Biomedical subjects

Bruce A Bamber

Publications and source records attributed to Bruce A Bamber.

5 recordsLinked to original sources

PubMed2006

Shaping cellular form and function by autophagy.

In addition to its familiar role in non-selective bulk degradation of cellular material, autophagy can also bring about specific changes in the structure and function of cells. Autophagy has been proposed to operate in a substrate-selective mode to carry out this function, although evidence to demonstrate selectivity has been lacking. A recent study of synapse formation in the nervous system of the nematode Caenorhabditis elegans now provides experimental evidence for substrate-selective autophagy. Synapses form when presynaptic cells contact their postsynaptic partners during development. This contact induces the assembly of synaptically-localized protein complexes in the postsynaptic cell that contain scaffolding proteins and neurotransmitter receptors. When presynaptic contact was blocked, autophagy in the postsynaptic cell was induced. Substrate selectivity was evident in this system: the gamma-aminobutyric acid type A receptor (GABA(A) receptor), an integral-membrane neurotransmitter receptor, trafficked from the cell surface to autophagosomes. By contrast, the acetylcholine receptor, a structurally-similar neurotransmitter receptor, remained on the cell surface. This result provides experimental support for the idea that autophagy can bring about changes in cell structure and behavior by degrading specific cellular proteins, particularly cell surface receptors that are often important for regulating cell growth, differentiation and function.

Animals↗

PubMed2006

Presynaptic terminals independently regulate synaptic clustering and autophagy of GABAA receptors in Caenorhabditis elegans.

Synaptic clustering of GABAA receptors is important for the function of inhibitory synapses, influencing synapse strength and, consequently, the balance of excitation and inhibition in the brain. Presynaptic terminals are known to induce GABAA receptor clustering during synaptogenesis, but the mechanisms of cluster formation and maintenance are not known. To study how presynaptic neurons direct the formation of GABAA receptor clusters, we have investigated GABAA receptor localization in postsynaptic cells that fail to receive presynaptic contacts in Caenorhabditis elegans. Postsynaptic muscles in C. elegans receive acetylcholine and GABA motor innervation, and GABAA receptors cluster opposite GABA terminals. Selective loss of GABA inputs caused GABAA receptors to be diffusely distributed at or near the muscle cell surface, confirming that GABA presynaptic terminals induce GABAA receptor clustering. In contrast, selective loss of acetylcholine innervation had no effect on GABAA receptor localization. However, loss of both GABA and acetylcholine inputs together caused GABAA receptors to traffic to intracellular autophagosomes. Autophagosomes normally transport bulk cytoplasm to the lysosome for degradation. However, we show that GABAA receptors traffic to autophagosomes after endocytic removal from the cell surface and that acetylcholine receptors in the same cells do not traffic to autophagosomes. Thus, autophagy can degrade cell-surface receptors and can do so selectively. Our results show that presynaptic terminals induce GABAA receptor clustering by independently controlling synaptic localization and surface stability of GABAA receptors. They also demonstrate a novel function for autophagy in GABAA receptor degradative trafficking.

Animals↗

PubMed2006

Residues in the first transmembrane domain of the Caenorhabditis elegans GABA(A) receptor confer sensitivity to the neurosteroid pregnenolone sulfate.

The GABA(A) receptor is a target of endogenous and synthetic neurosteroids. Little is known about the residues required for neurosteroid action on GABA(A) receptors. We have investigated pregnenolone sulfate (PS) inhibition of the Caenorhabditis elegans UNC-49 GABA receptor, a close homolog of the mammalian GABA(A) receptor. The UNC-49 locus encodes two GABA receptor subunits, UNC-49B and UNC-49C. UNC-49C is sensitive to PS but UNC-49B is not sensitive. By analyzing chimeric receptors and receptors containing site-directed mutations, we identified two regions required for PS inhibition. Four residues in the first transmembrane domain are required for the majority of the sensitivity to PS, but a charged extracellular residue at the end of the M2 helix also plays a role. Strikingly, mutation of one additional M1 residue reverses the effect of PS from an inhibitor to an enhancer of receptor function. Mutating the M1 domain had little effect on sensitivity to the inhibitor picrotoxin, suggesting that these residues may mediate neurosteroid action specifically, and not allosteric regulation in general.

