24 research outputs found
Kinetic properties of the alpha(2) homo-oligomeric glycine receptor impairs a proper synaptic functioning
Ionotropic glycine receptors (GlyRs) are present in the central nervous system well before the establishment of synaptic contacts. Immature nerve cells are known, at least in the spinal cord, to express alpha(2) homomeric GlyRs, the properties of which are relatively unknown compared to those of the adult synaptic form of the GlyR (mainly alpha(1)/beta heteromeres). Here, the kinetics properties of GlyRs at the single-channel level have been recorded in real-time by means of the patch-clamp technique in the outside-out configuration coupled with an ultra-fast flow application system (< 100 µs). Recordings were performed on chinese hamster ovary (CHO) cells stably transfected with the a, GlyR subunit. We show that the onset, the relaxation and the desensitisation of α(2) homomeric GlyR-mediated currents are slower by one or two orders of magnitude compared to synaptic mature GlyRs and to other ligand-gated ionotropic channels involved in fast synaptic transmission. First latency analysis performed on single GlyR channels revealed that their slow activation time course was due to delayed openings. When synaptic release of glycine was mimicked (1 mM glycine; 1 ms pulse duration), the opening probability of α(2) homomeric GlyRs was low (P-o ≈ 0.1) when compared to mature synaptic GlyRs (P-o = 0.9). This low P-o is likely to be a direct consequence of the relatively slow activation kinetics of α(2) homomeric GlyRs when compared to the activation kinetics of mature α(1)/β GlyRs. Such slow kinetics suggest that embryonic α(2) homomeric GlyRs cannot be activated by fast neurotransmitter release at mature synapses but rather could be suited for a non-synaptic paracrine-like release of agonist, which is known to occur in the embryo
Zn2+ inhibition of recombinant GABAA receptors: an allosteric, state-dependent mechanism determined by the γ-subunit
The γ-subunit in recombinant γ-aminobutyric acid (GABAA) receptors reduces the sensitivity of GABA-triggered Cl− currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non-competitive to competitive. To investigate underlying receptor function we studied Zn2+ effects on macroscopic and single-channel currents of recombinant α1β2 and α1β2γ2 receptors expressed heterologously in HEK-293 cells using the patch-clamp technique and rapid solution changes.Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 μM)-triggered currents of α1β2 receptors in a concentration-dependent manner (inhibition equation parameters: concentration at half-amplitude (IC50) = 0.94 μM; slope related to Hill coefficient, S= 0.7) that was unaffected by GABA concentration. The γ2 subunit (α1β2γ2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50= 51 μM, S= 0.86); increased GABA concentration (100 μM) antagonized inhibition by reducing apparent affinity (IC50= 322 μM, S= 0.79). Zn2+ slowed macroscopic gating of α1β2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For α1β2γ2 receptors, Zn2+ accelerated a fast component of apparent desensitization.Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of α1β2 receptors, but had little effect on currents from α1β2γ2 receptors.Steady-state fluctuation analysis of macroscopic α1β2γ2 currents (n= 5) resulted in control (2 μM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 ± 5.6 and 1.41 ± 0.15 ms, means ± s.e.m.). Zn2+ (200 μM) reduced the total power almost sixfold and accelerated the slow (23 ± 2.8 ms, P < 0.05) without altering the fast (1.40 ± 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 ± 0.55; Zn2+, 4.9 ± 0.37, P < 0.05).Zn2+ (500 μM) depression of previously activated current amplitudes (% control) for α1β2γ2 receptors was independent of GABA concentration (5 μM, 13.2 ± 0.72 %; 100 μM, 12.2 ± 2.9 %, P < 0.8, n= 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated α1β2 receptors with current depression more than fourfold less sensitive (5 μM GABA, IC50= 3.8 μM, S= 0.84) relative to that in constant Zn2+.The results lead us to propose a general model of Zn2+ inhibition of GABAA receptors in which Zn2+ binds to a single extracellular site, induces allosteric receptor inhibition involving two non-conducting states, site affinity is state-dependent, and the features of state dependence are determined by the γ-subunit
Milk productivity and exterior of holsteinized cows of the Kholmogory breed of different generations
The electrodeposition of copper-silver and copper-nickel alloy powders from aqueous ammoniacal solutions
Gallium-Doped Boehmite Nanotubes And Nanoribbons. A TEM, EDX, XRD, BET, And TG Study
Gallium doped boehmite nanostructures with varying gallium content have been prepared at low temperatures via a soft-chemistry route in the presence of poly (ethylene oxide) (PEO) surfactant. The effect of gallium content, hydrothermal temperature and mixing procedures on the growth of boehmite nanostructures was systematically studied. The resultant boehmite nanostructures were characterized by transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) microanalysis, X-ray diffraction (XRD), N2 adsorption and thermogravimetric analysis (TGA). Nanotubes with average length of ~90 nm, internal and external diameters of 2~5 nm and 3~7 nm respectively were formed when added gallium molar percentage ≤5 %; when added gallium percentage>10%, an amorphous phase dominated the sample with a mixture of nanosheets, nanotubes and nanoribbons was also formed. Synthesis at slightly higher temperatures (120 °C) for added gallium molar percentage ≤5 % resulted in longer nanotubes. For high gallium content boehmites large crystals are formed when hydrothermally treated at 120 °C. The detailed characterization of the resultant gallium doped boehmite nanostructures is presented