The functional significance of multimerization in ion channels.

Recently, the allosteric behavior of ion channels has been investigated by recording the individual steps leading to the complete activation of a cyclic nucleotide-gated ion channel. This information, in combination with recent studies on nicotinic acetylcholine receptor mutants, necessitates a modification of our current theories of the allosteric mechanisms of ion channels and gives new insights into the functional significance of multimerization in ion channels.

Electron microscopic evidence for the assembly of soluble pentameric extracellular domains of the nicotinic acetylcholine receptor.

Exploitation of soluble extracellular domains (ECDs) of the nicotinic acetylcholine receptor may provide a route to crystallographic studies aimed at exploring the structure and function of the intact receptor. The first step towards this goal is to manufacture and isolate soluble fragments that fold and assemble to form a functionally relevant complex. The baculovirus insect cell expression system was used to co-express soluble ECDs of all four muscle-type nicotinic acetylcholine receptor subunits (alpha, beta, gamma & delta-ECD) from Torpedo. Protein complexes were purified using either the conformationally sensitive monoclonal antibody mAb35, specific for a folded alpha subunit, or a NiNTA affinity resin, specific for a polyhistidine tag engineered on the delta-ECD. Western blotting with subunit specific antibodies confirmed the co-expression of each ECD and furthermore, indicated that the alpha, beta and gamma-ECDs were being co-purified with the polyhistidine-tagged delta-ECD. Chemical cross-linking was used to show that these co-purified proteins had indeed interacted specifically to form soluble oligomeric complexes. A low-resolution, three-dimensional image of these purified complexes, composed only of ECDs, was obtained by electron microscopy. They were shown to resemble the extracellular vestibule of the native receptor, having the same pseudo-pentameric symmetry, size and shape. Expression of incomplete sets of the four nicotinic acetylcholine receptor ECDs did not yield detectable complexes.

Differential Expression of Two Soybean (Glycine max L.) Proline-Rich Protein Genes after Wounding.

We have investigated the wound-induced expression of two members of the soybean (Glycine max L.) proline-rich cell wall protein gene family and show that SbPRP1 and SbPRP2 exhibit unique patterns of expression after physical damage. SbPRP1 mRNA can be detected in the hook of soybean seedlings within 2 h after wounding and is present at high levels in the hook and elongating hypocotyl 20 h after wounding. In contrast, SbPRP2 mRNA increases transiently and rapidly throughout the soybean seedling after wounding. SbPRP2 is also induced by wounding in soybean leaves, but the pattern of mRNA accumulation in leaves is distinct from that seen in seedlings and reaches high levels of expression 20 h after physical damage. SbPRP2 mRNA levels were also found to increase in the mature hypocotyl and roots of seedlings in response to treatment with 10 [mu]M indoleacetic acid and naphthalene-1-acetic acid. These data indicate that the wound-induced expression of PRPs in soybean is tissue specific and that the regulation of these genes after physical damage may operate through different signal transduction pathways.

Physicochemical and biological characterization of recombinant human inhibin A.

Recombinant human inhibin A was isolated from recombinant mammalian cell line culture media. Two forms of inhibin were identified with Mr of 34 and 31 Kd composed of subunits (alpha, beta) of 24 and 15 Kd and 21 and 15 Kd respectively. Both forms are bioactive in an inhibin in vitro bioassay and immunoactive with potencies comparable to or higher than purified bovine inhibin. Amino acid analyses and NH2-terminal sequences of each of the subunits are consistent with those predicted from their cDNA structures. The inhibin alpha- but not beta-subunit is glycosylated based on its binding to the lectins concanavalin A and wheat germ lectin. The difference in molecular weight of 31 and 34 Kd inhibin is attributed to variation in glycosylation of the alpha-subunit. The 31+34 Kd inhibin is heterogeneous on isoelectric focusing gels consisting of four isoforms in the pH range 6.2-7.6. Inhibition also exhibits in vivo biological activity by suppressing serum FSH but not LH in castrate male rats. These physicochemical and biological characteristics of recombinant human inhibin are similar to those described for native inhibin isolated from a variety of other species.

