Modification of cloned brain Na+ channels by batrachotoxin

The effects of batrachotoxin (BTX) on cloned α-subunit Na⁺ channels were examined in CHO-K1 cells (a chinese hamster ovary cell line) transfected with rat brain NaIIA cDNA. Under whole-cell patch clamp conditions, BTX shifted the voltage dependence of the activation process by about 45 mV towards the hyperpolarizing direction and eliminated the inactivating phase of Na⁺ currents. Repetitive depolarizations greatly facilitated the binding of BTX with NaIIA channels while the membrane was held at −100 mV. In chloramine-T-pretreated cells, the association rate of BTX binding with the NaIIA channel was 6.5-fold faster than that in untreated cells. The estimated association rate constant for BTX binding with the open form of NaIIA channel was 1.11×10⁶ mol⁻¹·s⁻¹ at room temperature. BTX-modified NaIIA channels were blocked by tetrodotoxin (TTX) in a complicated manner. First, the TTX binding to the closed state of BTX-modified NaIIA channels was not voltage dependent. The K _D value of TTX was measured at 8.9 nM, which was similar to that of unmodified channels (K _D=14.2 nM). Second, the block of the open state of BTX-modified NaIIA channels by TTX was voltage dependent; depolarization reduced the potency of TTX block between −20 mV to +50 mV. Below −30 mV, the TTX affinity began to level off, probably because of the increased presence of the closed state. Unexpectedly, steady-state inactivation of BTX-modified NaIIA channels was minimal as measured by the two-pulse protocol, a phenomenon distinctly different from that found in GH₃ cells. Neutral local anesthetic benzocaine, however, drastically enhanced the steady-state inactivation of BTX-modified NaIIA channels, with its maximal effect around −60 mV. We conclude that BTX can bind and modify the NaIIA α-subunit. However, a specific subtype of α-subunits and/or an unidentified modulating process may be required for the optimal steady-state inactivation of BTX-modified Na⁺ channels.     

Single-channel analysis of two types of Na+currents in rat dorsal root ganglia

The properties of voltage-gated Na⁺ channels were studied in neurones isolated from rat dorsal root ganglia using the outside-out configuration of the patch-clamp technique. Two types of single-channel currents were identified from the difference in unit amplitudes. Neither type was evoked in the medium in which extracellular Na⁺ ions were replaced by an equimolar amount of tetramethylammonium ions. The two types of single-channel currents differed in their sensitivity to tetrodotoxin (TTX). The smaller channel current was insensitive to 1 M TTX (referred to as TTX-I), while the larger channel current was blocked by 1 nM TTX (TTX-S). The unit amplitudes measured during a step depolarization to –30 mV (1.4 mM internal and 250 mM external Na⁺ concentrations) were 1.16 pA for TTX-S and 0.57 pA for TTX-I, respectively. The slope conductance measured at –30 mV was 16.3 pS for TTX-S and 8.5 pS for TTX-I. TTX-S could be activated by step depolarizations positive to –60 mV, while TTX-I could be activated at potentials positive to –40 mV. When the test pulse was preceded by a depolarizing prepulse, the prepulse positive to –50 mV preferentially inactivated TTX-S with a minimal effect on TTX-I. Activation and inactivation time courses of the averaged ensemble currents computed from TTX-S showed remarkable resemblances to the time courses of the macroscopic TTX-sensitive Na⁺ current. Similarly, the ensemble currents of TTX-I mimicked the macroscopic TTX-insensitive Na⁺ current. It was concluded that the two types of Na⁺ channels in rat dorsal root ganglia differ not only in their sensitivity to TTX, but also in their single-channel conductances.     

河豚毒素小分子拮抗肽的设计与鉴定

目的用计算机辅助分子设计技术,设计河豚毒素(TTX)小分子拮抗肽,分别用免疫学方法和动物实验进行验证。方法利用计算机辅助分子设计技术,对TTX的空间结构进行了优化;用分子对接确定了TTX关键位点;用分子模建设计了3个能与TTX结合的小分子拮抗肽;分别用竞争ELISA进行免疫学筛选;用动物实验进行中和活性测定。结果成功设计了3个针对TTX的小分子拮抗肽并进行了多肽合成,用竞争ELISA筛选到针对TTX的小分子拮抗肽P2,拮抗肽P2浓度与TTX的结合能力成正比。动物实验中,针对TTX的小分子拮抗肽P2对注射2.5倍半数致死剂量TTX昆明小鼠的保护率为25%,起到了一定解毒效果。结论获得了能与TTX特异性结合的中和性小分子拮抗肽,TTX浓度达到4.05μg/mL(LD50的2.5倍),TTX拮抗肽P2对实验组小鼠的保护率为25%。     

Evidence for the existence of a rapid sodium channel in the membrane of rabbit sinoatrial cells.

True intracellular pacemaker action potentials were recorded in isolated rabbit atria preparations at 37°C. It was verified that these action potentials were insensitive to tetrodotoxin (TTX). Hyperpolarization of the membrane using carbamylcholine (CCh) produced an increase in the rate of rise of the action potential. This increase was entirely suppressed by TTX. The insensitivity of the pacemaker potentials to TTX is generally attributed to the absence of a rapid sodium channel in the sinoatrial membrane. Our experiments strongly suggest that this insensitivity is attributable mainly to inactivation of rapid sodium conductance because of the low value of the take-off membrane potential of nodal cells.     

急性河豚鱼中毒患者的神经电生理测定的特点及意义

目的 分析急性河豚毒素(tetrodotoxin，TTX)中毒患者的神经电生理变化的特点与意义。方法 检测64例患者的运动神经传导速度(mote never conduction velocity，MCV)、感觉神经传导速度(sensory never conduction velocity，SCV)、F波、H反射和体感诱发电位(somatosensory evoked potentiols，SEP)。结果MCV、SCV均有减慢，以SCV减慢最为显著，MCV远段动作电位潜伏期明显延长，神经传导速度(NCV)的异常检出率高；F反应、H反射异常提示部分TIX中毒累及神经根；SEP的异常率达45．9％，说明TTX中毒伴有中枢神经的损害。结论 神经电生理检测可用来动态观察’FTX中毒患者神经系统损害程度、病稃，范围,亦旱，TTX中毒时早期检杏的重要手段之一。     

Temporary inactivation of the supramammillary area impairs spatial working memory and spatial reference memory retrieval

The aim of our study was to examine the supramammillary (SuM) area involvement in spatial memory. Sprague-Dawley rats with chronically implanted cannula in the supramammillary area were trained in two spatial memory tasks with different memory demands: reference and working memory. In the spatial reference memory task, the rats received microinjections in the SuM area of tetrodotoxin (TTX) (0.5 ng diluted in 0.5 microL of saline) or saline (0.5 microL). The microinjections were administered 30 min before the spatial training (day 4) (to assess the effect on acquisition) and on the following two days (days 5 and 6) the training was conducted without microinjections (to study the effect on consolidation). On the last training day (day 7), in order to assess the retrieval of spatial information, the rats received the microinjections 30 min before the spatial training. The spatial working memory used was a delayed-matching-to-position (DMTP) task. Spatial training was performed for seven days. During the first three days of the spatial training, the rats achieved a good spatial knowledge and learnt the working memory rule necessary to solve the spatial task. On days 4 and 6, the rats received microinjections to study involvement of the SuM area in working memory. The results showed that temporary inactivation of SuM area impairs both the rat's ability to solve a spatial working memory task with DMTP demands and the recovery of spatial information in a spatial reference memory task. We suggest that SuM area is involved in the rearrangement of spatial information during spatial working memory tasks with DMTP memory demands.     

河豚毒素处方前初步研究

目的 考察河豚毒素（TTX）在不同溶剂中的溶解性及稳定性，以及温度和pH值对稳定性的影响。方法 配制TTX不同介质的溶液，采用高效液相色谱法（HPLC）测定其在不同温度、不同pH缓冲液中的浓度，分析计算其溶解度及稳定性。结果 TTX在pH值为3.5时溶解度最大，随着pH值增加其溶解度逐渐降低。TTX在强碱条件下降解最为迅速，在70 ℃条件下、0.1 mol/L的氢氧化钠溶液中，20 min即完全降解。稳定性试验结果同样证明TTX在碱性条件下的稳定性最差，在37 ℃、pH值=7.4的磷酸缓冲盐溶液（PBS）中，TTX浓度在1~10 h时开始持续降低，28天降解率为88.07±0.27%。结论 TTX易溶于pH值=3.5的酸性水溶液，几乎不溶于碱性水溶液。其稳定性与温度、介质pH值密切相关，在酸性水溶液中较为稳定，在碱性条件下易降解，温度升高会加速其降解过程。     

Berberine ameliorates the neurological dysfunction of the gastric fundus by promoting calcium channels dependent release of ACh in STZ-induced diabetic rats

BackgroundIt has been reported diabetic gastroparesis is related to diabetic autonomic neuropathy of the gastrointestinal tract, and berberine (BBR) could ameliorate diabetic central and peripheral neuropathy. However, the influence of BBR on the function and motility of the gastric fundus nerve is unclear.MethodsA diabetic rat model was constructed, and HE staining was used to observe the morphological changes in the gastric fundus. The changes in cholinergic and nitrogen-related neurochemical indexes and the effects of BBR on them were measured using Elisa. The effects of BBR on the neural function and motility of gastric fundus were investigated by electric field stimulation (EFS) induced neurogenic response in vitro.ResultsIn the early stage of STZ-induced diabetic rats, the contractile response of gastric fundus induced by EFS was disorder, disturbance of contraction amplitude, and the cell bodies of neurons in the myenteric plexus of gastric fundus presented vacuolar lesions. Administration with BBR could improve the above symptoms. BBR further enhanced the contraction response in the presence of a NOS inhibitor or the case of inhibitory neurotransmitters removal. Interestingly, the activity of ACh could affect NO release directly and the enhancement of BBR on contractile response was canceled by calcium channel blockers completely.ConclusionsIn the early stage of STZ-induced diabetic rats, the neurogenic contractile response disorder of the gastric fundus is mainly related to cholinergic and nitrergic nerve dysfunction. BBR promotes the release of ACh mainly by affecting the calcium channel to improve the neurological dysfunction of the gastric fundus.