|Tumor necrosis factor alpha mediates GABA(A) receptor trafficking to the plasma membrane of spinal cord neurons in vivo. |
Stück, ED; Christensen, RN; Huie, JR; Tovar, CA; Miller, BA; Nout, YS; Bresnahan, JC; Beattie, MS; Ferguson, AR
The proinflammatory cytokine TNFα contributes to cell death in central nervous system (CNS) disorders by altering synaptic neurotransmission. TNFα contributes to excitotoxicity by increasing GluA2-lacking AMPA receptor (AMPAR) trafficking to the neuronal plasma membrane. In vitro, increased AMPAR on the neuronal surface after TNFα exposure is associated with a rapid internalization of GABA(A) receptors (GABA(A)Rs), suggesting complex timing and dose dependency of the CNS's response to TNFα. However, the effect of TNFα on GABA(A)R trafficking in vivo remains unclear. We assessed the effect of TNFα nanoinjection on rapid GABA(A)R changes in rats (N = 30) using subcellular fractionation, quantitative western blotting, and confocal microscopy. GABA(A)R protein levels in membrane fractions of TNFα and vehicle-treated subjects were not significantly different by Western Blot, yet high-resolution quantitative confocal imaging revealed that TNFα induces GABA(A)R trafficking to synapses in a dose-dependent manner by 60 min. TNFα-mediated GABA(A)R trafficking represents a novel target for CNS excitotoxicity.
|GABAA receptors are expressed and facilitate relaxation in airway smooth muscle. |
Mizuta, K; Xu, D; Pan, Y; Comas, G; Sonett, JR; Zhang, Y; Panettieri, RA; Yang, J; Emala, CW
American journal of physiology. Lung cellular and molecular physiology
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABA(A)) channels and metabotropic (GABA(B)) receptors. GABA(A) channels are ubiquitously expressed in neuronal tissues, and in mature neurons modulate an inward chloride current resulting in neuronal inhibition due to membrane hyperpolarization. In airway smooth muscle (ASM) cells, membrane hyperpolarization favors smooth muscle relaxation. Although GABA(A) channels and GABA(B) receptors have been functionally identified on peripheral nerves in the lung, GABA(A) channels have never been identified on ASM itself. We detected the mRNA encoding of the GABA(A) alpha(4)-, alpha(5)-, beta(3)-, delta-, gamma(1-3)-, pi-, and theta-subunits in total RNA isolated from native human and guinea pig ASM and from cultured human ASM cells. Selected immunoblots identified the GABA(A) alpha(4)-, alpha(5)-, beta(3)-, and gamma(2)-subunit proteins in native human and guinea pig ASM and cultured human ASM cells. The GABA(A) beta(3)-subunit protein was immunohistochemically localized to ASM in guinea pig tracheal rings. While muscimol, a specific GABA(A) channel agonist, did not affect the magnitude or the time to peak contractile effect of substance P, it directly concentration dependently relaxed a tachykinin-induced contraction in guinea pig tracheal rings, which was inhibited by the GABA(A)-selective antagonist gabazine. Muscimol also relaxed a contraction induced by an alternative contractile agonist histamine. These results demonstrate that functional GABA(A) channels are expressed on ASM and suggest a novel therapeutic target for the relaxation of ASM in diseases such as asthma and chronic obstructive lung disease.
|Stoichiometry and assembly of a recombinant GABAA receptor subtype. |
Tretter, V, et al.
J. Neurosci., 17: 2728-37 (1997)
GABAA receptors are ligand-gated chloride ion channels that are presumed to be pentamers composed of alpha, beta, and gamma subunits. The subunit stoichiometry, however, is controversial, and the subunit arrangement presently is not known. In this study the ratio of subunits in recombinant alpha1beta3gamma2 receptors was determined in Western blots from the relative signal intensities of antibodies directed against the N terminus or the cytoplasmic loop of different subunits after the relative reactivity of these antibodies had been determined with GABAA receptor subunit chimeras composed of the N-terminal domain of one and the remaining part of the other subunit. Via this method a subunit stoichiometry of two alpha subunits, two beta subunits, and one gamma subunit was derived. Similar experiments investigating the composition of alpha1beta3 receptors expressed on the surface of human embryonic kidney (HEK) 293 cells cotransfected with alpha1 and beta3 subunits resulted in a stoichiometry of two alpha and three beta subunits. Density gradient centrifugation studies indicated that combinations of alpha1beta3gamma2 or alpha1beta3 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of alpha1gamma2 or beta3gamma2 subunits predominantly form heterodimers. These results provide valuable information on the mechanism of GABAA receptor assembly and support the conclusion that GABAA receptors are pentamers in which a total of four alternating alpha and beta subunits are connected by a gamma subunit.