Immunisation against the amino-terminal peptide (alpha N) of the alpha 43 subunit of inhibin impairs fertility in sheep.

Processing of the 58 kDa to the 31 kDa form of inhibin (Inh) involves cleavage of the amino-terminal peptide (alpha N) from the alpha 43-subunit. We show that active immunisation of female sheep against a recombinant bovine alpha N impairs their fertility. In Exp 1, 5 treated (Group 1; 300 micrograms alpha N) and 6 control ewes (Group 2; adjuvant only) were immunized (Day 1) and given boosters on Days 22 and 56. In Group 1, mean +/- SEM binding of 125I-31 kDa Inh was less than 0.5% on Days 33 and 44, whereas binding of 125I-58 kDa Inh was 4.9 +/- 0.7 and 6.2 +/- 0.6%, respectively. In Group 2 binding of both tracers was less than 0.5%. The corpora lutea (CL)/ewe in Group 1 on Days 44 and 82 were 1.8 +/- 0.2 and 2.8 +/- 0.9, respectively, and were not different from those in Group 2 (1.7 +/- 0.3 and 1.5 +/- 0.2, respectively). One ewe in Group 1 versus 5/6 ewes in Group 2 were diagnosed pregnant. In Exp 2, 18 treated and 16 controls were immunized as in Exp 1. The binding of 125I-58 kDa Inh in treated ewes (2.4 +/- 0.3%) was greater than in controls (less than 0.5%) on Day 56. The CL/ewe in treated ewes (1.8 +/- 0.2) was similar to that in controls (2.0 +/- 0.1) on Day 76. All 16 control ewes but only 7/17 treated ewes were subsequently diagnosed pregnant. The plasma progesterone concentrations were similar in treated ewes which did (7.6 +/- 1.2 nmol/L) and did not (7.0 +/- 0.7) become pregnant. Neither basal nor GnRH-stimulated concentrations of LH, nor basal concentrations of Inh differed between treated and controls in Exp 2. Similarly, there were no differences in FSH, except that basal concentrations were higher in the luteal phase of treated ewes. We conclude that immunisation of ewes against alpha N results in a significant reduction in fertility.

A combination of human alpha 1 and beta 1 subunits is required for formation of detectable GABA-activated chloride channels in Sf9 cells.

The baculovirus expression system was used to produce alpha 1 and beta 1 subunits of the human GABAA receptor in Sf9 cells. In cells infected with both alpha 1 and beta 1 recombinant viruses, GABA elicited an outwardly rectifying chloride current that was blocked by bicuculline and potentiated by pentobarbitone. GABA did not produce detectable currents in cells infected with either alpha 1 or beta 1 recombinant viruses alone. In these cells, and in control (non-infected) Sf9 cells, pentobarbitone depressed the leakage current (Ki = 55 microM). Fluorescently labelled monoclonal antibodies to the alpha 1 subunit showed greater amounts of the alpha 1 subunit in cells infected with only the alpha 1 recombinant virus than in cells co-infected with the alpha 1 and beta 1 recombinant viruses. Fluorescence of the plasma membrane was seen in cells co-infected with the alpha 1 and beta 1 recombinant viruses, but was absent in cells infected with only the alpha 1 recombinant virus. It was concluded that the alpha 1 subunit normally interacts with the beta 1 subunit to be transported to the plasma membrane in Sf9 cells.

Direct vasoconstrictor action of homologous angiotensin II on isolated arterial ring preparations in an elasmobranch fish.

