Block of NMDA receptor channels by endogenous neurosteroids: implications for the agonist induced conformational states of the channel vestibule

N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system

GALVANIC MUD TREATMENT IN THE REHABILITATION OF PATIENTS WITH PAIN IN THE LOWER BACK

Purpose. Evaluati on of the effectiveness of complex restorati ve treatment with the inclusion of galvanic mud procedures in patients with pain in the lower back.Patients and methods. There were 30 patients (18 men and 12 women) with a diagnosis: lumbulgia or lumboeishalgia under observation. Patients underwent a course of rehabilitation on the basis of the Republican Hospital of Rehabilitation, Makhachkala. Against the background of standard therapy, galvanic mud procedures were included in the therapy.  The effectiveness of the treatment was assessed by the control of pain assessment according to the VAS scale, the body mass index, the psychoemoti onal state of the HAM test, the tests of Schober and Thomayer were carried out.Results. After the complex treatment in patients, clinical and functional indices of the state of the neuromuscular apparatus Were noted, varying depending on gender. The results of the study showed that the intensity of the pain syndrome according to the VAS score in the compared groups before the rehabilitation differed significantly (5.4 ± 0.1 points in men and 6.16 ± 0.3 in women, (p <0.05), after completion of the course of treatment, the VAS score decreased statistically in both groups: in men 2.0 ± 0.4 points and in women 1.8 ± 0.1 points (p <0.05), but between groups of significant differences in the quantitative assessment of pain the syndrome was not revealed. In determining the mobility of the lumbar spine, the results of the Schober test with lumbargia as in men, and women were unreliably reduced, after treatment restored within the norm: in men before treatment, 3.46 ± 1.47 cm, after 5.3 ± 0.73 cm (p < 0.05), in women before treatment 3.95 ± 0.5 cm, after 5.35 ± 0.55 cm (p < 0.05).The analysis of the dynamics of the test results of Tomayer revealed unreliably significant differences before treatment in men = 24.8 ± 2.4 cm, in women = 21,6 ± 6,2 cm. After the course of treatment in both groups there was a significant improvement in the indices: in men = 5.23 ± 1.44 cm (p <0.05), in women 1.45 ± 0.87 cm (p < 0,05), but at the same time a reliable difference in the indices (p < 0.01).Conclusion. The conducted research has shown that carrying out galvanic mud procedures in a complex of treatment in patients with lower back pain positively influences the restoration of lumbar vertebral segment motor activity, as well as the psychoemotional state of the patient. In this case, women are more actively reduced pain and increased motor activity, but less significant indicators of the change in the psychological test

Smooth muscle membrane potential modulates endothelium-dependent relaxation of rat basilar artery via myo-endothelial gap junctions

The release of endothelium-derived relaxing factors, such as nitric oxide (NO), is dependent on an increase in intracellular calcium levels ([Ca2+]i) within endothelial cells. Endothelial cell membrane potential plays a critical role in the regulation of [Ca2+]i in that calcium influx from the extracellular space is dependent on membrane hyperpolarization. In this study, the effect of inhibition of vascular smooth muscle delayed rectifier K+ (KDR) channels by 4-aminopyridine (4-AP) on endothelium-dependent relaxation of rat basilar artery to acetylcholine (ACh) was assessed. ACh-evoked endothelium-dependent relaxations were inhibited by N-(Ω)-nitro-l-arginine (l-NNA) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), confirming a role for NO and guanylyl cyclase. 4-AP (300 μm) also suppressed ACh-induced relaxation, with the maximal response reduced from ≈92 to ≈33 % (n = 11; P < 0.01). However, relaxations in response to exogenous NO, applied in the form of authentic NO, sodium nitroprusside or diethylamineNONOate (DEANONOate), were not affected by 4-AP treatment (n = 3-11). These data are not consistent with the view that 4-AP-sensitive KDR channels are mediators of vascular hyperpolarization and relaxation in response to endothelium-derived NO. Inhibition of ACh-evoked relaxation by 4-AP was reversed by pinacidil (0.5-1 μm; n = 5) or 18β-glycyrrhetinic acid (18βGA; 5 μm; n = 5), indicating that depolarization and electrical coupling of the smooth muscle to the endothelium were involved. 4-AP caused depolarization of both endothelial and vascular smooth muscle cells of isolated segments of basilar artery (mean change 11 ± 1 and 9 ± 2 mV, respectively; n = 15). Significantly, 18βGA almost completely prevented the depolarization of endothelial cells (n = 6), but not smooth muscle cells (n = 6) by 4-AP. ACh-induced hyperpolarization of endothelium and smooth muscle cells was also reduced by 4-AP, but this inhibition was not observed in the combined presence of 4-AP and 18βGA. These data indicate that 4-AP can induce an indirect inhibition of endothelium-dependent relaxation in the rat basilar artery by electrical coupling of smooth muscle membrane depolarization to the endothelium via myo-endothelial gap junctions

A steroid modulatory domain on NR2B controls N-methyl-d-aspartate receptor proton sensitivity

N-methyl-d-aspartate (NMDA) receptor function is modulated by several endogenous molecules, including zinc, polyamines, protons, and sulfated neurosteroids. Zinc, polyamines, and phenylethanolamines exert their respective modulatory effects by exacerbating or relieving tonic proton inhibition. Here, we report that pregnenolone sulfate (PS) uses a unique mechanism for enhancement of NMDA receptor function that is independent of the proton sensor. We identify a steroid modulatory domain, SMD1, on the NMDA receptor NR2B subunit that is critical for both PS enhancement and proton sensitivity. This domain includes the J/K helices in the S2 region of the glutamate recognition site and the fourth membrane transmembrane region (M4). A molecular model based on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor structure suggests that steroid modulatory domain 1 contributes residues to a hydrophobic pocket that is capable of accommodating PS. The results demonstrate that the J/K helices and the fourth membrane transmembrane region participate in transducing allosteric interactions induced by steroid and proton binding to their respective sites