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.

Phylogenetic conservation of disulfide-linked, dimeric acetylcholine receptor pentamers in southern ocean electric rays.

Intact acetylcholine receptors have been purified on a novel affinity resin from three electric fish endemic to Australian waters. Their binding properties and morphology are compared with those of their northern hemisphere homolog, Torpedo marmorata. All four exhibit apparent dissociation constants, Kd, in the nanomolar range for the snake neurotoxin alpha-bungarotoxin and have a distinctive rosette-like appearance when viewed in negative stain under the electron microscope. Furthermore, these rosettes are paired, indicating that acetylcholine receptors from southern ocean electric fish exist as dimers, in the same fashion as their northern hemisphere counterparts. The cDNAs of the receptor's four subunits were sequenced from Hypnos monopterigium and the northern hemisphere counterpart, Torpedo marmorata, while cDNAs from only two subunits, alpha and delta, were able to be sequenced from Narcine tasmaniensis. The penultimate amino acid in the delta subunit of each of the newly sequenced fish species is a cysteine residue. Its conservation suggests that the mechanism for the observed dimerization of acetylcholine receptors is disulfide bond formation between the delta subunit of adjacent receptors, analogous to acetylcholine receptor dimers observed in other electric fish. It appears that this mechanism for receptor clustering is unique to acetylcholine receptors packed and organized in the specialized organs of electric fish. Alignment of the deduced protein sequences with the equivalent sequences from Torpedo californica and humans reveals a high degree of homology.

Proline residues in two tightly coupled helices of the sulphate transporter, SHST1, are important for sulphate transport.

The sulphate transporter SHST1, from Stylosanthes hamata, features three tightly coupled transmembrane helices which include proline residues that are conserved in most related transporters. We used site-directed mutagenesis and expression of the mutant transporters in yeast to test whether these proline residues are important for function. Four proline residues were replaced by both alanine and leucine. Only one of these proline residues, Pro-144, was essential for sulphate transport. However, mutation of either Pro-133 or Pro-160 resulted in a severe decrease in sulphate transport activity; this was due more to a decrease in transport activity than to a decrease in the amount of mutant SHST1 in the plasma membrane. These results suggest that all three proline residues are important for transport, and that the conformation of the three tightly coupled helices may play a critical role in sulphate transport. We also show that SHST1 undergoes a post-translational modification that is required for trafficking to the plasma membrane.

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.

Angiotensin and angiotensin receptors in cartilaginous fishes.

In mammals, a principal bioactive component of the renin-angiotensin system (RAS), angiotensin II (ANG II), is known to be vasopressor, dipsogenic, a stimulant of adrenocortical secretion and to control glomerular and renal tubular function. Historically, a RAS analogous to that found in mammals was thought to have first evolved in the bony fishes. Recent research has identified the unusually structured elasmobranch [Asp(1)-Pro(3)-Ile(5)] ANG II. Physiological studies have demonstrated that ANG II in elasmobranchs is vasopressor, and stimulates interrenal gland production of the elasmobranch corticosteroid 1alpha-hydroxycorticosterone. The specific binding of ANG II in elasmobranchs has been reported in gills, heart, interrenal gland, gut and rectal gland. The precise osmoregulatory role ANG II plays in cartilaginous fishes is not yet known; however, putative evidence is emerging for a role in the control of drinking rate, rectal gland secretion, and kidney function.

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.

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.

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.

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.

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.

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 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.

Two threonine residues in the M2 segment of the alpha 1 beta 1 GABAA receptor are critical for ion channel function.

The role of three threonine residues in the M2 hydrophobic region of the GABAA receptor has been investigated by replacing these polar residues with alanine at the 6', 10' and 13' positions of M2 in the GABAA alpha 1, and beta 1 subunits and co-expressing the mutated subunits in the baculovirus Sf9 insect cell system. GABA did not elicit a current in cells expressing either the 6' or 13' threonine to the alanine mutants. The mutant subunits formed intact heteromeric GABAA receptors as judged by the binding of [3H] muscimol or the relative level of alpha 1 protein present in the plasma membrane. In contrast, a chloride current was generated by GABA in cells expressing the 10' mutant receptor. However, the current decayed more rapidly to baseline in the continued presence of GABA in the 10' mutant receptor than in the wild type receptor. The results are discussed in terms of the possible roles of the threonine residues in the ion conduction pathway.