Arterial rings were prepared from the branchial artery, coeliac artery and ventral aorta of the Japanese dogfish Triakis scyllia and used to determine arterial contraction in a myograph. Noradrenaline caused a dose-dependent contraction (10(-9)-3 x 10(-6) M) that was completely inhibited by pre-treatment with 10(-7) M phentolamine. Homologous dogfish angiotensin II (ANG II) ([Asn1, Pro3, Ile5]-ANG II) also caused dose-dependent contraction (10(-9)-3 x 10(-6) M), but phentolamine had no effect on this response. Administration of dogfish angiotensin I (ANG-I) ([Asn1, Pro3, Ile5, Gln9]-ANG I) resulted in a contraction similar to that produced by ANG II and the effect could be blocked with 10(-7) M captopril. The mammalian ANG II receptor antagonists [Sar1, Ile8]-ANG II and [Sar1, Ala8]-ANG II caused dose-dependent contractions of coeliac artery rings, but were less potent than homologous ANG I and ANG II. These results show that the contractile effect of [Asn1, Pro3, Ile5]-ANG II is not mediated by the alpha-adrenergic system and contractions of arterial rings by noradrenaline and elasmobranch ANG II are mediated by separate vascular receptors. The elasmobranch ANG II vascular receptor may have co-evolved with the unusual structure of this peptide.

A threonine residue in the M2 region of the beta1 subunit is needed for expression of functional alpha1beta1 GABA(A) receptors.

Although there is a high degree of homology in the M2 transmembrane segments of alpha1 and beta1 subunits, subunit-specific effects were observed in alpha1beta1 GABA(A) receptors expressed in Spodoptera frugipedra (Sf9) cells when the conserved 13' threonine residue in the M2 transmembrane region was mutated to alanine. When threonine 263 (13') was mutated to alanine in the beta1 subunit, high-affinity muscimol binding and the response to GABA were abolished. This did not occur when the threonine 263 (13') was mutated to alanine in the alpha1 subunit, but the rate of desensitisation increased and the effect of bicuculline, a competitive inhibitor, was reduced. The results show differential effects of subunits on receptor function and support a role for M2 in desensitisation.

A role for the 2' residue in the second transmembrane helix of the GABA A receptor gamma2S subunit in channel conductance and gating.

GABA(A) receptors composed of alpha, beta and gamma subunits display a significantly higher single-channel conductance than receptors comprised of only alpha and beta subunits. The pore of GABA(A) receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6', 10' and 13' positions. At the 2' position, however, a polar residue is present in the gamma subunit but not the alpha or beta subunits. As residues at the 2', 6' and 10' positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type alpha1beta1 and alpha1beta1gamma2S receptors with two mutant receptors, alphabetagamma(S2'A) and alphabetagamma(S2'V). We found that the single-channel conductance of both mutant alphabetagamma receptors was significantly decreased with respect to wild-type alphabetagamma, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant alphabetagamma receptors remained larger than wild-type alphabeta channels. This reduction in the conductance of mutant alphabetagamma receptors was observed at depolarized potentials only (E(Cl) = -1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the gamma2' residue. The substitutions at the gamma2' serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2' position in M2 of the gamma subunit affects both single-channel conductance and receptor kinetics.

Isolation and characterization of a proline-rich cell wall protein from soybean seedlings.

We have purified a cell wall protein from extracts of soybean (Glycine max) that was previously shown to be immunologically related to p33, a wound-induced carrot cell wall protein (Tierney ML, Wiechert J, Pluymers D [1988] Mol Gen Genet 211: 393-399). Amino acid composition analysis reveals that the protein is 36% proline and hydroxyproline and its amino-terminus was determined to be AsnTyrGluAsnProHypValTyrLysProHypThrGluLysProHypValTyr. In vivo [(3)H]proline labeling of soybean hypocotyl tissues and Western blot analysis indicate that the apical hook, a region characterized by active growth, is a predominant site for synthesis of this protein and that wounding does not significantly affect its synthesis or accumulation. Antibodies raised against the hook protein also cross-reacted with a higher molecular weight protein in cell wall extracts of wounded hook tissue. These data indicate that there are at least two related, proline-rich cell wall proteins in the hook region of soybean seedlings, one that appears to be developmentally regulated and another that is wound-inducible. RNA encoding the developmentally regulated seedling cell wall protein was detected in both the hook and elongating region of the hypocotyl. The presence of this RNA in the elongating region in the absence of detectable levels of the protein indicates either that the expression of this cell wall protein is subject to posttranscriptional control or that the rate of insolubilization of this protein increases in more mature regions of the hypocotyl.

