John Bracamontes

Bicuculline and Gabazine Are Allosteric Inhibitors of Channel Opening of the GABAA Receptor

Anesthetic drugs are known to interact with GABAAreceptors, both to potentiate the effects of low concentrations of GABA and to directly gate open the ion channel in the absence of GABA; however, the site(s) involved in direct gating by these drugs is not known. We have studied the ability of alphaxalone (an anesthetic steroid) and pentobarbital (an anesthetic barbiturate) to directly activate recombinant GABAA receptors containing the α1, β2, and γ2L subunits. Steroid gating was not affected when either of two mutated β2 subunits [β2(Y157S) and β2(Y205S)] are incorporated into the receptors, although these subunits greatly reduce the affinity of GABA binding. These observations indicate that steroid binding and subsequent channel gating do not require these particular residues, as already shown for barbiturates. Bicuculline or gabazine (two competitive antagonists of GABA binding) reduced the currents elicited by alphaxalone and pentobarbital from wild-type GABAA receptors; however, gabazine produced only a partial block of responses to pentobarbital or alphaxalone, and bicuculline only partially blocked responses to pentobarbital. These observations indicate that the blockers do not compete with alphaxalone or pentobarbital for a single class of sites on the GABAAreceptor. Finally, at receptors containing α1β2(Y157S)γ2L subunits, both bicuculline and gabazine showed weak agonist activity and actually potentiated responses to alphaxalone. These observations indicate that the blocking drugs can produce allosteric changes in GABAA receptors, at least those containing this mutated β2 subunit. We conclude that the sites for binding steroids and barbiturates do not overlap with the GABA-binding site. Furthermore, neither gabazine nor bicuculline competes for binding at the steroid or barbiturate sites. The data are consistent with a model in which both gabazine and bicuculline act as allosteric inhibitors of channel opening for the GABAA receptor after binding to the GABA-binding site.

Pregnenolone sulfate block of GABAA receptors: mechanism and involvement of a residue in the M2 region of the α subunit

1 Neurosteroids are produced in the brain, and can have rapid actions on membrane channels of neurons. Pregnenolone sulfate (PS) is a sulfated neurosteroid which reduces the responses of the γ‐aminobutyric acid A (GABAA) receptor. We analysed the actions of PS on single‐channel currents from recombinant GABAA receptors formed from α1, β2 and γ2L subunits. 2 Currents were elicited by a concentration of GABA eliciting a half‐maximal response (50 μm) and a saturating concentration (1 mm). PS reduced the duration of clusters of single‐channel activity at either concentration of GABA. 3 PS had no discernable effect on rapid processes: no effects were apparent on channel opening and closing, nor on GABA affinity, and a rapidly recovering desensitised state was not affected. Instead, PS produced a slowly developing block which occurred at a similar rate for receptors with open or closed channels and with one or two bound GABA molecules. 4 The rate of block was independent of membrane potential, implying that the charged sulfate moiety does not move through the membrane field. 5 Change in a specific residue near the intracellular end of the channel lining portion of the α1 subunit had a major effect on the rate of block. Mutation of the residue α1 V256S reduced the rate of block by 30‐fold. A mutation at the homologous position of the β2 subunit (β2 A252S) had no effect, nor did a complementary mutation in the γ2L subunit (γ2L S266A). It seems likely that this residue is involved in a conformational change underlying block by PS, instead of forming part of the binding site for PS.

