Biomedical subjects

Nevin A Lambert

Publications and source records attributed to Nevin A Lambert.

9 recordsLinked to original sources

PubMed2006

Rapid activation of inwardly rectifying potassium channels by immobile G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) mediate slow synaptic transmission and many other effects of small molecule and peptide neurotransmitters. In the standard model of GPCR signaling, receptors and G-proteins diffuse laterally within the plane of the plasma membrane and encounter each other by random collision. This model predicts that signaling will be most efficient if both GPCRs and G-proteins are free to diffuse, thus maximizing collision frequency. However, neuronal GPCRs are often recruited to and enriched at specific synaptic locations, suggesting receptor mobility is restricted in these cells. Here, we test the hypothesis that restricting GPCR mobility impairs signaling in neurons by limiting the frequency of collisions between receptors and G-proteins. Mu-opioid receptors (MORs) were immobilized on the surface of cerebellar granule neurons by avidin-mediated cross-linking, and inwardly rectifying potassium (GIRK) channels were used as rapid indicators of G-protein activation. Mobile and immobile MORs activated GIRK channels with the same onset kinetics and agonist sensitivity in these neurons. In a heterologous expression system, GFP (green fluorescent protein)-tagged G alpha(oA) subunits remained mobile after cross-linking, but their mobility was reduced in the presence of immobile MORs, suggesting that these receptors and subunits were transiently precoupled. In addition, channel activation could be reconstituted with immobile GPCRs, G-protein heterotrimers, and GIRK channels. These results show that collision frequency is not rate-limiting for G-protein activation in CNS neurons, and are consistent with the idea that signaling components are compartmentalized or preassembled.

Animals↗

PubMed2006

Some G protein heterotrimers physically dissociate in living cells.

Heterotrimeric G proteins mediate physiological processes ranging from phototransduction to cell migration. In the accepted model of G protein signaling, Galphabetagamma heterotrimers physically dissociate after activation, liberating free Galpha subunits and Gbetagamma dimers. This model is supported by evidence obtained in vitro with purified proteins, but its relevance in vivo has been questioned. Here, we show that at least some heterotrimeric G protein isoforms physically dissociate after activation in living cells. Galpha subunits extended with a transmembrane (TM) domain and cyan fluorescent protein (CFP) were immobilized in the plasma membrane by biotinylation and cross-linking with avidin. Immobile CFP-TM-Galpha greatly decreased the lateral mobility of intracellular Gbeta1gamma2-YFP, indicating the formation of stable heterotrimers. A GTPase-deficient (constitutively active) mutant of CFP-TM-GalphaoA lost the ability to restrict Gbeta1gamma2-YFP mobility, whereas GTPase-deficient mutants of CFP-TM-Galphai3 and CFP-TM-Galphas retained this ability. Activation of cognate G protein-coupled receptors partially relieved the constraint on Gbeta1gamma2-YFP mobility induced by immobile CFP-TM-GalphaoA and CFP-TM-Galphai3 but had no effect on the constraint induced by CFP-TM-Galphas. These results demonstrate the physical dissociation of heterotrimers containing GalphaoA and Galphai3 subunits in living cells, supporting the subunit dissociation model of G protein signaling for these subunits. However, these results are also consistent with the suggestion that G protein heterotrimers (e.g., Galphas) may signal without physically dissociating.

Animals↗

PubMed2005

Endogenous regulator of G-protein signaling proteins regulate the kinetics of Galphaq/11-mediated modulation of ion channels in central nervous system neurons.

