Mary Tierney

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.

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

Abstractα1 and β1 subunits of human GABAA 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 midto 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 μm GABA. The time course of Cl−} currents activated by saturating GABA concentrations in cells infected with α1β1recombinant 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.

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 α1 and β1 subunits of the human GABA receptor in Sf9 cells. In cells infected with both α1 and β1 recombinent 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 α1 or β1 recombinant viruses alone. In these cells, and in control (non-infected) Sf9 cells, pentobarbitone depressed the leakage current (Ki = 55 μM). Fluorescently labelled monoclonal antibodies to the α1 subunit showed greater amounts of the α1 subunit in cells infected with only the α1 recombinant virus than in cells co-infected with the α1 and β1 recombinant viruses. Fluorescence of the plasma membrane was seen in cells co-infected with the α1 and β1 recombinant viruses, but was absent in cells infected with only the α1 recombinant virus. It was concluded that the α1 subunit normally interacts with the β1 subunit to be transported to the plasma membrane in Sf9 cells.

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

GABAA receptors composed of α, β and γ subunits display a significantly higher single-channel conductance than receptors comprised of only α and β subunits. The pore of GABAA 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 γ subunit but not the α or β 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 α1β1 and α1β1γ2S receptors with two mutant receptors, αβγ(S2′A) and αβγ(S2′V). We found that the single-channel conductance of both mutant αβγ receptors was significantly decreased with respect to wild-type αβγ, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant αβγ receptors remained larger than wild-type αβ channels. This reduction in the conductance of mutant αβγ receptors was observed at depolarized potentials only (ECl = −1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the γ2′ residue. The substitutions at the γ2′ 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 γ subunit affects both single-channel conductance and receptor kinetics.

Insights into the biophysical properties of GABA A ion channels: Modulation of ion permeation by drugs and protein interactions

The fundamental properties of ion channels assure their selectivity for a particular ion, its rapid permeation through a central pore and that such electrical activity is modulated by factors that control the opening and closing (gating) of the channel. All cell types possess ion channels and their regulated flux of ions across the membrane play critical roles in all steps of life. An ion channel does not act alone to control cell excitability but rather forms part of larger protein complexes. The identification of protein interaction partners of ion channels and their influence on both the fundamental biophysical properties of the channel and its expression in the membrane are revealing the many ways in which electrical activity may be regulated. Highlighted here is the novel use of the patch clamp method to dissect out the influence of protein interactions on the activity of individual GABA(A) receptors. The studies demonstrate that ion conduction is a dynamic property of a channel and that protein interactions in a cytoplasmic domain underlie the channels ability to alter ion permeation. A structural model describing a reorganisation of the conserved cytoplasmic gondola domain and the influence of drugs on this process are presented.

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

SUMMARY 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 α-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 receptors four subunits were sequenced from Hypnos monopterigium and the northern hemisphere counterpart, Torpedo marmorata, while cDNAs from only two subunits,α and δ, were able to be sequenced from Narcine tasmaniensis. The penultimate amino acid in the δ 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 δ 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.

Selective modulation of different GABAA receptor isoforms by diazepam and etomidate in hippocampal neurons.

Diazepam modulation of native γ2-containing GABA(A) (γGABA(A)) receptors increases channel conductance by facilitating protein interactions involving the γ2-subunit amphipathic (MA) region, which is found in the cytoplasmic loop between transmembrane domains 3 and 4 (Everitt et al., 2009). However, many drugs, predicted to act on different GABA(A) receptor subtypes, increase channel conductance leading us to hypothesize that conductance variation in GABA(A) receptors may be a general property, mediated by protein interactions involving the cytoplasmic MA stretch of amino acids. In this study we have tested this hypothesis by potentiating extrasynaptic GABA(A) currents with etomidate and examining the ability of peptides mimicking either the γ2- or δ-subunit MA region to affect conductance. In inside-out hippocampal patches from newborn rats the general anesthetic etomidate potentiated GABA currents, producing either an increase in open probability and single-channel conductance or an increase in open probability, as described previously (Seymour et al., 2009). In patches displaying high conductance channels application of a δ((392-422)) MA peptide, but not a scrambled version or the equivalent γ2((381-403)) MA peptide, reduced the potentiating effects of etomidate, significantly reducing single-channel conductance. In contrast, when GABA currents were potentiated by the γ2-specific drug diazepam the δ MA peptide had no effect. These data reveal that diazepam and etomidate potentiate different extrasynaptic GABA(A) receptor subtypes but both drugs modulate conductance similarly. One interpretation of the data is that these drugs elicit potentiation through protein interactions and that the MA peptides compete with these interactions to disrupt this process.