Tai Kubo

Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence

The complete amino acid sequence of the porcine cardiac muscarinic acetylcholine receptor has been deduced by cloning and sequencing the cDNA. The tissue location of the RNA hybridizing with the cDNA suggests that this muscarinic receptor species represents the M2 subtype.

Tissue distribution of mRNAs encoding muscarinic acetylcholine receptor subtypes

The tissue distribution of the mRNAs encoding muscarinic acetylcholine receptors (mAChRs) I, II, III and IV has been investigated by blot hybridization analysis with specific probes. This study indicates that exocrine glands contain both mAChR I and III mRNAs, whereas smooth muscles contain both mAChR II and III mRNAs. All four mAChR mRNAs are present in cerebrum, whereas only mAChR II MRNA is found in heart.

Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle

The nicotinic acetylcholine receptor (AChR) from fish electric organ has a subunit structure of α2βγδ, and this is thought to be also the case for the mammalian skeletal muscle AChR1–3. By cloning and sequencing the complementary or genomic DNAs, we have previously elucidated the primary structures of all four sub-units of the Torpedo californica electroplax4–6 and calf muscle AChR7–10 and of the α- and γ-subunits of the human muscle AChR7,11; the primary structures of the γ-subunit of the T. californien AChR12 and the α-subunit of the Torpedo marmorata AChR13,14 have also been deduced elsewhere. We have now cloned DNA complementary to the calf muscle messenger RNA encoding a novel polypeptide (the ε-subunit) whose deduced amino-acid sequence has features characteristic of the AChR subunits and which shows higher sequence homology with the γ-subunit than with the other subunits. cDNA expression studies indicate that the calf ε-subunit, as well as the calf γ-subunit, can replace the Torpedo γ-subunit to form the functional receptor in combination with the Torpedo α-, β- and δ-subunits.

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.

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.

Intracellular calcium release mediated by two muscarinic receptor subtypes

Four subtypes of muscarinic acetylcholine receptor (mAChR) were stably expressed in neuroblastoma‐glioma hybrid cells (NG108‐15). By combining fluorescent indicator dye (fura‐2) studies with electrophysiological measurements it is shown that stimulation of mAChR I and mAChR III readily leads to release of calcium from intracellular stores and to associated conductance changes, whereas stimulation of mAChR II and mAChR IV exerts no such effect. Dose‐response curves describing the amplitude or the delay of the calcium rise induced by acetylcholine suggest that the apparent affinity of mAChR III for its agonist is higher by about one order of magnitude than that of mAChR I. Ionic substitution experiments and current fluctuation analysis indicate that calcium activates a K+‐specific conductance of ‘small’ single‐channel amplitude similar to the SK type [1]. Furthermore, an outward current (M current) suppressed by activation of mAChR I and mAChR III has a single‐channel amplitude corresponding to a conductance of approximately 3 pS.

Different sensitivities to agonist of muscarinic acetylcholine receptor subtypes

Muscarinic acetylcholine receptor (mAChR) III expressed in Xenopus oocytes, like mAChR I, mediates activation of a Ca2+‐dependent Cl− current, whereas mAChR IV, like mAChR II, principally induces activation of Na+ and K+ currents in a Ca2+‐independent manner. mAChR III has a sensitivity to agonist of about one order of magnitude higher than that of mAChR I in mediating the Ca2+‐dependent current response in Xenopus oocytes and in stimulating phosphoinositide hydrolysis in NG108‐15 neuroblastoma‐glioma hybrid cells. The agonist‐binding affinity of mAChR III is also about one order of magnitude higher than that of mAChR I.