ABSTRACT In mammalian cells, one-third of all polypeptides is transported into or through the ER-membrane via the Sec61-channel. While the Sec61-complex facilitates the transport of all polypeptides with amino-terminal signal peptides (SP) or SP-equivalent transmembrane helices (TMH), the translocating chain-associated membrane protein (now termed TRAM1) was proposed to support transport of a subset of precursors. To identify possible determinants of TRAM1 substrate specificity, we systematically identified TRAM1-dependent precursors by analyzing cellular protein abundance changes upon TRAM1 depletion in HeLa cells using quantitative label-free proteomics. In contrast to previous analysis after TRAP depletion, SP and TMH analysis of TRAM1 clients did not reveal any distinguishing features that could explain its putative substrate specificity. To further address the TRAM1 mechanism, live-cell calcium imaging was carried out after TRAM1 depletion in HeLa cells. In additional contrast to previous analysis after TRAP depletion, TRAM1 depletion did not affect calcium leakage from the ER. Thus, TRAM1 does not appear to act as SP- or TMH-receptor on the ER-membrane’s cytosolic face and does not appear to affect the open probability of the Sec61-channel. It may rather play a supportive role in protein transport, such as making the phospholipid bilayer conducive for accepting SP and TMH in the vicinity of the lateral gate of the Sec61-channel. Abbreviations: ER, endoplasmic reticulum; OST, oligosaccharyltransferase; RAMP, ribosome-associated membrane protein; SP, signal peptide; SR, SRP-receptor; SRP, signal recognition particle; TMH, signal peptide-equivalent transmembrane helix; TRAM, translocating chain-associated membrane protein; TRAP, translocon-associated protein.

ABSTRACT Neuropathic pain is clinically unsatisfactorily treated because of unclear mechanisms. The present study aims to explore the concrete mechanisms underlying the alleviation of resveratrol-activated silent information regulator 1 (Sirt1) to chronic constriction injury (CCI)-induced neuropathic pain. CCI surgery was conducted to the unilateral sciatic nerve of male Sprague-Dawley rats to induce neuropathic pain experimentally. Resveratrol with or without miR-182 antagomir were administered to CCI rats via intrathecal catheter. Behavioral tests including paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were conducted to explore mechanical allodynia and thermal hyperalgesia. Western blot, qRT-PCR were used to detect the expression levels of Sirt1, miR-182, and Nav1.7 in CCI dorsal root ganglions (DRGs). CCI rats displayed lower PWT and PWL compared with the sham control. Also, the CCI DRGs displayed lower Sirt1 and miR-182 expression as well as higher Nav1.7 expression, which would be almost reversed by resveratrol treatment for 4 successive days. We also found that miR-182 expression inhibition erased the analgesia effect of resveratrol to CCI–induced neuropathic pain possibly through upregulating Nav1.7 expression. In summary, resveratrol alleviated CCI–induced neuropathic pain, possibly through activating Sirt1 to suppress Nav1.7 expression via upregulating miR-182 expression in CCI DRGs.

ABSTRACT Cytosolic Ca2+ concentration ([Ca2+ ]) has an important role in spermatozoa and hence it regulates fertilization. In male germinal cells, there are indirect evidences that this ion could regulate physiological processes in spermatogenesis. Since little is known about Ca 2+ homeostasis in spermatogenic cells, in this work we propose a mathematical model that accounts for experimental [Ca2+ ] dynamics triggered by blockade of the SERCA transport ATPase with thapsigargin in round rat spermatids, without external Ca2+ and with different extracellular lactate concentrations. The model included three homogeneous calcium compartments and Ca2+-ATPase activities sensitive and insensitive to thapsigargin, and it adjusted satisfactorily the experimental calcium dynamic data. Moreover, an extended version of the model satisfactorily adjusted the stationary states of calcium modulated by extracellular lactate, which is consistent with the participation of a low affinity lactate transporter and further lactate metabolism in these cells. Further studies and modeling would be necessary to shed some light into the relation between Ca 2+-lactate-ATP homeostasis and cell–cell interactions in the seminiferous tubules that are expected to modulate Ca 2+ dynamics by hormonal factors or energetic substrates in meiotic and postmeiotic spermatogenic cells.

ABSTRACT Residue Thr1604 in the CaV1.2 channel is a Ca2+/calmodulin dependent protein kinase II (CaMKII) phosphorylation site, and its phosphorylation status maintains the basic activity of the channel. However, the role of CaV1.2 phosphorylation at Thr1604 in myocardial hypertrophy is incompletely understood. Isoproterenol (ISO) was used to induce cardiomyocyte hypertrophy, and autocamtide-2-related inhibitory peptide (AIP) was added as a treatment. Rats in a myocardial hypertrophy development model were subcutaneously injected with ISO for two or three weeks. The heart and left ventricle weights, each of which were normalized to the body weight and cross-sectional area of the myocardial cells, were used to describe the degree of hypertrophy. Protein expression levels were detected by western blotting. CaMKII-induced CaV1.2 (Thr1604) phosphorylation (p-CaV1.2) was assayed by coimmunoprecipitation. The results showed that CaMKII, HDAC, MEF2 C, and atrial natriuretic peptide (ANP) expression was increased in the ISO group and downregulated by AIP treatment in vitro. There was no difference in the expression of these proteins between the ISO 2-week group and the ISO 3-week group in vivo. CaV1.2 channel expression did not change, but p-CaV1.2 expression was increased after ISO stimulation and decreased by AIP. In the rat model, p-CaV1.2 levels and CaMKII activity were much higher in the ISO 3-week group than in the ISO 2-week group. CaMKII-induced CaV1.2 channel phosphorylation at residue Thr1604 may be one of the key features of myocardial hypertrophy and disease development.Abbreviations: CaMKII: Ca2+/calmodulin dependent protein kinase II; p-CaMKII: autophosphorylated Ca2+/calmodulin dependent protein kinase II; CaM: calmodulin; AIP: autocamtide-2-related inhibitory peptide; ECC: excitation-contraction coupling; ISO: isoproterenol; BW: body weight; HW: heart weight; LVW: left ventricle weight; HDAC: histone deacetylase; p-HDAC: phosphorylated histone deacetylase; MEF2C: myocyte-specific enhancer factor 2C; ANP: atrial natriuretic peptide; PKC: protein kinase C

