Joksovic Pavle M, Doctor Allan, Gaston Benjamin, Todorovic Slobodan M
Dept. of Anesthesiology, University of Virginia Health System, Mail Box 800710, Charlottesville, VA 22908-0710, USA.
J Neurophysiol. 2007 Apr;97(4):2712-21. doi: 10.1152/jn.00926.2006. Epub 2007 Feb 7.
Although T-type Ca(2+) channels in the reticular thalamic nucleus (nRT) have a central function in tuning neuronal excitability and are implicated in sensory processing, sleep, and epilepsy, the mechanisms involved in their regulation are poorly understood. Here we recorded T-type Ca(2+) currents from intact nRT neurons in brain slices from young rats and investigated the mechanisms of T-type channel modulation by S-nitrosothiols (SNOs). We found that extracellular application of S-nitrosoglutathione (GSNO), S-nitrosocysteine (CSNO) and S-nitroso-N-acetyl-penicillamin (SNAP) rapidly and reversibly reduced T-type currents. The effects of SNOs are strongly stereoselective at physiological concentrations: (L)-CSNO was fourfold more effective in inhibiting T-type current than was (D)-CSNO. The effects of GSNO were abolished if cells had been treated with free hemoglobin or N-ethylmaleimide, an irreversible alkylating agent but not by 8-bromoguanosine-3',5'-cyclomonophosphate sodium salt, a membrane-permeant cGMP analogue or 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one, a specific soluble guanylyl cyclase inhibitor. In addition, bath applications of GSNO inhibited T-type currents in nucleated outside-out patches and whole cell recordings to a similar extent, with minimal effect on cell-attached recordings, suggesting a direct effect of GSNO on putative extracellular thiol residues on T-type channels. Biophysical studies indicate that GSNO decreased the availability of T-type channels at physiological potentials by modifying gating and stabilizing inactive states of the channels. In current-clamp experiments, GSNO diminished the amplitude of low-threshold calcium spikes and frequency of spike firing with minimal effects on the passive membrane properties. Collectively, the results indicate that SNOs may be a class of endogenous agents that control the functional states of the thalamus.
尽管丘脑网状核（nRT）中的T型Ca(2+)通道在调节神经元兴奋性方面具有核心作用，并与感觉处理、睡眠和癫痫有关，但其调节机制仍知之甚少。在此，我们记录了幼鼠脑片中完整nRT神经元的T型Ca(2+)电流，并研究了S-亚硝基硫醇（SNOs）对T型通道的调节机制。我们发现，细胞外应用亚硝基谷胱甘肽（GSNO）、亚硝基半胱氨酸（CSNO）和亚硝基-N-乙酰青霉胺（SNAP）可快速、可逆地降低T型电流。在生理浓度下，SNOs的作用具有强烈的立体选择性：（L）-CSNO抑制T型电流的效果比（D）-CSNO强四倍。如果细胞用游离血红蛋白或N-乙基马来酰亚胺（一种不可逆的烷基化剂）处理，GSNO的作用就会消失，但用膜通透性cGMP类似物8-溴鸟苷-3',5'-环一磷酸钠盐或特异性可溶性鸟苷酸环化酶抑制剂1H-(1,2,4)恶二唑并(4,3-a)喹喔啉-1-酮处理则不会。此外，在核内向外膜片和全细胞记录中，浴用GSNO对T型电流的抑制程度相似，对细胞贴附记录的影响最小，这表明GSNO对T型通道假定的细胞外硫醇残基有直接作用。生物物理研究表明，GSNO通过改变门控和稳定通道的失活状态，降低了生理电位下T型通道的可用性。在电流钳实验中，GSNO减小了低阈值钙峰的幅度和峰发放频率，对被动膜特性的影响最小。总的来说，这些结果表明SNOs可能是一类控制丘脑功能状态的内源性物质。