The extensins.

The plant cell was includes a matrix which is species specific and which chages in composition during growth and development. Characterization of the protein component of the wall matrix has resulted in the purification of extensin and the genes which encode it. Analysis of the protein sequences for the extensins has provided clues about the types of interactions which may occur as the chemistry and architecture of the cell wall accommodate growth and development.

Expression of AtPRP3, a proline-rich structural cell wall protein from Arabidopsis, is regulated by cell-type-specific developmental pathways involved in root hair formation.

The tightly regulated expression patterns of structural cell wall proteins in several plant species indicate that they play a crucial role in determining the extracellular matrix structure for specific cell types. We demonstrate that AtPRP3, a proline-rich cell wall protein in Arabidopsis, is expressed in root-hair-bearing epidermal cells at the root/shoot junction and within the root differentiation zone of light-grown seedlings. Several lines of evidence support a direct relationship between AtPRP3 expression and root hair development. AtPRP3/beta-glucuronidase (GUS) expression increased in roots of transgenic seedlings treated with either 1-aminocyclopropane-1-carboxylic acid (ACC) or alpha-naphthaleneacetic acid (alpha-NAA), compounds known to promote root hair formation. In the presence of 1-alpha-(2-aminoethoxyvinyl)glycine (AVG), an inhibitor of ethylene biosynthesis, AtPRP3/GUS expression was strongly reduced, but could be rescued by co-addition of ACC or alpha-NAA to the growth medium. In addition, AtPRP3/GUS activity was enhanced in ttg and gl2 mutant backgrounds that exhibit ectopic root hairs, but was reduced in rhd6 and 35S-R root-hair-less mutant seedlings. These results indicate that AtPRP3 is regulated by developmental pathways involved in root hair formation, and are consistent with AtPRP3's contributing to cell wall structure in Arabidopsis root hairs.

Deletion analysis and localization of SbPRP1, a soybean cell wall protein gene, in roots of transgenic tobacco and cowpea.

SbPRP1 is a member of the soybean (Glycine max L. Merr) proline-rich cell wall protein family and is expressed at high levels in root tissue. To characterize the sequences required for this expression, we have fused 1.1 kb of upstream flanking DNA sequence from an SbPRP1 genomic clone to a gene encoding beta-glucuronidase (GUS). This construct was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. Histochemical staining of GUS activity in transgenic tobacco indicated that SbPRP1 is expressed in the apical and elongating region of both primary and lateral roots, most strongly in the epidermis. A similar localization pattern was found in transformed hairy roots when this construct was introduced into cowpea (Vigna aconitifolia) using Agrobacterium rhizogenes-mediated transformation. Nested 5'-deletion analysis of the SbPRP1 promoter indicated that a minimal promoter for SbPRP1 expression in roots is located within the first 262 bases of upstream flanking DNA and that the region between -1080 and -262 is required for maximal expression of this gene. Gel retardation assays showed that nuclear factors can be detected in soybean roots which specifically bind to sequences located between -1080 and -623, a region which is needed for maximal expression of the SbPRP1 promoter. Northern hybridization analysis was also used to show that little SbPRP1 mRNA was present in roots during the first 24 h after imbibition. These studies indicate that SbPRP1 expression is localized to the actively growing region of the root and that this expression is temporally regulated during very early stages of seedling growth.

Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression.

Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to soybean suspension cultures also increased mRNA levels for three wound-responsive genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) suggesting a role for MeJA/JA in the mediation of several changes in gene expression associated with the plants' response to wounding.

The role of the renin-angiotensin system in the control of blood pressure and drinking in the European eel, Anguilla anguilla.

The role of the renin-angiotensin system (RAS) in the control of blood pressure and drinking was investigated in fresh water (FW)- and seawater (SW)-adapted eels, Anguilla anguilla, by comparing the effects of pharmacological manipulation through the use of papaverine (stimulator) and captopril (inhibitor) on the endogenous system. In SW eels basal blood pressure levels were lower (23.3 +/- 0.8 mm Hg) with correspondingly higher basal drinking rates (0.51 +/- 0.07 ml/kg/hr) and plasma AII concentrations (32.89 +/- 4.19 fmol/ml) compared to FW eels (33.8 +/- 1.3 mm Hg, 0.06 +/- 0.02 ml/kg/hr, 9.72 +/- 0.60 fmol/ml, respectively). In FW eels papaverine caused immediate hypotension with full recovery, decrease in plasma osmolality, and increase in drinking rate and plasma AII concentration, but in SW eels, hypotension with full recovery and an increase in plasma osmolality, drinking rate, and plasma AII concentration occurred. In FW eels captopril had no effect on the parameters measured, but in SW eels it caused a sustained decrease in blood pressure and a decline in the basal drinking rate and plasma AII concentration. Papaverine was also administered 15 min after captopril. In FW eels this manipulation caused hypotension only after the papaverine injection, followed by a partial recovery. Osmolality was unaffected, the previously observed papaverine-induced dipsogenic response was blocked, and the rise in plasma AII concentrations was smaller than with papaverine only. In SW eels there was an immediate hypotension after captopril administration with full recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and expression of four proline-rich cell wall protein genes in Arabidopsis encoding two distinct subsets of multiple domain proteins.

We have characterized the molecular organization and expression of four proline-rich protein genes from Arabidopsis (AtPRPs). These genes predict two classes of cell wall proteins based on DNA sequence identity, repetitive motifs, and domain organization. AtPRP1 and AtPRP3 encode proteins containing an N-terminal PRP-like domain followed by a C-terminal domain that is biased toward P, T, Y, and K. AtPRP2 and AtPRP4 represent a second, novel group of PRP genes that encode two-domain proteins containing a non-repetitive N-terminal domain followed by a PRP-like region rich in P, V, K, and C. Northern hybridization analysis indicated that AtPRP1 and AtPRP3 are exclusively expressed in roots, while transcripts encoding AtPRP2 and AtPRP4 were most abundant in aerial organs of the plant. Histochemical analyses of promoter/beta-glucuronidase fusions localized AtPRP3 expression to regions of the root containing root hairs. AtPRP2 and AtPRP4 expression was detected in expanding leaves, stems, flowers, and siliques. In addition, AtPRP4 expression was detected in stipules and during the early stages of lateral root formation. These studies support a model for involvement of PRPs in specifying cell-type-specific wall structures, and provide the basis for a genetic approach to dissect the function of PRPs during growth and development.

A radioimmunoassay for the determination of angiotensin II in elasmobranch fish.

A homologous radioimmunoassay was developed to determine the concentration of angiotensin II (Asn1, Pro3, Ile5)-Ang II) in elasmobranchs. Cross-reactivity with elasmobranch angiotensin I and other heterologous angiotensins was high and therefore all potentially cross-reacting angiotensins were separated by high performance liquid chromatography after prior extraction with Sep-Pak C18 cartridges. The validity of the assay for the determination of elasmobranch Ang II was demonstrated by parallelism with a series of Ang II standards with serially diluted elasmobranch plasma extracts. Overall recovery of elasmobranch Ang II added to a plasma pool was 75.1 +/- 5.2%. Plasma Ang II concentrations measured by our RIA were similar in fish adapted to 70, 100, or 120% SW at 139 +/- 20.1, 109 +/- 15.3, and 119 +/- 16.3 fmol . ml-1, respectively.

