Biomedical subjects

M T Bianchi

Publications and source records attributed to M T Bianchi.

5 recordsLinked to original sources

PubMed2002

Alpha1 and alpha6 subunits specify distinct desensitization, deactivation and neurosteroid modulation of GABA(A) receptors containing the delta subunit.

GABA(A) receptor alpha subunit subtypes have distinct CNS distributions and confer different pharmacological and biophysical properties to alphabetagamma receptor isoforms. However, the alpha subtype-dependent properties of alphabetadelta receptor isoforms that may be targeted to extrasynaptic sites remain poorly understood. We investigated the properties of alpha1beta3delta and alpha6beta3delta receptor currents evoked by concentration jumps using a saturating GABA concentration (1 mM). alpha1beta3delta receptor currents desensitized slowly, deactivated rapidly and displayed voltage-dependence only of peak amplitude. In contrast, alpha6beta3delta receptor currents had voltage-dependent increased desensitization and slower deactivation, but did not show rectification. The neurosteroid THDOC (1 microM) enhanced alpha1beta3delta more than alpha6beta3delta currents, but increased the extent of desensitization and prolonged deactivation for both receptor isoforms. alpha1-alpha6 and alpha6-alpha1 chimeras (spliced in transmembrane domain 1) suggested that differences in deactivation rate and its voltage-dependence correlated with N-terminal domains, while the extent of desensitization and its voltage-dependence correlated with C-terminal domains. Both chimeras showed outward rectification. alpha1 subunit-like THDOC enhancement was observed with the alpha1-alpha6 chimera, but the alpha6-alpha1 chimera did not confer alpha6 subunit-like enhancement, suggesting that multiple alpha1 subunit domains contributed to neurosteroid efficacy. Thus, alpha subunit subtypes may regulate the kinetic and pharmacological properties of tonic neuronal inhibition.

Amino Acid Sequence↗

PubMed2001

Agonist Trapping by GABAA Receptor Channels.

GABAergic IPSCs have a relatively slow decay (deactivation) that appears to result from GABA(A) receptor channel openings that occur well beyond the predicted duration of free GABA at central synapses. Open and desensitized states have been suggested to prevent dissociation of agonist from the receptor, thus prolonging deactivation. However, simultaneous assessment of GABA binding and channel gating has not been possible. We developed a functional assay for occupancy of the GABA binding site or sites to test the GABA "trapping" hypothesis. Deactivation currents were compared in the absence and presence of bicuculline, a competitive antagonist that also allosterically inhibits GABA(A) receptors. This provided a model-independent, functional test of the hypothesis that GABA is trapped on the receptor during gating: bicuculline could only inhibit the channel if it was open but unbound by GABA. Although bicuculline inhibited spontaneous and neurosteroid-activated GABA(A) receptor currents, it failed to alter the deactivation time course of GABA-activated GABA(A) receptor currents. Protection of deactivation current from bicuculline block indicated that GABA remained bound to the receptors while the channel was open, thus suggesting that all open states, as well as all closed and desensitized states from which channel opening can occur, must be GABA liganded states. Trapping may be specific to agonists, because the positive allosteric modulator diazepam unbound from GABA(A) receptors independent of GABA binding and channel activity.

Animals↗

PubMed2001

Structural determinants of fast desensitization and desensitization-deactivation coupling in GABAa receptors.

Fast IPSCs in the brain are predominantly caused by presynaptic release of GABA that activates GABA(A) receptor (GABA(A)R) channels. The IPSCs are shaped by the gating and desensitization properties of postsynaptic GABA(A)Rs. Specifically, fast desensitization has been suggested to decrease IPSC amplitude and to increase IPSC duration by slowing deactivation; however, the mechanisms underlying desensitization, deactivation, and their coupling are poorly understood. Consistent with this suggestion, alpha1beta3gamma2L GABA(A)Rs desensitize with a prominent fast phase and deactivate slowly, whereas alpha1beta3delta GABA(A)Rs desensitize without a fast phase and deactivate rapidly. Using the concentration-jump technique applied to excised patches, we studied GABA(A)Rs containing chimeras or exchange mutants between delta and gamma2L subunits to gain insight into the structural bases for fast desensitization and its coupling to deactivation. We demonstrated that the N terminus and two adjacent residues (V233, Y234) in the first transmembrane domain (TM1) of the delta subunit were both required to abolish fast desensitization. Additionally, these residues in TM1 of the gamma2L subunit (Y235, F236) were critical for desensitized states to prolong deactivation after removal of GABA, because mutations resulted in accelerated deactivation despite unaltered desensitization time course. Interestingly, control of desensitization and deactivation was independent of the identity (gamma2L or delta subunit sequence) of TM2, indicating that structures related to the putative channel gate may play a less direct role in desensitization than previously suggested.

Animals↗

PubMed2001

Mutation of the 9' leucine in the GABA(A) receptor gamma2L subunit produces an apparent decrease in desensitization by stabilizing open states without altering desensitized states.

A conserved leucine near the middle (9' position) of the second transmembrane domain of ligand-gated ion channels has been implicated in both gating and desensitization. Specifically, L9'S and L9'T mutations decreased agonist EC50, decreased apparent desensitization and prolonged deactivation in members of the LGIC superfamily, suggesting that this residue may regulate channel properties including desensitization. GABA(A) receptors desensitize in three phases, but in previous 9' leucine studies, only slow phases of desensitization were resolved. We used excised patches containing alpha1beta3gamma2L or alpha1beta3gamma2L(L9'S) GABA(A) receptors and combined single channel recording and concentration jump techniques to reevaluate the effects of this mutation on desensitization. Although desensitization extent was decreased in mutated channels, desensitization still occurred in three phases, suggesting that desensitized states may be intact. Interestingly, deactivation rate was slowed by the mutation, opposite to that expected if desensitization was attenuated. alpha1beta3gamma2L(L9'S) receptor single channels had increased open durations. Simulations revealed that stabilizing the open state (by decreasing the channel closing rate) could account for multiple macroscopic findings: left-shifted GABA EC50, smaller extent of desensitization, slower desensitization rate, and longer deactivation. We concluded that changes in efficacy can alter macroscopic desensitization without affecting desensitized states per se.

Amino Acid Substitution↗

PubMed1989

Psychosocial factors in women who undergo cholecystectomy. A case-control study.

Thirty women admitted to hospital for cholecystectomy were compared with a healthy community control group. Univariate comparisons showed that patients had significantly more social problems, manifested greater psychological distress and reported poorer social support. Logistic multiple regression analysis indicated that patient status was best predicted by manual occupational class and the interaction between employment and presence/absence of young children. The findings are discussed within the framework of illness behaviour.

Adult↗