ channel activity of the DHPR (Nakai, J., Dirksen, R. T., Nguyen, H. T., Pessah, I. N., Beam, K. G., and Allen, P. D. (1996) Nature 380, 72–76). A similar kind of retrograde signaling (from RyRs tol-type Ca2+ channels) has also been reported in neurons (Chavis, P., Fagni, L., Lansman, J. B., and Bockaert, J. (1996) Nature 382, 719–722). To investigate the molecular mechanism of reciprocal signaling, we constructed cDNAs encoding chimeras of RyR-1 and RyR-2 (the predominant cardiac RyR isoform) and expressed them in dyspedic myotubes, which lack an endogenous RyR-1. We found that a chimera that contained residues 1,635–2,636 of RyR-1 both mediated skeletal-type EC coupling and enhanced Ca2+channel function, whereas a chimera containing adjacent RyR-1 residues (2,659–3,720) was only able to enhance Ca2+ channel function. These results demonstrate that two distinct regions are involved in the reciprocal interactions of RyR-1 with the skeletal DHPR.

Localization in the II-III Loop of the Dihydropyridine Receptor of a Sequence Critical for Excitation-Contraction Coupling

Skeletal and cardiac muscles express distinct isoforms of the dihydropyridine receptor (DHPR), a type of voltage-gated Ca2+ channel that is important for excitation-contraction (EC) coupling. However, entry of Ca2+ through the channel is not required for skeletal muscle-type EC coupling. Previous work (Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S. (1990) Nature346, 567–569) revealed that the loop between repeats II and III (II-III loop) is an important determinant of skeletal-type EC coupling. In the present study we have further dissected the regions of the II-III loop critical for skeletal-type EC coupling by expression of cDNA constructs in dysgenic myotubes. Because Ser687 of the skeletal II-III loop has been reported to be rapidly phosphorylatedin vitro, we substituted this serine with alanine, the corresponding cardiac residue. This alanine-substituted skeletal DHPR retained the ability to mediate skeletal-type EC coupling. Weak skeletal-type EC coupling was produced by a chimeric DHPR, which was entirely cardiac except for a small amount of skeletal sequence (residues 725–742) in the II-III loop. Skeletal-type coupling was stronger when both residues 725–742 and adjacent residues were skeletal (e.g. a chimera containing skeletal residues 711–765). However, residues 725–742 appeared to be critical because skeletal-type coupling was not produced either by a chimera with skeletal residues 711–732 or by one with skeletal residues 734–765.

Location of a region of the muscarinic acetylcholine receptor involved in selective effector coupling

Chimaeric muscarinic acetylcholine receptors (mAChR) in which corresponding portions of mAChR I and mAChR II are replaced with each other have been produced in Xenopus oocytes by expression of cDNA constructs encoding them. Functional analysis of the chimaeric mAChRs indicates that a region mostly comprising the putative cytoplasmic portion between the proposed transmembrane segments V and VI is involved in selective coupling of mAChR I and mAChR II with different effector systems. In contrast, the exchange of this region between mAChR I and mAChR II does not significantly affect the antagonist binding properties of the two mAChR subtypes.

Rings of Anionic Amino Acids as Structural Determinants of Ion Selectivity in the Acetylcholine Receptor Channel

To gain an insight into the molecular basis of the weak but significant selectivity among alkali metal cations of the nicotinic acetylcholine receptor (AChR) channel, we have determined single-channel conductance and permeability ratios for alkali metal cations on specifically mutated Torpedo californica AChR channels expressed in Xenopus oocytes. The mutations involved charged and polar side chains in the three anionic rings (extracellular, interm ediate and cytoplasmic ring) which have previously been found to determine the rate of K + transport through the AChR channel. The results obtained reveal that mutations in the intermediate ring exert much stronger effects on ion selectivity than do mutations in the extracellular and the cytoplasmic ring. The experimental results, together with simulations of the channel’s energy profile, suggest that the amino acid residues forming the intermediate ring come into close contact with permeating cations and possibly represent part of the physical correlate of the postulated selectivity filter in the AChR channel.

A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor

The channel pore of the nicotinic acetylcholine receptor (AChR) has been investigated by analysing single‐channel conductances of systematically mutatedTorpedo receptors expressed in Xenopus oocytes. The mutations mainly alter the size and polarity of uncharged polar amino acid residues of the acetylcholine receptor subunits positioned between the cytoplasmic ring and the extracellular ring. From the results obtained, we conclude that a ring of uncharged polar residues comprising threonine 244 of the α‐subunit (αT244), βS250, γT253 and δS258 (referred to as the central ring) and the anionic intermediate ring, which are adjacent to each other in the assumed α‐helical configuration of the M2‐containing transmembrane segment, together form a narrow channel constriction of short length, located close to the cytoplasmic side of the membrane. Our results also suggest that individual subunits, particularly the γ‐subunit, are asymmetrically positioned at the channel constriction.

Primary structure of porcine muscarinic acetylcholine receptor III and antagonist binding studies

The complete amino acid sequence of porcine muscarinic acetylcholine receptor III has been deduced by cloning and sequencing the genomic DNA. The antagonist binding properties of muscarinic acetylcholine receptor III expressed from the cloned DNA in Xenopus oocytes correspond most closely to those of the pharmacologically defined M2 glandular (III) subtype.

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.