Renin-angiotensin system in elasmobranch fish: A review.

The renin-angiotensin system (RAS) has been identified recently in elasmobranch fish, and the structure of angiotensin II (ANG II) is unusual ([Asp(1),Pro(3),Ile(5)]-ANG II) compared to other vertebrates. Receptors for ANG II have been identified in blood vessels and in a variety of osmoregulatory tissues including the gill, kidney and rectal gland. In addition, there is considerable binding to the interrenal gland and the stimulation of 1alpha-hydroxycorticosterone production in vitro suggests a physiological role in corticosteroidogenesis. ANG II is a potent vasoconstrictor and this effect does not appear to be mediated by sympathetic activation or catecholamine release. Although the RAS may not be involved in maintaining basal blood pressure, it may be important in situations in which blood pressure is reduced. Understanding of the role of ANG II as an osmoregulatory hormone is only just emerging with putative roles in the control of gill, rectal gland and perhaps, drinking. In addition, the stimulation of corticosteroid secretion may provide another means of controlling osmoregulation. J. Exp. Zool. 284:526-534, 1999.

Expression of DcPRP1 is linked to carrot storage root formation and is induced by wounding and auxin treatment.

A carrot (Daucus carota, L.) genomic clone (DcPRP1) was isolated on the basis of its homology to previously described cDNAs encoding a wound-inducible, proline-rich cell wall protein. DNA sequence analysis showed that DcPRP1 contains a single open-reading frame encoding a 235-amino acid protein that is colinear with that predicted from the cDNA sequence with the exception of four amino acids at the N terminus and a 60-nucleotide insertion present within the genomic clone. Genomic Southern hybridization analysis showed that the cloned sequence hybridized with a single restriction enzyme fragment using several restriction enzymes. Primer extension and northern hybridization analysis indicated that the expression of DcPRP1 is developmentally regulated and linked to the formation of storage roots, where this gene is expressed at high levels after wounding. The level of DcPRP1 mRNA was greatest in tissue immediately adjacent to the wound site. Treatment of unwounded carrot storage roots with 10 microM 2,4-dichlorophenoxy-acetic acid, indoleacetic acid, or naphthalene-1-acetic acid also resulted in the accumulation of DcPRP1 transcripts to a level equal to that seen in wounded tissue.

Angiotensin II binding sites in the heart of Scyliorhinus canicula: an autoradiographic study.

Dogfish (125)I [Asn(1), Pro(3), Ile(5)] angiotensin II ((125)I dfANG II) was used to establish the specific binding patterns of the different cardiac regions of the elasmobranch Scyliorhinus canicula by in vitro autoradiography. In the ventricular myocardium Scatchard analysis of saturation and displacement binding data revealed two classes of high- and low-affinity dfANG II binding sites (K(d) = 53 +/- 10 and 1300 +/- 900 pM). Two classes of dfANG II binding sites were also detected in the atrium (K(d) = 47 +/- 13 and 4690 +/- 930 pM) and in the outer layer of the conus arteriosus (K(d) = 16 +/- 9 and 398 +/- 83 pM). Conversely, the ventricular endocardium and the inner conal layer were characterized by a single class of dfANG II binding sites with affinity values of 48 +/- 11 and 106 +/- 3.3 pM, respectively. Competition experiments with either cold dfANG II or CV11974 or CGP42112 (specific ligands for mammalian AT(1) and AT(2) receptors, respectively) demonstrated a prevalence of CGP42112-selective dfANG II binding sites in both the inner and the outer conal layers. In the atrium, the ventricular myocardium, and the outer conal layer, dfANG II high-affinity binding sites poorly discriminated among the cold ligands. These results suggest that the dogfish heart may be a target organ of ANG II with distinct ANG II receptor subtype distributions.