A new non-voltage-dependent,epithelial-like Na+ channel in vascular smooth muscle cells

A new type of Na⁺ channel was identified in smooth muscle cells of the rat aortic cell line A7r5, and in smooth muscle cells cultured from rat aorta and rat portal vein. The channel is highly selective for Na⁺ (P _Na/P _K>11). It is active in cell-attached patches, and independent of the trans-patch membrane potential. The single channel conductance is low (10.7 pS). Two substates were identified. This channel is insensitive to effectors of other types of Na⁺ channels, such as amiloride (100 M) or tetrodotoxin (100 M). It is inhibited by phenamil at high concentrations (>10 M). The mean open state probability P(O) varied from patch to patch (0.05–0.88). Kinetics analysis reveals a complex behaviour: open times separate in short (₁ = 84 ms) and long (₂ = 845 ms) openings and closed times separate into short (₁ = 60 ms) and long closures (₂ = 272 –3130 ms). Short openings and long closures are preponderant at a low P(O). Long openings are absent in the presence of phenamil (50 M) and are unaffected by amiloride (100 M). Fluctuations of the channel activity in cell-attached patches and the fast disappearance after excision suggest that this channel is under metabolic control. This vascular smooth muscle channel appears to be a potentially important Na⁺ entry pathway for vascular cells and an amiloride-resistant homologue of the epithelial Na⁺ channel.     

Nimodipine and nifedipine enhance transmission at the Schaffer collateral CA1 pyramidal neuron synapse

Summary Intracellular recording in the in vitro hippocampal slice was utilized to examine the effects of nimodipine and nifedipine on CA1 pyramidal cell excitability. The excitatory postsynaptic potential (EPSP) elicited by a single stimulus in stratum radiatum was enhanced by nifedipine as evidenced by increases in EPSP amplitude, area and slope. Threshold for synaptically-evoked somatic action potentials was decreased following either nifedipine or nimodipine application, often resulting in spontaneous action potential activity. A secondary, late EPSP-like event appeared in the intracellular recordings during and following bath application of nimodipine, and was associated with burst-like activity in field potential recordings. In accordance with the hydrophobic nature of these compounds, extensive washout in normal Krebs' solution failed to reverse their effects, but nifedipine's actions were photolabile. These results indicate that dihydropyridines can enhance synaptic efficacy in the CA1 region of the hippocampus. Offprint requests to: Neuroscience Research Laboratory/127A     

Potassium conductance of smooth muscle cells from rabbit aorta in primary culture

Vascular smooth muscle cells were obtained from rabbit aorta and were studied in primary culture on days 1–7 after seeding with electrophysiological techniques. In impalement experiments a mean membrane potential difference (PD) of –50±0.3 mV (n=387) was obtained with Ringer-type solution in the bath. PD was depolarized by 6±0.3 mV (n=45) and 16±2 mV (n= 5) when the bath K⁺ concentration was increased from the control value of 3.6 mmol/l to 13.6 and 23.6 mmol/l, respectively. Ba²⁺ (0.1–1 mmol/l) depolarized PD. Tetraethylammonium (TEA, 10 mmol/l) depolarized PD only slightly but significantly. Verapamil (0.1 mmol/l) and charybdotoxin (10 nmol/l) had no effect on PD. The conductance properties of these cells were further examined with the patch-clamp technique. K⁺ channels were spontaneously present in cell-attached patches. When the pipette was filled with 145 mmol/l KCl, a mean conductance (g _K) of 209.6±4.6 mV (n=17) was read from the current/voltage curves at a clamp voltage (V _c) of 0 mV. After excision K⁺ channels were found in 129 patches with inside-out and in 50 with outside-out configuration. With KCl on one and NaCl on the other side the mean g _K at a V _c of 0 mV was 134.6±3.9 pS (n=179). The mean permeability was 0.89±0.03×10^–12 cm³/s. With symmetrical KCl solution the mean g _K was 227±6 pS (n=17). The conductance sequence was g _K g _Rb= g _Cs=g _Na=0. TEA blocked dose-dependently only from the outside.(1–10 mmol/l). Lidocaine (5 mmol/l) quinidine (0.01–1 mmol/l) and quinine (0.01–1 mmol/l) blocked from both sides. Charybdotoxin (0.5–5 nmol/l) blocked only from the extracellular side. Ba²⁺ blocked from the cytosolic side and the inhibition was increased by depolarization and reduced by hyperpolarization. At a V _c of 0 mV a half-maximal inhibition (IC₅₀) of 2 mol/l was obtained. Verapamil and diltiazem blocked from both sides, verapamil with an IC₅₀ of 2 mol/l and diltiazem with an IC₅₀ of 10 mol/l. The open probability of this channel was increased by Ca²⁺ on the cytosolic side at activities > 0.1 mol/l. Half-maximal activation occurred at Ca²⁺ activities exceeding 1 mol/l. The present data indicate that the vascular smooth muscle cells of rabbit aorta in primary culture possess a K⁺ conductance. In excised patches only a maxi K⁺ channel was detected. This channel has properties different from the macroscopic K⁺ conductance. Hence, it is likely that the K⁺ conductance of the intact cell is dominated by yet another and thus far not detected K⁺ channel.Supported by DFG Gr 480/10     