ABSTRACT CACNA1 C, which codes for the Cav1.2 isoform of L-type Ca2+ channels (LTCCs), is a prominent risk gene in neuropsychiatric and neurodegenerative conditions. A role forLTCCs, and Cav1.2 in particular, in transcription-dependent late long-term potentiation (LTP) has long been known. Here, we report that elimination of Cav1.2 channels in glutamatergic neurons also impairs theta burst stimulation (TBS)-induced LTP in the hippocampus, known to be transcription-independent and dependent on N-methyl D-aspartate receptors (NMDARs) and local protein synthesis at synapses. Our expansion of the established role of Cav1.2channels in LTP broadens understanding of synaptic plasticity and identifies a new cellular phenotype for exploring treatment strategies for cognitive dysfunction.

ABSTRACT Congenital hyperinsulinemic hypoglycemia is the most frequent cause of persistent and recurrent hypoglycemia in the first years of life and in many patients rare genetic variants can be identified. Recently a case of congenital hyperinsulinemic hypoglycemia and a severe neurodevelopmental syndrome due to a mutation in the voltage-gated Cav1.3 Ca2+ channel CACNA1D gene has been reported which required long-term treatment with diazoxide. This suggested CACNA1D variants as a potential cause for this condition. Here we support this observation by presenting the case of a female child with congential hyperinsulinemic hypoglycemia and primary hyperaldosteronism, aortic insufficiency, pronounced developmental delay, muscle hypotonia, and facial dysmorphias but without seizures. Sequencing of the exome of the child and its parents identified a novel de novo CACNA1D missense mutation p.L271 H, replacing a highly conserved residue in a functionally relevant region of the voltage-gated Cav1.3 Ca2+ channel. The patient was treated with diazoxide and nifedipine with adequate control of glucose metabolism and blood pressure, and with improvement in muscle tone. Our findings further confirm the pathogenic role of CACNA1D for congentital hyperinsulinemic hypoglycemia and primary aldosteronism. Moreover, we provide evidence that the dihydropyridine Ca2+ channel blocker nifedipine, although not considered a first-line treatment for congenital hyperinsulinism, may be beneficial to control blood pressure and neurological symptoms in patients with CACNA1D mutations.

ABSTRACT Transient receptor potential vanilloid subfamily member 1 (TRPV1) is a nonselective cation channel, that is mainly distributed in sensory nerve endings and can release a variety of neurotransmitters after activation. Early studies showed that it mainly conducts pain sensation, but research has demonstrated that it also plays an important role in cardiovascular diseases. Notably, in atherosclerosis, the activation of TRPV1 can regulate lipid metabolism, reduce foam cell formation, protect endothelial cells, inhibit smooth muscle cell proliferation and inhibit inflammation and oxidation. In this review, the role of the TRPV1 channel in atherosclerosis was discussed to provide new ideas for the prevention and treatment of atherosclerotic diseases.

ABSTRACTAtomic resolution structures have provided significant insight into the gating and permeation mechanisms of various ion channels, including potassium channels. However, ion channels may also be regulated by numerous factors, including the physiochemical properties of the membrane in which they are embedded. For example, the matching of the bilayer’s hydrophobic region to the hydrophobic external surface of the ion channel is thought to minimize the energetic penalty needed to solvate hydrophobic residues or exposed lipid tails. To understand the molecular basis of such regulation by hydrophobic matching requires examining channels in the presence of the lipid membrane. Here we examine the role of hydrophobic matching in regulating the activity of the model potassium channel, KcsA. 86Rb+ influx assays and single-channel recordings indicate that the non-inactivating E71A KcsA channel is most active in thin bilayers (<diC18:1PC). Bilayer thickness affects the open probability of KcsA and not its unitary conductance. Molecular dynamics simulations indicate that the bilayer can sufficiently modify its dimensions to accommodate KcsA channels without major perturbations in the protein helical packing within the nanosecond timescale. Based on experimental results and MD simulations, we present a model in which bilayer thickness influences the stability of the open and closed conformations of the intracellular gate of KcsA, with minimal impact on the stability of the selectivity filter of the non-inactivating mutant, E71A.

ABSTRACTThe voltage-gated calcium channel CaV1.1a primarily functions as voltage-sensor in skeletal muscle excitation-contraction (EC) coupling. In embryonic muscle the splice variant CaV1.1e, which lacks exon 29, additionally function as a genuine L-type calcium channel. Because previous work in most laboratories used a CaV1.1 expression plasmid containing a single amino acid substitution (R165K) of a critical gating charge in the first voltage-sensing domain (VSD), we corrected this substitution and analyzed its effects on the gating properties of the L-type calcium currents in dysgenic myotubes. Reverting K165 to R right-shifted the voltage-dependence of activation by ~12 mV in both CaV1.1 splice variants without changing their current amplitudes or kinetics. This demonstrates the exquisite sensitivity of the voltage-sensor function to changes in the specific amino acid side chains independent of their charge. Our results further indicate the cooperativity of VSDs I and IV in determining the voltage-sensitivity of CaV1.1 channel gating.

KEYWORDS: TMEM16anoctaminlipid scramblasegnathodiaphyseal dysplasiamuscular dystrophyphosphatidylserine