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.

Nature of the 5' residue in the M2 domain affects function of the human alpha 1 beta 1 GABAA receptor.

The effects on the functional properties of the alpha 1 beta 1 GABAA receptor when the 5' (alpha 1 Val260; beta 1 Ile255) hydrophobic amino acids in the second transmembrane (M2) region were changed to threonine were examined. In response to a saturating concentration of GABA, the current evoked in mutant receptors showed a decreased rate of desensitization and at equilibrium was a greater fraction of the peak current than in wild-type receptors. The half-saturation concentration of the peak current response to GABA in mutant receptors was comparable to that in wild-type receptors, but the Hill coefficient was reduced to less than one. It was concluded that the 5' amino acids in the M2 region have a role in the conformational changes that occur within the alpha 1 beta 1 GABAA receptor in response to GABA.

A structural determinant of desensitization and allosteric regulation by pentobarbitone of the GABAA receptor.

Functional properties of the alpha1beta1 GABAA receptor changes in a subunit-specific manner when a threonine residue in the M2 region at the 12' position was mutated to glutamine. The rate and extent of desensitization increased in all mutants but the rate of activation was faster in the beta1 mutants. A negligible plateau current and abolition of potentiation by pentobarbitone of the GABA-activated current depended on the Thr 12' Gln mutation being present in the beta1 subunit. The Hill coefficient of the peak current response to GABA was reduced to less than one also in a beta1 subunit-specific manner. It was concluded that the beta1 subunit dominated conformational changes activated by GABA.

Effects of mutating leucine to threonine in the M2 segment of alpha1 and beta1 subunits of GABAA alpha1beta1 receptors.

The conserved leucine residues at the 9' positions in the M2 segments of alpha1 (L264) and beta1 (L259) subunits of the human GABAA receptor were replaced with threonine. Normal or mutant alpha1 subunits were co-expressed with normal or mutant beta1 subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher "resting" chloride conductance than cells expressing wild-type alpha1beta1 receptors. This chloride conductance was blocked by 10 mM penicillin, a recognized blocker of GABAA channels, but not by bicuculline (100 microm) or picrotoxin (100 microm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 microM). In cells expressing wild-type beta1 with mutated alpha1 subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type alpha1beta1 receptors. In cells expressing mutated beta1 subunits with wild-type or mutated alpha1 subunits (alphabeta(L9'T) and alpha(L9'T)beta(L9'T)), no response to GABA could be elicited: this was not due to an absence of GABAA receptors in the plasmalemma because the cells bound [3H]-muscimol. It was concluded that in GABAA channels containing the L9'T mutation in the beta1 subunit, GABA-binding does not cause opening of channels, and that the L9'T mutation in either or both subunits gives an open-channel state of the GABAA receptor in the absence of ligand.

Rapid desensitization of alpha 1 beta 1 GABA A receptors expressed in Sf9 cells under optimized conditions.

alpha 1 and beta 1 subunits of human GABA A receptors were expressed in Sf9 cells using the Sf9-baculovirus system. Better expression was obtained by manipulating the system. Cell growth phase at the time of infection determined the practical range of virus titre, the period postinfection during which cells were useful for signal detection and the maximal current obtained. Cells in the early exponential phase were relatively insensitive to multiplicity of infection (MOI) whereas cells in the mid- to late-exponential phase were highly dependent on MOI and they responded with the largest Cl- current generated by GABA. Channels activated by GABA were chloride-selective. Half the maximum peak whole-cell current was obtained with 11 microM GABA. The time course of Cl- currents activated by saturating GABA concentrations in cells infected with alpha 1 beta 1-recombinant viruses was examined employing a rapid perfusion system which allowed whole-cell solution exchange in less than 1 msec. The current decay could be fitted by 3 to 4 exponentials for the first 8 sec. The initial fast current decrease had a time constant of about 23 msec. No voltage dependence of time constants was detected but the whole-cell IV relation showed outward rectification. Currents were depressed by bicuculline, penicillin and picrotoxin and potentiated by pentobarbitone.

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)

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.