Characterization Of Voltage-gated Sodium Channels Research Articles

The voltage-gated sodium channel Nav1.7 associated with endometrial cancer.

Background: Endometrial cancer is the most common gynecologic malignancy in women in the developed countries. Despite recent progress in functional characterization of voltage-gated sodium channel (Nav) in multiple cancers, very little was known about the expression of Nav in human endometrial cancer. The present study sought to determine the role of Nav and molecular nature of this channel in the endometrial cancer.Methods: PCR approach was introduced to determine expression level of Nav subunits in endometrial cancer specimens. Pharmacological agents were used to investigate Nav function in endometrial cancer cells. Flow cytometry were used to test cancer apoptosis, and invasion assays were applied to test tumor metastasis.Results: Transcriptional levels of the all Nav α and β subunits were determined by real time-PCR in endometrial cancer with pair tissues of carcinoma and adjacent nonneoplastic tissue, Nav1.7 was the most highly expressed Nav subtype in endometrial cancer tissues. Nav1.7 level was closely associated with tumor size, local lymph node metastasis, and 5-year and 10-year survival ratio. Inhibition of this channel by Nav1.7 blocker PF-05089771, promoted cancer apoptosis and attenuated cancer cell invasion.Conclusion: These results establish a relationship between voltage-gated sodium channel protein and endometrial cancer, and suggest that Nav1.7 is a potential prognostic biomarker and could serve as a novel therapeutic target for endometrial cancer.

Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea gigas.

Paralytic shellfish toxins (PST) bind to voltage-gated sodium channels (Nav) and block conduction of action potential in excitable cells. This study aimed to (i) characterize Nav sequences in Crassostrea gigas and (ii) investigate a putative relation between Nav and PST-bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gigas: a Nav1 (CgNav1) and a Nav2 (CgNav2) with sequence properties of sodium-selective and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of CgNav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junctions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The PST binding site (domain II) of CgNav1 variants possess an amino acid Q that could potentially confer a partial saxitoxin (STX)-resistance to the channel. The CgNav1 genotype or alternative splicing would not be the key point determining PST bioaccumulation level in oysters.

Identification and functional characterization of voltage-gated sodium channels in lymphocytes

A variety of ion channels has been discovered in lymphocytes. RT-PCR and real-time PCR analysis revealed that ALL (acute lymphocytic leukemia) cell lines and human peripheral blood mononuclear cells mainly expressed TTX (tetrodotoxin)-sensitive voltage-gated sodium channels (VGSCs). Expression of VGSC protein was confirmed by western blots and Immunofluorescence. Whole-cell patch-clamp recordings showed that a sub-population (20%) of MOLT-4 cells expressed functional VGSCs, which were TTX-sensitive. Importantly, 2 μM TTX decreased the invasion of Jurkat and MOLT-4 cells ∼90%. These results indicate that the activity of VGSCs could represent a novel mechanism potentiating the invasive capacity of these cells.

ATP Regulates Sodium Channel Kinetics in Pancreatic Islet Beta Cells

Pancreatic beta cells act as glucose sensors, in which intracellular ATP ([ATP](i)) are altered with glucose concentration change. The characterization of voltage-gated sodium channels under different [ATP](i) remains unclear. Here, we demonstrated that increasing [ATP](i) within a certain range of concentrations (2-8mM) significantly enhanced the voltage-gated sodium channel currents, compared with 2mM cytosolic ATP. This enhancement was attenuated by even high intracellular ATP (12mM). Furthermore, elevated ATP modulated the sodium channel kinetics in a dose-dependent manner. Increased [ATP](i) shifted both the current-voltage curve and the voltage-dependent inactivation curve of sodium channel to the right. Finally, the sodium channel recovery from inactivation was significantly faster when the intracellular ATP level was increased, especially in 8mM [ATP](i), which is an attainable concentration by the high glucose stimulation. In summary, our data suggested that elevated cytosolic ATP enhanced the activity of Na(+) channels, which may play essential roles in modulating β cell excitability and insulin release when blood glucose concentration increases.