Slow synaptic potentials are generated when metabotropic G-protein-coupled receptors activate heterotrimeric G-proteins, which in turn modulate ion channels. Many neurons generate excitatory postsynaptic potentials mediated by G-proteins of the Galphaq/11 family, which in turn activate phospholipase C-beta. Accessory GTPase-activating proteins (GAPs) are thought to be required to accelerate GTP hydrolysis and rapidly turn off G-proteins, but the involvement of GAPs in neuronal Galphaq/11 signaling has not been examined. Here, we show that regulator of G-protein signaling (RGS) proteins provide necessary GAP activity at neuronal Galphaq/11 subunits. We reconstituted inhibition of native 2-pore domain potassium channels in cerebellar granule neurons by expressing chimeric Galpha subunits that are activated by Galphai/o-coupled receptors, thus bypassing endogenous Galphaq/11 subunits. RGS-insensitive variants of these chimeras mediated inhibition of potassium channels that developed and recovered more slowly than inhibition mediated by RGS-sensitive (wild-type) chimeras or native Galphaq/11 subunits. These changes were not accompanied by a change in agonist sensitivity, as might be expected if RGS proteins acted primarily as effector antagonists. The slowed recovery from potassium channel inhibition was largely reversed by an additional mutation that mimics the RGS-bound state. These results suggest that endogenous RGS proteins regulate the kinetics of rapid Galphaq/11-mediated signals in central nervous system neurons by providing GAP activity.

Animals↗

PubMed2004

Endogenous RGS proteins regulate presynaptic and postsynaptic function: functional expression of RGS-insensitive Galpha subunits in central nervous system neurons.

Regulators of G-protein signaling (RGS)-insensitive (RGSi) G-protein alpha subunits can be used to indirectly determine the function of endogenous RGS proteins in native cells. This article describes the application of RGSi Galpha subunits to the study of endogenous RGS function in central nervous system (CNS) neurons. Presynaptic inhibition of neurotransmitter release was reconstituted in primary neurons using RGSi Galpha(i/o) subunits, whereas postsynaptic regulation of potassium channels was reconstituted using RGSi chimeras of Galpha(q) and Galpha(i). These studies have shown that endogenous RGS proteins are essential for the rapid termination of some G-protein-mediated signals in CNS neurons, whereas these proteins are much less important for the regulation of other signals. Together, these techniques have helped reveal the complexity of RGS regulation of CNS function.

Adenoviridae↗

PubMed2004

Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.

The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.

Animals↗

PubMed2003

Differentiation of rhesus embryonic stem cells to neural progenitors and neurons.

Embryonic stem (ES) cells are pluripotent cells capable of differentiating into cell lineages derived from all primary germ layers including neural cells. In this study we describe an efficient method for differentiating rhesus monkey ES cells to neural lineages and the subsequent isolation of an enriched population of Nestin and Musashi positive neural progenitor (NP) cells. Upon differentiation, these cells exhibit electrophysiological characteristics resembling cultured primary neurons. Embryoid bodies (EBs) were formed in ES growth medium supplemented with 50% MEDII. After 7 days in suspension culture, EBs were transferred to adherent culture and either differentiated in serum containing medium or expanded in serum free medium. Immunocytochemistry on differentiating cells derived from EBs revealed large networks of MAP-2 and NF200 positive neurons. DAPI staining showed that the center of the MEDII-treated EBs was filled with rosettes. NPs isolated from adherent EB cultures expanded in serum free medium were passaged and maintained in an undifferentiated state by culture in serum free N2 with 50% MEDII and bFGF. Differentiating neurons derived from NPs fired action potentials in response to depolarizing current injection and expressed functional ionotropic receptors for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). NPs derived in this way could serve as models for cellular replacement therapy in primate models of neurodegenerative disease, a source of neural cells for toxicity and drug testing, and as a model of the developing primate nervous system.

Action Potentials↗

PubMed2002

GABA(B) receptor activation desensitizes postsynaptic GABA(B) and A(1) adenosine responses in rat hippocampal neurones.