Neuroactive steroids have multiple actions to potentiate GABAA receptors

The effects of neuroactive steroids on the function of GABAA receptors were studied using cell‐attached records of single channel activity recorded from HEK293 cells transfected with α1 β2 γ2L subunits. Activity was elicited with a half‐maximal (50 μm) concentration of GABA. Two steroids were studied in detail: ACN ((3α,5α,17β)‐3‐hydroxyandrostane‐17‐carbonitrile) and B285 ((3α,5β,17β)‐3‐hydroxy‐18‐norandrostane‐17‐carbonitrile). Four effects on channel activity were seen, two on open time distributions and two on closed times. When clusters of openings were elicited in the absence of steroid, the open time distribution contained three components. ACN produced concentration‐dependent alterations in the open time distribution. The prevalence of the longest duration class of open times was increased from about 15% to about 40% (EC50 about 180 nm ACN), while the duration of the longest class increased from 7.4 ms to 27 ms (EC50 about 35 nm ACN). B285 also increased the prevalence of the longest duration open times (EC50 about 18 nm B285) but increased the duration only at concentrations close to 10 μm. The differences in the actions of these two steroids suggest that the effects on proportion and duration of the long duration open time component are produced by independent mechanisms and that there are separate recognition sites for the steroids which are associated with the two functional actions. The closed time distributions also showed three components in the absence of steroid. The rate of occurrence of the two brief duration closed time components decreased with increasing ACN, with an EC50 of about 50 nm ACN. In contrast, B285 did not reduce the rate of occurrence of the brief closings until high concentrations were applied. However, both B285 and ACN reduced the rate of occurrence of the activation‐related closed state selectively, with comparable IC50 concentrations (about 40 nm ACN, 20 nm B285). As in the case for action on open times these data suggest that there are two recognition sites and two independent mechanisms, perhaps the sites and mechanisms associated with actions on open times. The presence of  1 μm ACN had no effect on the estimated channel opening rate or on the apparent affinity of the receptor for GABA. Mutation of the carboxy terminus of the γ2 subunit, but not the α1 or β2 subunits, abolished the ability of ACN to increase the duration of OT3 but had no effect on the reduction of the rate of occurrence of the activation‐related closed state. These observations are also consistent with the idea that there is more than one distinguishable steroid recognition site on the GABAA receptor.

Structural domains of the human GABAA receptor β3 subunit involved in the actions of pentobarbital

1 This study was conducted to search for the residues of the β3 subunit which affect pentobarbital action on the γ‐aminobutyric acid type A (GABAA) receptor. Three chimeras were constructed by joining the GABAA receptor β3 subunit to the ρ1 subunit. For each chimera, the N‐terminal sequence was derived from the β3 subunit and the C‐terminal sequence from the ρ1 subunit, with junctions located between the membrane‐spanning regions M2 and M3, in the middle of M2, or in M1, respectively. 2 In receptors obtained by the coexpression of α1 with the chimeric subunits, in contrast with those obtained by the coexpression of α1 and β3, pentobarbital exhibited lower potentiation of GABA‐evoked responses, and in the direct gating of Cl− currents, an increase in the EC50 together with a marked decrease in the relative maximal efficacy compared with that of GABA. 3 Estimates of the channel opening probability through variance analysis and single‐channel recordings of one chimeric subunit showed that the reduced relative efficacy for gating largely resulted from an increase in gating by GABA, with little change in efficacy of pentobarbital. 4 A fit of the time course of the response by the predictions of a class of reaction schemes is consistent with the conclusion that the change in the concentration dependence of activation by pentobarbital is due to a change in pentobarbital affinity for the receptor. Therefore, the data suggest that residues of the β3 subunit involved in pentobarbital binding to GABAA receptors are located downstream from the middle of the M2 region.

Activation of GABAA receptors containing the α4 subunit by GABA and pentobarbital

The activation properties of GABAA receptors containing α4β2γ2 and α4β2δ subunits were examined in the presence of GABA or pentobarbital. The receptors were expressed transiently in HEK 293 cells, and the electrophysiological experiments were carried out using cell‐attached single‐channel patch clamp or whole‐cell macroscopic recordings. The data show that GABA is a stronger activator of α4β2γ2 receptors than α4β2δ receptors. Single‐channel clusters were recorded from α4β2γ2 receptors in the presence of 10–5000 μm GABA. The maximal intracluster open probability was 0.35, with a half‐maximal response elicited by 32 μm GABA. Simultaneous kinetic analysis of single‐channel currents obtained at various GABA concentrations yields a channel opening rate constant of 250 s−1, and a KD of 20 μm. In contrast, only isolated openings were observed in the presence of GABA for the α4β2δ receptor. Pentobarbital was a strong activator of both α4β2γ2 and α4β2δ receptors. The maximal cluster open probability, recorded in the presence of 100 μm pentobarbital, was 0.7. At higher pentobarbital concentrations, the cluster open probability was reduced, probably due to channel block. The results from single‐channel experiments were confirmed by macroscopic recordings from HEK cells in the presence of GABA or pentobarbital.