Amino Acid Sequence↗

PubMed2005

The composition of the GABA receptor at the Caenorhabditis elegans neuromuscular junction.

1. The unc-49 gene of the nematode Caenorhabditis elegans encodes three gamma-aminobutyric acid type A (GABA(A)) receptor subunits. Two of these, UNC-49B and UNC-49C, are expressed at high abundance and co-localize at the neuromuscular junction. 2. The UNC-49B subunit is sufficient to form a GABA(A) receptor in vitro and in vivo. Furthermore, all loss-of-function unc-49 alleles lack functional UNC-49B. No mutations specifically inactivate UNC-49C. Thus, UNC-49C appears to be dispensable for receptor function; however, UNC-49C has been conserved among different nematode species, suggesting it plays a necessary role. 3. To ascertain whether UNC-49C is part of the GABA(A) receptor in vivo, we performed patch-clamp electrophysiology on C. elegans muscle cells. Sensitivity to GABA, and to the antagonists picrotoxin and pregnenolone sulfate, matched the UNC-49B/C heteromer rather than the UNC-49B homomer, for both exogenous and synaptically-released GABA. 4. The synaptic localization of UNC-49C requires the presence of UNC-49B, indicative of a physical association between the two subunits in vivo. Thus, the in vivo receptor is an UNC-49B/C heteromer. 5. UNC-49C plays a negative modulatory role. Using the rapid ligand-exchange technique in vitro, we determined that UNC-49C causes accelerated receptor desensitization. Previously, UNC-49C was shown to reduce single-channel conductance in UNC-49B/C heteromers. Thus, the function of UNC-49B is to provide GABA responsiveness and localization to synapses, while the function of UNC-49C is to negatively modulate receptor function and precisely shape inhibitory postsynaptic currents.

Alternative Splicing↗

PubMed2003

Pharmacological characterization of the homomeric and heteromeric UNC-49 GABA receptors in C. elegans.

(1) UNC-49B and UNC-49C are gamma-aminobutyric acid (GABA) receptor subunits encoded by the Caenorhabditis elegans unc-49 gene. UNC-49B forms a homomeric GABA receptor, or can co-assemble with UNC-49C to form a heteromeric receptor. The pharmacological properties of UNC-49B homomers and UNC-49B/C heteromers were investigated in Xenopus oocytes. (2) The UNC-49 subunits are most closely related to the bicuculline- and benzodiazepine-insensitive RDL GABA receptors of insects. Consistent with this classification, bicuculline (10 micro M) did not inhibit, nor did diazepam (10 micro M) enhance UNC-49B homomeric or UNC-49B/C heteromeric receptors. (3) The UNC-49C subunit strongly affects the pharmacology of UNC-49B/C heteromeric receptors. UNC-49B homomers were much more picrotoxin sensitive than UNC-49B/C heteromers (IC(50)=0.9+/-0.2 micro M and 166+/-42 micro M, respectively). Pentobarbitone enhancement was greater for UNC-49B homomers compared to UNC-49B/C heteromers. Propofol (50 micro M) slightly enhanced UNC-49B homomers but slightly inhibited UNC-49B/C heteromers. Penicillin G (10 mM) inhibited UNC-49B homomers less strongly than UNC-49B/C heteromers (30% compared to 53% inhibition, respectively). (4) Several aspects of UNC-49 pharmacology were unusual. Picrotoxin sensitivity strongly correlates with dieldrin sensitivity, yet UNC-49B homomers were highly dieldrin resistant. The enhancing neurosteroid pregnanolone (5beta-pregnan-3alpha-ol-20-one; 10 micro M) strongly inhibited both UNC-49 receptors. Alphaxalone (10 micro M), another enhancing neurosteroid, did not affect UNC-49B homomers, but slightly inhibited UNC-49B/C heteromers. (5) UNC-49 subunits and mammalian GABA(A) receptor alpha, beta, and gamma subunit classes all share roughly the same degree of sequence similarity. Thus, although they are most similar to other invertebrate GABA receptors, the UNC-49 receptors share significant structural and pharmacological overlap with mammalian GABA(A) receptors.

Amino Acid Sequence↗