Masayoshi Mishina

Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels

Abstract1.Functional acetylcholine receptor (AChR) and sodium channels were expressed in the membrane ofXenopus laevis oocytes following injection with poly(A)+-mRNA extracted from denervated rat leg muscle. Wholecell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle. Oocytes injected with specific mRNA, transcribed from cDNA templates and coding for the AChR ofTorpedo electric organ, expressed functional AChR channels at a much higher density.2.Single-channel currents were recorded from the oocyte plasma membrane following removal of the follicle cell layer and the vitelline membrane from the oocyte. The follicle cell layer was removed enzymatically with collagenase. The vitelline membrane was removed either mechanically after briefly exposing the oocyte to a hypertonic solution, or by enzyme treatment with pronase.3.Stretch activated (s.a.) currents were observed in most recordings from cell-attached patches obtained with standard patch pipettes. S.a.-currents were evoked by negative or positive pressure (≥5 mbar) applied to the inside of the pipette, and were observed in both normal and mRNA injected oocytes indicating that they are endogenous to the oocyte membrane.4.The s.a.-channels are cation selective and their conductance is 28 pS in normal frog Ringers solution (20±1°C). Their gating is voltage dependent, and their open probability increases toward more positive membrane potentials.5.The density of s.a.-channels is estimated to be 0.5–2 channels per μm2 of oocyte plasma membrane. In cell-attached patches s.a.-currents are observed much less frequently when current measurement is restricted to smaller patches of 3–5 μm2 area using thick-walled pipettes with narrow tips. In outside-out patches s.a.-currents occur much less frequently than in cell-attached or inside-out patches.6.AChR-channel and sodium channel currents were observed only in a minority of patches from oocytes injected with poly(A)+-mRNA from rat muscle. AChR-channel currents were seen in all patches of oocytes injected with specific mRNA coding forTorpedo AChR. In normal frog Ringers solution (20±2°C) the conductance of implanted rat muscle AChR-channels was 38 pS and that of sodium channels 20 pS. The conductance of implantedTorpedo AChR channels was 40 pS. The conductance of implanted channels was similar in cell-attached and in cell-free patches.7.The conductances of rat muscle AChR and sodium channels implanted into the oocyte membrane were similar to those of channels in their native muscle membrane, suggesting that important functional properties of these channels are determined by their primary amino acid sequence.

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.

Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate

The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1‐specific mRNA and the dose‐response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate‐gated ionic channels are hetero‐oligomers of different 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.

Expression of functional (Na+ + K+)-ATPase from cloned cDNAs

Functional (Na+ + K+)‐ATPase is formed in Xenopus oocytes injected with α‐ and β‐subunit‐specific mRNAs derived from cloned Torpedo californica cDNAs. Both the mRNAs are required for the expression of functional (Na+ + K+)‐ATPase.

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.

Functional properties of nicotinic acetylcholine receptor subunits expressed in various combinations

The four kinds of subunits of the Torpedo californica nicotinic acetylcholine receptor have been produced in various combinations by injecting Xenopus oocytes with the corresponding subunit‐specific mRNAs synthesized by transcription in vitro of the cloned cDNAs. Functional analysis suggests that association of the α‐subunit with either the γ‐ or the δ‐subunit is a prerequisite for generating the conformation necessary for agonist binding. The acetylcholine receptor devoid of either the β‐, γ‐ or δ‐subunit exhibits weak channel activity.

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.

Isolation and characterization of the mouse corticotropin-β-lipotropin precursor gene and a related pseudogene

Two mouse genomic DNA sequences homologous with human corticotropin‐β‐lipotropin precursor gene sequences have been cloned. One of them represents the functional corticotropin‐β‐lipotropin precursor gene, which exhibits a structural organization similar to those of its bovine and human counterparts. The other represents a pseudogene that corresponds to the functional mouse gene sequence encoding the carboxy‐terminal 143 amino acid residues (including corticotropin and β‐lipotropin) and the 3′‐untranslated region.