Toshihide Nukada

Primary structure of the α-subunit of bovine adenylate cyclase-stimulating G-protein deduced from the cDNA sequence

The primary structure of the α‐subunit of the adenylate cyclase‐stimulating G‐protein (Gs) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. Comparison of the amino acid sequences of the α‐subunits of Gs and transducin reveals that some of the highly conserved regions show sequence homology with elongation factor‐Tu and ras p21 proteins and correspond to functional regions of guanine nucleotide‐binding proteins.

A region of the muscarinic-gated atrial K+ channel critical for activation by G protein βγ subunits

Abstract Complementary DNAs encoding two types of inwardly rectifying K + channels, GIRK1 and IRK1, have been cloned from rat atrium and mouse macrophage, respectively. GIRK1 expressed in Xenopus oocytes was activated by acetylcholine when m2 muscarinic acetylcholine receptor was coexpressed. The acetylcholine-induced activation of GIRK1 was enhanced by coexpression with the G protein β1γ2 subunit but not the β1γ1 or a subunits. Deletion of the C-terminus of GIRK1 impaired the channel activation associated with the β1γ2 subunit. Moreover, replacement of the C-terminus of IRK1 with that of GIRK1 produced a chimera channel that was activated by the β1γ2 subunit, whereas intact IRK1 was not activated by the S1γ2 subunit. These findings define the C-terminus of GIRK1 as a regulatory region for the G protein βγ subunit.

Voltage and pH dependent block of cloned N-type Ca2+ channels by amlodipine

Two types of Ca2+ channel α1‐subunits were co‐expressed in Xenopus oocytes with the Ca2+ channel α2‐ and β1‐subunits. The Ba2+ current through the α1Cα2β and the α1Bα2β channels had electrophysiological and pharmacological properties of L‐ and N‐type Ca2+ channels, respectively. Amlodipine had a strong blocking action on both the L‐type and N‐type Ca2+ channels expressed in the oocyte. The potency of the amlodipine block on the N‐type Ca2+ channel was comparable to that on the L‐type Ca2+ channel. At −100mV holding potential, the IC50 values for amlodipine block on the L‐type and N‐type Ca2+ channel were 2.4 and 5.8μm, respectively. The blocking action of amlodipine on the N‐type Ca2+ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L‐type Ca2+ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine. The time course of block development by amlodipine was similar for L‐type and N‐type Ca2+ channels. However, it was slower than the time course of block development by nifedipine for the L‐type Ca2+ channel.

DIFFERENTIAL INTERACTIONS OF THE C TERMINUS AND THE CYTOPLASMIC I-II LOOP OF NEURONAL CA2+ CHANNELS WITH G-PROTEIN ALPHA AND BETA GAMMA SUBUNITS. II . EVIDENCE FOR DIRECT BINDING

The present study was designed to obtain evidence for direct interactions of G-protein α (Gα) and βγ subunits (Gβγ) with N- (α1B) and P/Q-type (α1A) Ca2+ channels, using synthetic peptides and fusion proteins derived from loop 1 (cytoplasmic loop between repeat I and II) and the C terminus of these channels. For N-type, prepulse facilitation as mediated by Gβγ was impaired when a synthetic loop 1 peptide was applied intracellularly. Receptor agonist-induced inhibition of N-type as mediated by Gα was also impaired by the loop 1 peptide but only when applied in combination with a C-terminal peptide. For P/Q-type channels, by contrast, the Gα-mediated inhibition was diminished by application of a C-terminal peptide alone. Moreover, in vitro binding analysis for N- and P/Q-type channels revealed direct interaction of Gα with C-terminal fusion proteins as well as direct interaction of Gβγ with loop 1 fusion proteins. These findings define loop 1 of N- and P/Q-type Ca2+ channels as an interaction site for Gβγ and the C termini for Gα.

Amino acid residues in the transmembrane domain of the type 1 sigma receptor critical for ligand binding

The type 1 sigma receptor expressed in Xenopus oocytes showed binding abilities for the sigma‐1 ligands, [3H](+)pentazocine and [3H]NE‐100, with similar kinetic properties as observed in native tissue membranes. Amino acid substitutions (Ser99Ala, Tyr103Phe and di‐Leu105,106di‐Ala) in the transmembrane domain did not alter the expression levels of the type 1 sigma receptor as determined by immunoblot analysis using an anti‐type 1 sigma receptor antiserum. By contrast, ligand binding was significantly suppressed by the substitutions. These findings provide evidence that the transmembrane domain of the type 1 sigma receptor plays a critical role in ligand binding of this receptor.

Differential blocking action of dihydropyridine Ca2+ antagonists on a T-type Ca2+ channel (alpha1G) expressed in Xenopus oocytes.

Recent reports show that efonidipine, a dihydropyridine Ca2+ antagonist, has blocking action on T-type Ca2+ channels, which may produce favorable actions on cardiovascular systems. However, the effects of other dihydropyridine Ca2+ antagonists on T-type Ca2+ channels have not been investigated yet. Therefore, in this study, we examined the effects of dihydropyridine compounds clinically used for treatment of hypertension on a T-type Ca2+ channel subtype, α1G, expressed in Xenopus oocytes. These effects were compared with those on T-type Ca2+ channel. Rabbit L-type (α1Cα2/δβ1a) or rat T-type (α1G) Ca2+ channel was expressed in Xenopus oocytes by injection of cRNA for each subunit. The Ba2+ currents through expressed channels were measured by conventional 2-microelectrode voltage-clamp methods. Twelve DHPs (amlodipine, barnidipine, benidipine, cilnidipine, efonidipine, felodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nitrendipine) and mibefradil were tested. Cilnidipine, felodipine, nifedipine, nilvadipine, minodipine, and nitrendipine had little effect on the T-type channel. The blocks by drugs at 10 μM were less than 10% at a holding potential of −100 mV. The remaining 6 drugs had blocking action on the T-type channel comparable to that on the L-type channel. The blocking actions were also comparable to that by mibefradil. These results show that many dihydropyridine Ca2+ antagonists have blocking action on the α1G channel subtype. The action of dihydropyridine Ca2+ antagonists in clinical treatment should be evaluated on the basis of subtype selectivity.