Whole-cell recordings of EPSCs and G-protein-activated inwardly rectifying (GIRK) currents were made from cultured hippocampal neurones to determine the effect of long-term agonist treatment on the presynaptic and postsynaptic responses mediated by GABA(B) receptors (GABA(B)Rs). GABA(B)R-mediated presynaptic inhibition was unaffected by agonist (baclofen) treatment for up to 48 h, and was desensitized by about one-half after 96 h. In contrast, GABA(B)R-mediated GIRK currents were desensitized by a similar amount after only 2 h of agonist treatment. In addition, presynaptic inhibition mediated by A(1) adenosine receptors (A(1)Rs) was unaffected by prolonged GABA(B)R activation, whereas A(1)R-mediated GIRK currents were desensitized. Desensitization of postsynaptic GABA(B)R and A(1)R responses was blocked by the GABA(B)R antagonist (1-(S)-3,4-dichlorophenylethyl)amino-2-(S) hydroxypropyl-p-benzyl-phosphonic acid (CGP 55845A), but not by the A(1)R antagonist cyclopentyldipropylxanthine (DPCPX). GIRK current amplitude could be partially restored after baclofen treatment by either coapplication of baclofen and adenosine, or intracellular infusion of the non-hydrolysable GTP analog 5'-guanylylimidodiphosphate (Gpp(NH)p). Short-term (4-24 h) baclofen treatment also significantly desensitized the inhibition of postsynaptic voltage-gated calcium channels by activation of GABA(B)Rs or A(1)Rs. These results show that responses mediated by GABA(B)Rs and A(1)Rs desensitize differently in presynaptic and postsynaptic compartments, and demonstrate the heterologous desensitization of postsynaptic A1R responses.

Animals↗

PubMed2002

Differential desensitization of responses mediated by presynaptic and postsynaptic A1 adenosine receptors.

G-protein-coupled receptors (GPCRs) often desensitize during continuous activation, but it is not known whether desensitization is influenced by subcellular location. In hippocampal neurons, activation of adenosine A1 receptors (A1Rs) or GABA(B) receptors on synaptic terminals inhibits neurotransmitter release, whereas activation of the same receptors on cell bodies and dendrites decreases excitability by activating inwardly rectifying potassium (GIRK) channels. Here we report that responses mediated by presynaptic A1Rs desensitize more slowly than responses mediated by postsynaptic (somatodendritic) A1Rs in cultured neurons. Agonist treatment for 2 hr has no effect on adenosine-induced presynaptic inhibition, whereas such treatment nearly abolishes adenosine-induced activation of postsynaptic GIRK channels. Agonist treatment for longer periods (>12 hr) eventually desensitizes A1R-mediated presynaptic inhibition. Presynaptic and postsynaptic responses both recover from desensitization after agonist removal, but recovery of presynaptic inhibition requires more time. Desensitization of postsynaptic responses apparently occurs at the level of the receptor, because postsynaptic G-proteins and GIRK channels appear to be fully functional. Inhibition of voltage-gated calcium channels by postsynaptic A1Rs also desensitizes rapidly, although this desensitization is less complete than is observed for activation of postsynaptic GIRK channels. Comparison of concentration-response curves for presynaptic and postsynaptic responses suggests that a receptor reserve exists for presynaptic inhibition, but that the magnitude of this reserve is insufficient to account for the absence of presynaptic desensitization after brief agonist exposure. These results suggest that agonist-induced desensitization of responses mediated by neuronal GPCRs may depend on the subcellular location of the receptors.

2-Chloroadenosine↗

PubMed2002

Low pH inhibits compensatory endocytosis at a step between depolarization and calcium influx.

Cell function can be modulated by the insertion and removal of ion channels from the cell surface. The mechanism used to keep channels quiescent prior to delivery to the cell surface is not known. In eggs, cortical vesicle exocytosis inserts voltage-gated calcium channels into the cell surface. Calcium influx through these channels triggers compensatory endocytosis. Secretory vesicles contain high concentrations of calcium and hydrogen ions. We propose that lumenal hydrogen ions inhibit vesicular calcium channel gating prior to exocytosis, discharge of lumenal protons upon vesicle-plasma membrane fusion enables calcium channel gating. Consistent with this hypothesis we find that cortical vesicle lumens are acidic, and exocytosis releases lumenal hydrogen ions. Acidic extracellular pH reversibly blocks endocytosis, and the windows of opportunity for inhibition with a calcium-channel blocker or hydrogen ions are indistinguishable. Calcium ionophore treatment circumvents the low pH block, suggesting that calcium influx, or an upstream step, is obstructed. Inhibition of calcium influx by preventing membrane depolarization is unlikely, as elevation of the extracellular potassium concentration failed to overcome the pH block, and low extracellular pH was found to depolarize the membrane potential. We conclude that low pH inhibits endocytosis at a step between membrane depolarization and calcium influx.

Animals↗