Dysfunctional astrocytic and synaptic regulation of hypothalamic glutamatergic transmission in a mouse model of early-life adversity: relevance to neurosteroids and programming of the stress response.

Adverse early-life experiences, such as poor maternal care, program an abnormal stress response that may involve an altered balance between excitatory and inhibitory signals. Here, we explored how early-life stress (ELS) affects excitatory and inhibitory transmission in corticotrophin-releasing factor (CRF)-expressing dorsal-medial (mpd) neurons of the neonatal mouse hypothalamus. We report that ELS associates with enhanced excitatory glutamatergic transmission that is manifested as an increased frequency of synaptic events and increased extrasynaptic conductance, with the latter associated with dysfunctional astrocytic regulation of glutamate levels.

Mutagenic analysis of the intracellular portals of the human 5-HT3A receptor.

Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg(436) and Arg(440)) within the MA helix of 5-hydroxytryptamine type 3A (5-HT3A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT3A receptor model.

Rings of charge within the extracellular vestibule influence ion permeation of the 5-HT3A receptor.

The determinants of single channel conductance (γ) and ion selectivity within eukaryotic pentameric ligand-gated ion channels have traditionally been ascribed to amino acid residues within the second transmembrane domain and flanking sequences of their component subunits. However, recent evidence suggests that γ is additionally controlled by residues within the intracellular and extracellular domains. We examined the influence of two anionic residues (Asp(113) and Asp(127)) within the extracellular vestibule of a high conductance human mutant 5-hydroxytryptamine type-3A (5-HT(3)A) receptor (5-HT(3)A(QDA)) upon γ, modulation of the latter by extracellular Ca(2+), and the permeability of Ca(2+) with respect to Cs(+) (P(Ca)/P(Cs)).

Novel structural determinants of single channel conductance and ion selectivity in 5-hydroxytryptamine type 3 and nicotinic acetylcholine receptors.

Nicotinic acetylcholine (nACh) and 5-hydroxytryptamine type 3 (5-HT(3)) receptors are cation-selective ion channels of the pentameric ligand-gated ion channel (pLGIC) superfamily. Multiple lines of evidence adduced over the last 30 years indicate that the lining of the channel of such receptors is formed by the alpha-helical second transmembrane (TM2) domain and flanking sequences contributed by each of the five subunits present within the receptor complex. Specific amino acid residues within, and adjacent to, the TM2 domain influence single channel conductance, ion selectivity, and other aspects of receptor function that include gating and desensitization.

Neurosteroids: endogenous allosteric modulators of GABA(A) receptors.

In the mammalian central nervous system activation of the ionotropic GABA(A) receptor by the neurotransmitter GABA plays a crucial role in controlling neuronal excitability. This essential form of neuronal regulation may be subject to "fine tuning" by particular metabolites of progesterone and deoxycorticosterone, which bind directly to the GABA(A) receptor to enhance the actions of GABA. Originally such steroids were considered to act as endocrine messengers, being synthesised in peripheral glands such as the adrenals and ovaries and crossing the blood brain barrier to influence neuronal signalling.

Characterization of the effects of charged residues in the intracellular loop on ion permeation in alpha1 glycine receptor channels.

The Cys loop receptor channels mediate fast synaptic transmission in the nervous system. The M2-demarcated transmembrane pore is an important determinant of their ion permeation properties. Portals within the intracellular domain are also part of the permeation pathway in cationic Cys loop receptors, with charged residues in a helical MA stretch partially lining these openings profoundly affecting channel conductance.

Structural determinants of Ca2+ permeability and conduction in the human 5-hydroxytryptamine type 3A receptor.

Cation-selective cysteine (Cys)-loop transmitter-gated ion channels provide an important pathway for Ca2+ entry into neurones. We examined the influence on Ca2+ permeation of amino acids located at intra- and extracellular ends of the conduction pathway of the human 5-hydroxytryptamine type 3A (5-HT3A) receptor. Mutation of cytoplasmic arginine residues 432, 436, and 440 to glutamine, aspartate, and alanine (the aligned residues of the human 5-HT3B subunit (yielding 5-HT3A(QDA)) increased PCa/PCs from 1.

Dynamic modification of a mutant cytoplasmic cysteine residue modulates the conductance of the human 5-HT3A receptor.

Structural models suggest that Arg(436) lies within five cytoplasmic portals of the 5-HT(3A) receptor. We tested both the accessibility of residue 436 and the influence of its charge on single channel conductance (gamma) by substituting Arg(436) with Cys and examining the effect of methanethiosulfonate (MTS) reagents on gamma. Inclusion of positively charged 2-aminoethyl-MTS (MTSEA) within the electrode solution reduced gamma of 5-HT(3A)(R436C) receptors in outside-out patches from 7.