Identification of R(−)‐isomer of efonidipine as a selective blocker of T‐type Ca2+ channels

Efonidipine, a derivative of dihydropyridine Ca2+ antagonist, is known to block both L‐ and T‐type Ca2+ channels. It remains to be clarified, however, whether efonidipine affects other voltage‐dependent Ca2+ channel subtypes such as N‐, P/Q‐ and R‐types, and whether the optical isomers of efonidipine have different selectivities in blocking these Ca2+ channels, including L‐ and T‐types. To address these issues, the effects of efonidipine and its R(−)‐ and S(+)‐isomers on these Ca2+ channel subtypes were examined electrophysiologically in the expression systems using Xenopus oocytes and baby hamster kidney cells (BHK tk‐ts13). Efonidipine, a mixture of R(−)‐ and S(+)‐isomers, exerted blocking actions on L‐ and T‐types, but no effects on N‐, P/Q‐ and R‐type Ca2+ channels. The selective blocking actions on L‐ and T‐type channels were reproduced by the S(+)‐efonidipine isomer. By contrast, the R(−)‐efonidipine isomer preferentially blocked T‐type channels. The blocking actions of efonidipine and its enantiomers were dependent on holding potentials. These findings indicate that the R(−)‐isomer of efonidipine is a specific blocker of the T‐type Ca2+ channel.

Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. I. Molecular determination.

Interactions of G-protein α (Gα) and βγ subunits (Gβγ) with N- (α1B) and P/Q-type (α1A) Ca2+ channels were investigated using the Xenopus oocyte expression system. Gi3α was found to inhibit both N- and P/Q-type channels by receptor agonists, whereas Gβ1γ2 was responsible for prepulse facilitation of N-type channels. L-type channels (α1C) were not regulated by Gα or Gβγ. For N-type, prepulse facilitation mediated via Gβγ was impaired when the cytoplasmic I-II loop (loop 1) was deleted or replaced with the α1C loop 1. Gα-mediated inhibitions were also impaired by substitution of the α1C loop 1, but only when the C terminus was deleted. For P/Q-type, by contrast, deletion of the C terminus alone diminished Gα-mediated inhibition. Moreover, a chimera of L-type with the α1B loop 1 gained Gβγ-dependent facilitation, whereas an L-type chimera with the N- or P/Q-type C terminus gained Gα-mediated inhibition. These findings provide evidence that loop 1 of N-type channels is a regulatory site for Gβγ and the C termini of P/Q- and N-types for Gα.

Five different profiles of dihydropyridines in blocking T-type Ca2+ channel subtypes (Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I)) expressed in Xenopus oocytes☆

1,4-dihydropyridine (DHP) Ca(2+) antagonists have recently been shown to block T-type Ca(2+) channels, which may render favorable actions on cardiovascular systems. However, this evaluation remains to be done systematically for each T-type Ca(2+) channel subtype except for the Ca(v)3.1 (alpha(1G)) subtype. To address this issue at the molecular level, blocking effects of 14 kinds of DHPs (amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, efonidipine, felodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nitrendipine), which are clinically used for treatments of hypertension, on 3 subtypes of T-type Ca(2+) channels [Ca(v)3.2 (alpha(1H)), Ca(v)3.3 (alpha(1I)), and Ca(v)3.1 (alpha(1G))] were investigated in the Xenopus oocyte expression system using the two-microelectrode voltage-clamp technique. These 3 kinds (alpha(1H), alpha(1I) and alpha(1G)) of T-type channels were blocked by amlodipine, manidipine and nicardipine. On the other hand, azelnidipine, barnidipine, benidipine and efonidipine significantly blocked alpha(1H) and alpha(1G), but not alpha(1I) channels, while nilvadipine and nimodipine apparently blocked alpha(1H) and alpha(1I), but not alpha(1G) channels. Moreover, aranidipine blocked only alpha(1H) channels. By contrast, cilnidipine, felodipine, nifedipine and nitrendipine had little effects on these subtypes of T-type channels. The result indicates that the blockade of T-type Ca(2+) channels by derivatives of DHP Ca(2+) antagonist was selective for the channel subtype. Therefore, these selectivities of DHPs in blocking T-type Ca(2+) channel subtypes would provide useful pharmacological and clinical information on the mode of action of the drugs including side-effects and adverse effects.

A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K+ channels by G-protein βγ-subunits

To determine the interaction site(s) of ATP‐sensitive K+ (KATP) channels for G‐proteins, sulfonylurea receptor (SUR2A or SUR1) and pore‐forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS‐7. Intracellular application of the G‐protein βγ2‐subunits (Gβγ2) caused a reduction of ATP‐induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. Gβγ2 bound in vitro to both intracellular (loop‐NBD) and C‐terminal segments of SUR2A, each containing a nucleotide‐binding domain (NBD). Furthermore, a single amino acid substitution in the loop‐NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the Gβγ2‐dependent alteration of the channel activities. These findings provide evidence that Gβγ modulates KATP channels through a direct interaction with the loop‐NBD of SUR.