Function Of Receptor Complexes Research Articles

Two for all: receptor-associated kinases SOBIR1 and BAK1

Leucine-rich repeat-receptor-like proteins (LRR-RLPs) are ubiquitous cell surface receptors lacking a cytoplasmic signalling domain. For most of these LRR-RLPs, it remained enigmatic how they activate cellular responses upon ligand perception. Recently, the LRR-receptor-like kinase (LRR-RLK) SUPPRESSOR OF BIR1-1 (SOBIR1) was shown to be essential for triggering defence responses by certain LRR-RLPs that act as immune receptors. In addition to SOBIR1, the regulatory LRR-RLK BRI1-ASSOCIATED KINASE-1 (BAK1) is also required for LRR-RLP function. Here, we compare the roles of SOBIR1 and BAK1 as regulatory LRR-RLKs in immunity and development. BAK1 has a general regulatory role in plasma membrane-associated receptor complexes comprising LRR-RLPs and/or LRR-RLKs. By contrast, SOBIR1 appears to be specifically required for the function of receptor complexes containing LRR-RLPs.

Conformational Mobility of S1-M1 and S2-M4 Linkers Influence NMDA Receptor Gating

NMDA receptors are tetrameric glutamate-gated ion channels pivotal to excitatory neurotransmission in the mammalian central nervous system. Each subunit of the NMDA receptor harbors an extracellular ligand-binding domain (LBD) connected by three linkers to the transmembrane segments that form the ion channel. Activation gating of NMDAR involves the propagation of ligand-induced conformational changes in the LBD into ion channel pore opening via linker peptide segments. The M3-S2 linker, which is attached to the pore-lining M3 transmembrane segment, has been shown to be a critical gating element. Constraining the conformational freedom of M3-S2 relative to the adjacent S2-M4 linker of the same subunit drastically reduced the efficacy of gating with a 30-75-fold reduction in channel open probability. Here we report the gating effects of limiting the relative conformational mobility of the S1-M1 and S2-M4 linkers with crosslinking disulfide bonds. Both intra- and inter- subunit crosslinking strategies in GluN1/GluN2A-containing receptors were used to dissect the linkers’ roles in gating transduction. We found that although the S1-M1 and S2-M4 linkers are connected respectively to the M1 and M4 transmembrane segment located peripheral to the pore, constraining their relative movements significantly hampered gating efficacy, as assayed using single channel recording. These results suggest that all the linkers in a single receptor complex function as an intricate unit in the gating transduction process and have important implications for potential drug targeting as unique noncompetitive antagonists are known to bind to the S1-M1 and S2-M4 linkers of certain glutamate receptor subtypes, although their mechanisms of action are yet undefined.

Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes.

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C = 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E = 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.

Ganaxolone, a selective, high-affinity steroid modulator of the gamma-aminobutyric acid-A receptor, exacerbates seizures in animal models of absence.

Ganaxolone (3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one) is a novel neurosteroid which has anticonvulsant properties in a number of seizure models as well as the ability to enhance function of the gamma-aminobutyric acid-A (GABA(A)) receptor complex via a neurosteroid binding site. The object of these experiments was to ascertain the efficacy of ganaxolone against absence seizures. Ganaxolone was assessed in the low-dose pentylenetetrazol (PTZ) and the gamma-hydroxybutyric acid (GHB) model of absence seizures in rats. Ganaxolone pretreatment resulted in a significant prolongation of absence seizure in both the PTZ and GHB models. Further, ganaxolone in doses above 20 mg/kg alone produced bilaterally synchronous spike wave discharges (SWDs) associated with behavioral arrest. These data suggest that augmentation of GABA(A) receptor complex function by neurosteroids has the potential to result in or exacerbate absence seizures.

Susceptibility of adult rats to lead-induced changes in NMDA receptor complex function.

Because cognitive impairments can occur with occupational Pb exposures, changes in NMDA receptor complex function might be expected to occur in adult rats treated with Pb. Using drug discrimination procedures, MK-801 sensitivity was determined in adult rats at three time points: after chronic exposure to 0, 50, or 150 ppm Pb acetate; again after exposure levels were increased to 500 and 1000 ppm; and 6 months after termination of Pb exposure. Changes in blood (PbB) and brain Pb levels, and in MK-801 and CGP-39653 binding, were examined in additional groups of nonbehaviorally tested rats. Pb decreased MK-801 sensitivity after Pb exposure levels were increased, but only at 500 ppm, indicating biphasic effects and precluding any correspondence between behavioral changes and biomarkers of exposure. Associated PbBs were higher, but brain Pbs similar to those associated with MK-801 sensitivity changes following postnatal and postweaning exposures. Neither MK-801 nor CGP-39653 binding was systematically affected by chronic Pb exposure in adults. Although adult Pb exposures do produce changes in NMDA function, at least as indicated by changes in MK-801 sensitivity, vulnerability to such effects is clearly less pronounced than with exposures occurring earlier in development.

Effects of desipramine and alprazolam on forced swimming behaviour of adult rats exposed to prenatal diazepam

Pregnant rats were treated with a single daily s.c. injection of diazepam (2 mg/kg) over gestation days 14–20. This treatment led to a reduction in GABA receptor complex function since adult male offspring showed a strong decrease in electrographic hippocampal responses to alprazolam and a strongly increased response to picrotoxin after intra-locus coeruleus injection of the two compounds. No difference in immobility time in the forced swimming test and in spontaneous motor activity was observed between prenatally vehicle- and diazepam-exposed offspring. Conversely, prenatal exposure to diazepam potentiated the anti-immobility effect of subchronic desipramine (10 mg/kg i.p.) and made active a dose of desipramine (5 mg/kg i.p.) that was ineffective in prenatally vehicle-exposed rats. This effect was observed only in pretested rats. Prenatal exposure to diazepam blocked the anti-immobility effect of subchronic alprazolam (15 mg/kg i.p.) in both non-pretested and pretested rats. Spontaneous motor activity was strongly reduced in all groups. These findings suggest that a persistent reduction in GABA receptor complex function, induced by prenatal exposure to diazepam, does not alter the mobility of adult progeny in the forced swimming test, but it may have consequences when drugs acting on the GABA receptor complex are used.