Expression and Biophysical Characterization of Voltage-Gated Sodium Channels in Axons and Growth Cones of the Regenerating Optic Nerve

Successful regeneration and re-establishment of synaptic connections in the adult central nervous system is a complex process determined by both the exterior environment and the endogenous neural activity of the regenerating growth cones. The purpose of this study was to determine the expression and properties of voltage-gated sodium channels (Na(v)) expressed by regenerating growth cones. Na(v) channels were studied in an organotypic explant culture of the adult rat retina by immunocytochemistry and whole-cell, patch-clamp recordings. Regenerating axons and growth cones, but not glial processes, expressed Na(v) channels. Whole-cell, current-clamp recordings from growth cones displayed a high input resistance of 1.29 GOmega and a resting membrane potential of -69.0 mV. All growth cones responded to depolarizing voltage steps with fast, transient, inward currents mediated by Na(+) ions, followed by slow, sustained outward K(+) currents. Half-maximum activation clustered in two groups, suggesting the presence of at least two Na(v) channel isoforms. Steady state inactivation and recovery from fast inactivation were characterized by a half-maximum value of -69.7 mV and by a time constant of 3.64 ms, respectively. Injection of depolarizing current steps larger than threshold (-29.3 mV) consistently induced a single action potential, whereas ganglion cell bodies responded to above-threshold stimulation with a series of fast, all-or-none action potentials. These experiments describe for the first time the biophysical properties of Na(v) channels recorded from the growth cones of regenerating retinal ganglion cells and contrast their properties with those of adult retinal ganglion cells.

Molecular and functional characterization of voltage-gated sodium channels in human sperm

BackgroundWe have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility.MethodsFreshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA). Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2.ResultsThe mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9) were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels.ConclusionThis research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function.

Identification and characterization of voltage-gated sodium channels in mouse taste bud cells

Identification and characterization of voltage-gated sodium channels in mouse taste bud cells

Functional and Molecular Characterization of Voltage-Gated Sodium Channels in Uteri from Nonpregnant Rats1

We investigated the function and expression of voltage-gated Na(+) channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca(2+)] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50-60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca(2+)](i), which were abolished by treatment with 0.1 microM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.

Interactions of the C-11 Hydroxyl of Tetrodotoxin with the Sodium Channel Outer Vestibule

The highly selective sodium channel blocker, tetrodotoxin (TTX) has been instrumental in characterization of voltage-gated sodium channels. TTX occludes the ion-permeation pathway at the outer vestibule of the channel. In addition to a critical guanidinium group, TTX possesses six hydroxyl groups, which appear to be important for toxin block. The nature of their interactions with the outer vestibule remains debatable, however. The C-11 hydroxyl (C-11 OH) has been proposed to interact with the channel through a hydrogen bond to a carboxyl group, possibly from domain IV. On the other hand, previous experiments suggest that TTX interacts most strongly with pore loops of domains I and II. Energetic localization of the C-11 OH was undertaken by thermodynamic mutant cycle analysis assessing the dependence of the effects of mutations of the adult rat skeletal muscle Na + channel (rNa v1.4) and the presence of C-11 OH on toxin IC 50. Xenopus oocytes were injected with the mutant or native Na + channel mRNA, and currents were measured by two-electrode voltage clamp. Toxin blocking efficacy was determined by recording the reduction in current upon toxin exposure. Mutant cycle analysis revealed that the maximum interaction of the C-11 OH was with domain IV residue D1532 (ΔΔ G: 1.0 kcal/mol). Furthermore, C-11 OH had significantly less interaction with several domain I, II, and III residues. The pattern of interactions suggested that C-11 was closest to domain IV, probably involved in a hydrogen bond with the domain IV carboxyl group. Incorporating this data, a new molecular model of TTX binding is proposed.

Molecular characterization of voltage-gated sodium channels in human gliomas.

Voltage-sensitive sodium channels appear to be an electrophysiological hallmark of gliomas. However, the expression of channel subtypes is unclear in these tumors. In this study different gliomas were investigated for the expression of sodium channel subtypes Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4, Na(v)1.6, and Na(x)(Na(v)2.1) using RT-PCR. At least one subtype of channels could be detected in each tumor. High-grade gliomas expressed fewer sodium channel subtypes and these at weaker levels than low-grade tumors. Expression of Na(v)1.6, the most abundant isoform in the CNS, was almost absent in the gliomas except the pilocytic variant. Our study gives clear evidence for a differential expression of sodium channel subtypes in gliomas and indicates a predominant expression of channels related to malignancy grades.