Shosaku Numa

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.

Isolation and structural organization of the human preproenkephalin B gene.

Recently, we have elucidated the primary structure of bovine adrenal preproenkephalin by determining the nucleotide sequence of cloned DNA complementary to its mRNA1. The structure of most of this precursor molecule has also been deduced by Gubler et al.2 using cDNA sequencing in conjunction with protein sequencing. Bovine preproenkephalin contains four copies of methionine-enkephalin3 (Met-enkephalin) and one copy each of leucine-enkephalin3 (Leu-enkephalin), Met-enkephalin-Arg6-Phe7 (ref. 4) and Met-enkephalin-Arg6-Gly7-Leu8 (refs 1, 2, 5). The region containing the repeated enkephalin and extended enkephalin sequences, which are each bounded by paired basic amino acid residues, is connected with a cysteine-containing amino-terminal sequence preceded by a signal peptide6. We have now studied the relationship between the repetitive structure of preproenkephalin and the structural organization of its gene by cloning a human genomic DNA segment containing the entire gene. We find that the general organization of the preproenkephalin gene is strikingly similar to that of the gene encoding the common precursor of corticotropin (ACTH) and β-lipotropin (β-LPH)7–9 (alternatively designated preproopiomelanocortin), another multi-hormone precursor. Furthermore, the complete mRNA and amino acid sequences of human preproenkephalin have been deduced from the corresponding gene sequence.

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.

Cloning and sequence analysis of cDNA for rat corticotropin-releasing factor precursor.

DNA complementary to the rat hypothalamic mRNA coding for the corticotropin‐releasing factor precursor (prepro‐CRF) has been cloned by screening a cDNA library with a human genomic DNA probe. Nucleotide sequence analysis of the cloned cDNA has revealed that rat prepro‐CRF consists of 187 amino acid residues including a putative signal peptide. The CRF and putative signal peptide regions are more highly conserved among rat, human and ovine prepro‐CRF than is the cryptic portion.

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.

Patch clamp characterization of sodium channels expressed from rat brain cDNA

Sodium currents, INa, were recorded from Xenopus laevis oocytes which had been injected with mRNA synthesized by in vitro transcription of the rat brain sodium channel II cDNA (Noda et al. 1986 a,b). Patch pipettes were used to apply depolarizing voltage steps and to record macroscopic sodium currents of between 50 and 750 pA from cellattached patches of the oocyte membrane. With a combination of whole-cell and patch clamp recording, the properties of the implanted sodium channels could be studied in detail. They were analyzed according to the model of Hodgkin and Huxley (1952a) assuming three activation gates. The activation of the sodium currents is characterized by an equilibrium potential of-29 mV and an apparent gating charge of 8.7 e0. At-64 mV half of the sodium currents were inactivated. From singlechennel current recordings, an elementary sodium channel conductance of 19 pS and an average open time of 0.43 ms were obtained at-32 mV membrane potential and 16°C. The single-channel and activation properties of rat brain sodium channel II are therefore comparable to those found in peripheral nerve and skeletal muscle, but inactivation occurs at less negative potentials. This could be a specific property of the brain sodium channels and may underlie the maintained inward sodium currents reported in brain neurones (French and Gage 1985).

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.

Leumorphin is a novel endogenous opioid peptide derived from preproenkephalin B

Using synthetic leumorphin, we obtained antisera for leumorphin and set up two radioimmunoassays (RIAs) with different specificities. Gel exclusion chromatography coupled with the two RIAs showed the existence of a considerable amount of leumorphin-like peptide in water extracts from porcine neuro-intermediate pituitaries. Reverse phase high performance liquid chromatography revealed that leumorphin-like peptide in the water extracts was indistinguishable from synthetic leumorphin. These results along with potent opioid activity of leumorphin indicate that leumorphin is a novel endogenous opioid peptide derived from preproenkephalin B.

Structure and Function of Sodium Channel

The primary structures of the Electrophorus electroplax sodium channel and two distinct sodium channel large polypeptides from rat brain have been deduced by cloning and sequencing the cDNAs. The sodium channel molecule contains four internal repeats with homologous amino acid sequences, which are oriented presumably in a pseudosymmetric fashion across the membrane, thus forming the channel. The mRNAs generated by transcription of the cloned cDNAs encoding the rat brain sodium channel large polypeptides, when injected into Xenopus oocytes, direct the formation of functional sodium channels. The transmembrane topology of the sodium channel molecule and the structure that may be involved in the voltage-dependent gating of the channel are discussed.

Sequence requirement for transcription in vivo of the human preproenkephalin A gene.

The nucleotide sequence of the 5′‐flanking region of the cloned human preproenkephalin A gene, extending to 949 bp upstream of the capping site, has been determined. The preproenkephalin A gene, when joined with an SV40 vector and introduced into COS monkey cells, is efficiently transcribed from its own promoter. To assess the DNA sequence required for promoter function, we have constructed a series of 5′‐deletion mutants of a fusion gene that consists of the 949‐bp 5′‐flanking sequence and capping site of the preproenkephalin A gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The deletions up to 757‐172 bp upstream of the capping site exert essentially no effect on the expression of the fusion gene, whereas the deletions up to 145, 111, 81 and 67 bp upstream of the capping site result in a gradual decrease in the transcriptional efficiency. No detectable amount of the fusion gene transcript is produced with the mutants having deletions up to 67, 43 and 28 bp upstream of the capping site. These results indicate that a functional promoter of the preproenkephalin A gene lies between 67 and 171 bp upstream of the capping site. This promoter region corresponds to a highly GC‐rich segment with short repeated sequences and palindromes.