Lead exposure potentiates the effects of NMDA on repeated learning

Several studies now suggest that Pb exposure disrupts NMDA receptor complex function, findings which may have implications for understanding the basis of Pb-induced learning impairments. To further evaluate this possibility, the behavioral properties of the glutamate agonist NMDA were compared in rats that had been chronically exposed to 0, 50, or 250 ppm Pb acetate in drinking water from weaning. Acute administration of NMDA (20–50 mg/kg IP) decreased accuracy in both the repeated acquisition (RA) and performance (P) components of this multiple schedule with a selective effect on the learning component in the second half of the session. Analyses of error patterns revealed that the disruption of RA accuracy derived from initial perseverative errors followed by errors of skipping forward and backwards in the 3-member response sequence. Response rates in both RA and P were suppressed by NMDA. Pb-exposure potentiated the accuracy-impairing effects of NMDA by further increasing the frequencies of these error classes, and likewise potentiated the rate-suppressing effects of NMDA. These findings add further support to the possible involvement of Pb with the NMDA receptor complex.

Subsensitivity of lead-exposed rats to the accuracy-impairing and rate-altering effects of MK-801 on a multiple schedule of repeated learning and performance

Exposure to lead (Pb) has been reported to inhibit MK-801 binding and to alter other NMDA receptor complex-associated functions. These reported changes are provocative since both NMDA receptor antagonism and Pb exposure are known to impair learning processes. Whether the Pb-induced changes in NMDA function relate to the learning impairments associated with Pb exposure, however, has not been explored. The contention of this study was that if changes in NMDA function produced by Pb serve as the basis of Pb-associated learning impairments, then such changes should be of sufficient biological magnitude and clinical relevance to induce alterations in sensitivity at the level of the whole animal, i.e., changes in behavioral sensitivity to glutamatergic compounds. Thus, in this study, dose-effect curves of control and Pb-treated rats working on a multiple schedule of repeated learning (repeated acquisition, RA) and performance (P) were compared following acute administration of MK-801, the non-competitive NMDA antagonist. Based on the nature of the reported effects of Pb on NMDA systems, it was expected that the curves of Pb-exposed rats would be right-shifted relative to controls, if differential behavioral sensitivity was evident. Rats were chronically exposed to 0, 50 or 250 ppm Pb acetate in drinking water from weaning and trained on the multiple RA and P schedule beginning at 55 days old. The RA component required the rat to learn a new 3-member sequence of responses during each experimental session (center right left, RLC, CLR, RCL, or LRC), while the correct sequence of responses for the P component was constant across sessions (LCR), requiring performance of an already learned response. Acute administration of MK-801 (0.05–0.3 mg/k, i.p.) resulted in decrements in accuracy in both the RA and P components of the schedule, indicative of non-specific effects on behavior rather than selective effects on learning. The declines in accuracy during the RA component of the schedule were primarily the result of increased perseverative responding, i.e., repetitive responding on a single lever. Both the decline in RA accuracy and the increases in perseverative responding produced by MK-801 were attenuated by Pb exposure. Moreover, dose-effect curves relating MK-801 dose to changes in rates of responding were significantly shifted to the right in Pb-exposed rats relative to controls. Taken together, these data demonstrate a subsensitivity of Pb-exposed rats to both the accuracy-impairing and response rate-altering properties of MK-801. Furthermore, they suggest that Pb-induced changes in NMDA receptor complex function could be involved in the learning impairments that result from exposures to Pb.

GAEA A receptor complex function in frontal cortex membranes from control and neurological patients

The functional integrity of the GABA A receptof-benzodiazepine (BZ) recognition site-Cl − ionophore complex was assessed by means of [ 35S]TBPS (t-butylbicyclophosphorothionate) binding to frontal cortex membranes prepared from frozen postmortem brain tissue taken from control ( n = 4), Alzheimer ( n = 7), Parkinson ( n = 3) and Huntington's chorea ( n = 2) patients. Specific [ 35S]TBPS binding was similar in control, Parkinson's disease and Huntington's chorea brains, but was significantly reduced (78% control, P < 0.01) in frontal cortex membranes from Alzheimer's patients. The linkage between the BZ recognition sites and the GABA A receptor-linked Cl − ionophore was functionally intact in these membranes as BZ site agonists (zolpidem, alpidem, flunitrazepam and clonazepam) enhanced [ 35S]TBPS binding under the conditions used (well-washed membranes in the presence of 1.0 M NaCl). Zolpidem (BZ 1 selective) exhibited a biphasic enhancement in control membranes whereas the other compounds induced a bell-shaped concentration-response curve. The enhancement of [ 35S]TBPS binding by alpidem, flunitrazepam and clonazepam was greater in frontal cortex membranes from Alzheimer's patients than in controls whereas it tended to be reduced in membranes from the brains of Huntington's chorea patients. These studies demonstrate the functional integrity of the GABA A receptor macromolecular complex and also the usefulness of [ 35S]TBPS binding in the study of human postmortem tissue.