Mutations of the GABA-A receptor α1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids

Neuroactive steroids are among the most efficacious modulators of the mammalian GABA-A receptor. Previous work has proposed that receptor potentiation is mediated by steroid interactions with a site defined by the residues α1Asn407/Tyr410 in the M4 transmembrane domain and residue α1Gln241 in the M1 domain. We examined the role of residues in the α1 subunit M1 domain in the modulation of the rat α1β2γ2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the α1Q241W or α1Q241L mutations were insensitive to (3α,5α)-3-hydroxypregnan-20-one (3α5αP), albeit with different underlying mechanisms. The α1Q241S mutant was potentiated by 3α5αP, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the α1Q241L mutation had no effect on channel potentiation by (3α,5α)-3-hydroxymethyl-pregnan-20-one, but mutation of the neighboring residue, α1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5α-pregnan-20-one) potentiated the wild-type receptor but not the α1Q241L mutant. The findings are consistent with a model in which the α1Ser240 and α1Gln241 residues shape the surface to which steroid molecules bind.

Mutations in MLH1 are more frequent than in MSH2 in sporadic colorectal cancers with microsatellite instability

The microsatellite instability that is a feature of tumors in patients with hereditary nonpolyposis colorectal cancer (HNPCC) is a consequence of defective DNA mismatch repair. Mutations in the DNA mismatch repair genes MSH2 and MLH1 may account for up to 90% of HNPCC kindreds. Microsatellite instability is also seen in 10–16% of sporadic colorectal cancers. A limited number of MSH2 and MLH1 mutations have been described for sporadic colorectal cancers. In this study, we screened 12 primary sporadic colorectal cancers with microsatellite instability for mutations in MSH2 and MLH1 by using reverse transcription‐polymerase chain reaction (RT‐PCR) and single‐strand‐conformation‐variant (SSCV) analysis. Eight mutations were identified in six tumors. One mutation in MLH1 was found to be present in the patients germline DNA. Four tumors had somatic mutations in MLH1, and, in two of these tumors, two different mutations were identified. A single tumor had a somatic MSH2 mutation. Our observations suggest that MLH1 is mutated more frequently than MSH2 in sporadic colorectal cancers with microsatellite instability. Genes Chromosom. Cancer 18:42–49, 1997.

Characteristics of concatemeric GABAA receptors containing α4/δ subunits expressed in Xenopus oocytes

BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems.

Natural and enantiomeric etiocholanolone interact with distinct sites on the rat α1β2γ2l GABAA receptor

We have studied the ability of the androgen etiocholanolone and its enantiomer (ent-etiocholanolone) to modulate rat α1β2γ2L GABAA receptor function transiently expressed in human embryonic kidney cells. Studies on steroid enantiomer pairs can yield powerful new information on the pharmacology of steroid interactions with the GABAA receptor. Both steroids enhance currents elicited by GABA, but ent-etiocholanolone is much more powerful than etiocholanolone at producing potentiation. At a low GABA concentration (0.5 μM, <EC5), the presence of 10 μM ent-etiocholanolone potentiates whole-cell currents by almost 30-fold, whereas 10 μM etiocholanolone merely doubles the peak response. At higher GABA concentration (5 μM, ∼EC25), the potentiation curve for ent-etiocholanolone is positioned at lower concentrations than that for etiocholanolone. Single-channel kinetic analysis shows that exposure to etiocholanolone has a single effect on currents: the relative frequency of long openings is increased in the presence of steroid. But exposure to ent-etiocholanolone produces two kinetic effects: an increase in the relative frequency of long openings and a decrease in the frequency of long closed times. The presence of etiocholanolone does not inhibit potentiation by ent-etiocholanolone, suggesting that etiocholanolone is unable to interact with the sites through which ent-etiocholanolone modifies receptor function. The double mutation α1(N407A/Y410F) prevents potentiation by etiocholanolone but not by ent-etiocholanolone, and the α1(Q241A) and α1(I238N) point mutations fully abolish potentiation by etiocholanolone but not by ent-etiocholanolone. We conclude that etiocholanolone and its enantiomer interact with distinct sites on the α1β2γ2L GABAA receptor.

γ-aminobutyric acid type A α4, β2, and δ subunits assemble to produce more than one functionally distinct receptor type.

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1–3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes.