The new target of Rapamycin: lysosomal calcium channel TRPML1 / 生理学报

Rapamycin (Rap) is an immunosuppressant, which is mainly used in the anti-rejection of organ transplantation. Meanwhile, it also shows great potential in the fields of anticancer, neuroprotection and anti-aging. Rap can inhibit the activity of mammalian target of Rap (mTOR). It activates the transcription factor EB (TFEB) to up-regulate lysosomal function and eliminates the inhibitory effect of mTOR on ULK1 (unc-51 like autophagy activating kinase 1) to promote autophagy. Recent research showed that Rap can directly activate the lysosomal cation channel TRPML1 in an mTOR-independent manner. TRPML1 activation releases lysosomal calcium. Calcineurin functions as the sensor of the lysosomal calcium signal and activates TFEB, thus promoting lysosome function and autophagy. This finding has greatly broadened and deepened our understanding of the pharmacological roles of Rap. In this review, we briefly introduce the canonical Rap-mTOR-ULK1/TFEB signaling pathway, and then discuss the discovery of TRPML1 as a new target of Rap and the pharmacological potential of this novel Rap-TRPML1-Calcineurin-TFEB pathway.

Cancer pain, a serious threat to patientsmemory / 生理学报

A large number of cancer patients suffer from pain. Growing evidence suggested that pain might be a serious risk factor for cancer patients. The shared modulators and modulation pathways between neural system and tumor cells, such as various neurotransmitters and neurogenic cytokines, provide essential basis for the effect of pain on tumor. In this article, we reviewed some possible mechanism of this process from two aspects: the systematic regulation of central nervous system on endocrine and immunity, and the regional regulation of peripheral nerves on tumor cells. The aim of this review is to provide more innovative knowledge about pain and cancer and to emphasize the importance of anti-pain in the therapy of cancer.

Lipid bilayer modification alters the gating properties and pharmacological sensitivity of voltage-gated sodium channel / 生理学报

Voltage-gated sodium channels (VGSCs) are widely distributed in most cells and tissues, performing many physiological functions. As one kind of membrane proteins in the lipid bilayer, whether lipid composition plays a role in the gating and pharmacological sensitivity of VGSCs still remains unknown. Through the application of sphingomyelinase D (SMaseD), the gating and pharmacological sensitivity of the endogenous VGSCs in neuroblastoma ND7-23 cell line to BmK I and BmK AS, two sodium channel-specific modulators from the venom of Buthus martensi Karsch (BmK), were assessed before and after lipid modification. The results showed that, in ND7-23 cells, SMaseD did not change the gating properties of VGSCs. However, SMaseD application altered the slope factor of activation with the treatment of 30 nmol/L BmK I, but caused no significant effects at 100 and 500 nmol/L BmK I. With low concentration of BmK I (30 and 100 nmol/L) treatment, the application of SMaseD exerted hyperpolarizing effects on both slow-inactivation and steady-state inactivation, and increased the recovery time constant, whereas total inactivation and recovery remained unaltered at 500 nmol/L BmK I. Meanwhile, SMaseD modulation hyperpolarized the voltage dependence of slow-inactivation at 0.1 nmol/L BmK AS and altered the slope factor of slow-inactivation at 10 nmol/L BmK AS, whereas other parameters remained unchanged. These results indicated a possibility that the lipid bilayer would disturb the pharmacological sensitivity of VGSCs for the first time, which might open a new way of developing new drugs for treating sodium channelopathies.

Spinal 5-HT3AR contributes to BmK I-induced inflammatory pain in rats / 生理学报

Subcutaneous injection of BmK I could be adopted to well establish a novel pain model. Moreover, 5-hydroxytryptamine (serotonin, 5-HT) receptor is involved in regulating animal pain-related behaviors. However, the underlying mechanism of 5-HT3R on BmK I-induced pain remains unclear. Animal behavioral testing, RT-PCR and Western blotting were used to yield the following results: first, intraplantar (i.pl.) injection of BmK I (10 μg) induced elevated mRNA and protein levels of 5-HT3AR in bilateral L4-L5 spinal cord; Second, intrathecal (i.t.) injection of ondansetron (a specific antagonist of 5-HT3AR) reduced spontaneous pain responses, attenuated unilateral thermal and bilateral mechanical hypersensitivity elicited by BmK I; Microglia could be activated by BmK I (i.pl.) in both sides of L4-L5 spinal cord, and this effect was reversed by intrathecal pre-treatment with 5-HT3AR antagonist. Meanwhile, the 5-HT3AR in L4-L5 spinal cord was almost co-localized with NeuN (a marker of nerve cell), but not co-expressed with Iba-1 (a marker of microglia). Finally, the expression level of CX3CL1 and CX3CR1 was reduced by intrathecal pre-treatment with ondansetron. Our results indicate that both 5-HT3AR signaling pathway and microglia are activated in the process of induction and maintenance of BmK I-induced pain nociception. Meanwhile, our results suggest that the neuronal 5-HT3AR may communicate with microglia indirectly via CX3CL1 which is involved in regulating the BmK I-induced hyperalgesia and sensitization.

Martentoxin: a unique ligand of BK channels / 生理学报

The large-conductance calcium-activated potassium (BK) channels distributed in both excitable and non-excitable cells are key participants in a variety of physiological functions. By employing numerous high-affinity natural toxins originated from scorpion venoms the pharmacological and structural characteristics of these channels tend to be approached. A 37-residue short-chain peptide, named as martentoxin, arising from the venom of the East-Asian scorpion (Buthus martensi Karsch) has been investigated with a comparatively higher preference for BK channels over other voltage-gated potassium (Kv) channels. Up to now, since the specific drug tool probing for clarifying structure-function of BK channel subtypes and related pathology remain scarce, it is of importance to illuminate the underlying mechanism of molecular interaction between martentoxin and BK channels. As for it, the current review will address the recent progress on the studies of pharmacological characterizations and molecular determinants of martentoxin targeting on BK channels.

Exploring the obscure profiles of pharmacological binding sites on voltage-gated sodium channels by BmK neurotoxins

Diverse subtypes of voltage-gated sodium channels (VGSCs) have been found throughout tissues of the brain, muscles and the heart. Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch (BmK) act as sodium channel-specific modulators and have therefore been widely used to study VGSCs. α-type neurotoxins, named BmK I, BmK αIV and BmK abT, bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs. In contrast, β-type neurotoxins, named BmK AS, BmK AS-1, BmK IT and BmK IT2, occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels. Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs, however, indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simple α-type and β-type neurotoxin distinction. Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region- and/or species-specific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs. In this review, we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3- or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.

Slow rise of intracellular Ca(2+) concentration in rat primary sensory neurons triggered by loureirin B / 生理学报

In the present study, the intracellular free calcium concentration ([Ca(2+)](i)) in acutely isolated rat dorsal root ganglia (DRG) neurons modulated by loureirin B, an active component of "dragon's blood" which is a kind of Chinese herbal medicine, was determined by the means of Fura-2 based microfluorimetry. It was found that loureirin B could evoke the elevation of [Ca(2+)](i) in a dose-dependent manner. However, the elevation of [Ca(2+)](i) evoked in the calcium free solution was much smaller than that in the standard external cell solution, suggesting that most change of [Ca(2+)](i) was generated by the influx of extracellular Ca(2+), not by the activities of intracellular organelles like Ca(2+) stores and mitochondria. In addition, the mixture of loureirin B and caffeine also induced [Ca(2+)](i) rise, but the peak of [Ca(2+)](i) rise induced by the mixture was significantly lower than that by caffeine alone, which means the triggering pathway and the targets of caffeine are probably involved in loureirin B-induced [Ca(2+)](i) rise. Moreover, compared to the transients induced by caffeine, KCl and capsaicin, the loureirin B-induced [Ca(2+)](i) rise is much slower and more stable. These results indicate that the capability of loureirin B of inducing the [Ca(2+)](i) rise is solid and unique.

Deglycosylation altered the gating properties of rNav1.3: glycosylation/deglycosylation homeostasis probably complicates the functional regulation of voltage-gated sodium channel / 神经科学通报·英文版

<p><b>OBJECTIVE</b>To examine the effect of deglycosylation on gating properties of rNav1.3.</p><p><b>METHODS</b>rNav1.3 was expressed in Xenopus oocyte, with glycosylation inhibition by using tunicamycin. Two-electrode voltage clamp was employed to record the whole-cell sodium current and data were analyzed by Origin software. Those of glycosylated rNav1.3 were kept as control.</p><p><b>RESULTS</b>Compared with glycosylated ones, the steady-state activation curve of deglycosylated rNav1.3 was positively shifted by about 10 mV, while inactivation curve was negatively shifted by about 8 mV.</p><p><b>CONCLUSION</b>Glycosylation altered the gating properties of rNav1.3 and contributed to the functional diversity.</p>

The study of sodium channels involved in pain responses using specific modulators / 生理学报

Voltage-gated sodium channels (VGSCs) are transmembrane proteins responsible for generation and conduction of action potentials in excitable cells. Physiological and pharmacological studies have demonstrated that VGSCs play a critical role in chronic pain associated with tissue or nerve injury. Many long-chain peptide toxins (60-76 amino acid residues) purified from the venom of Asian scorpion Buthus martensii Karsch (BmK) are investigated to be sodium channel-specific modulators. The alpha-like neurotoxins that can bind to receptor site 3 of sodium channels, named as BmK I and BmK abT, could induce nociceptive effects in rats. On the contrast, the beta-like neurotoxins that can bind to receptor site 4 of sodium channels, named as BmK AS, BmK AS-1 and BmK IT2, could produce potent anti-nociceptive effects in animal pain models. BmK I could strongly prolong the fast inactivation of tetrodotoxin (TTX)-sensitive Na(+) currents on the rat dorsal root ganglia (DRG) neurons together with the augmentation of peak current amplitude. However, BmK IT2 and BmK ASs, potently suppressed both the peak TTX-resistant and TTX-sensitive Na(+) currents on rat small DRG neurons. Moreover, BmK ASs could decrease the excitability of small DRG neurons. Thus, the nociception/anti-nociception induced by scorpion neurotoxins may attribute to their distinct modulation on sodium channels in primary afferent sensory neurons. Therefore, the sodium channel-specific modulators from BmK venom could be used as not only pharmacological tools for better understanding the roles of VGSCs in pain signal conduction, but also lead molecules in the development of ideal analgesics targeting VGSCs.

BmK I, an alpha-like scorpion neurotoxin, specifically modulates isolated rat cardiac mechanical and electrical activity / 生理学报

In this study, cardiotonic and cardiotoxic effects of Buthus martensi Karsch (BmK) I, a modulator of voltage-gated sodium channels, were investigated on the isolated rat hearts. The results showed that BmK I evoked complex effects characterized by a change in both cardiac mechanical and electrical activity. Langendorff perfusion showed that: (1) maximal left ventricular developed pressure (LVDP(max)) and dp/dt(max) were markedly increased by BmK I (0.5-10 micromol/L) in a dose-dependent manner (n=6, P<0.05), positive chronotropic effects were also induced by BmK I (n=6, P<0.05); (2) negative inotropic action and bradycardia could be elicited at a larger dose of BmK I (20 micromol/L); (3) the coronary flow varied inversely with the positive inotropic effects, coronary flow reduced during positive inotropic effects from 14.5 to 8.6 ml/min after administration of 500 nmol/L BmK I (n=6, P<0.05). In addition, tachycardia and complex cardiac arrhythmias were induced by BmK I (0.5-10 micromol/L). The modulating of BmK I on the heart mechanical, electrical activity could be partially recovered after washing. As propranolol was applied to block the release of catecholamines before administration of BmK I, suggesting that the changes in cardiac mechanical and electrical activity induced by BmK I might not due to catecholamine release from the nerve terminal and subsequent stimulation of the beta-adrenoceptor but attributable to the modulation of BmK I on cardiac voltage-gated sodium channels.