The Association between the Polymorphisms in a Sodium Channel Gene SCN7A and Essential Hypertension: A Case‐Control Study in the Northern Han Chinese

Na_x, an α‐subunit of the sodium channel encoded by the SCN7A gene, has been deemed to be a sensor of the concentration of sodium in the brain and may be involved in salt intake behavior. We inferred that Na_x/SCN7A may participate in the regulation of blood pressure and the pathogenesis of essential hypertension (EH). The present case‐control study involving 615 hypertensives and 617 normotensives was performed to investigate the association between SCN7A polymorphisms and EH in the Northern Han Chinese population. The three common single nucleotide polymorphisms (SNPs) (rs3791251, rs6738031, rs7565062) in the exons of SCN7A were genotyped with the TaqMan assay. Significant association between SNP rs7565062 and EH was found under the addictive and dominant genetic models (P = 0.024, OR = 1.283, 95%CI [1.033–1.592]; P = 0.013, OR = 1.203, 95%CI [1.040–1.392]; respectively). The three SNPs were in close pair‐wise linkage disequilibrium with each other and the haplotype analyses indicated that haplotype G–A–T was significantly associated with increased risk of EH (P = 0.023, OR = 1.290). In conclusion, our data showed that SNP rs7565062 of SCN7A was significantly associated with EH and the allele T of rs7565062 or the related haplotype G–A–T will be a genetic risk factor for EH in the Northern Han Chinese population.     

Regulation of Na+ excretion and arterial blood pressure by purinergic signalling intrinsic to the distal nephron: consequences and mechanisms

Discretionary control of Na⁺ excretion is a key component of the regulation of arterial blood pressure in mammals. Sodium excretion is fine‐tuned in the aldosterone‐sensitive distal nephron by the activity of the epithelial Na⁺ channel (ENaC). Here, ENaC functions as a final effector of the renin–angiotensin–aldosterone system (RAAS) during negative feedback control of blood pressure. Mutations affecting ENaC activity and abnormal regulation of this channel affect blood pressure through pathological changes to Na⁺ excretion. Recent evidence demonstrates that powerful signalling pathways function in parallel with the RAAS to modulate ENaC activity and blood pressure. An inclusive paradigm is emerging with respect to regulation of blood pressure where ENaC serves as a critical point of convergence for several important signalling systems that affect renal Na⁺ excretion. A robust inhibitory purinergic signalling system intrinsic to the distal nephron dynamically regulates ENaC through paracrine ATP signalling via the metabotropic P2Y₂ purinergic receptor to properly match urinary Na⁺ excretion to dietary Na⁺ intake. This enables blood pressure to be maintained within a normal range despite broad changes in dietary Na⁺ consumption. Loss of purinergic inhibition of ENaC increases blood pressure by causing inappropriate Na⁺ excretion. In contrast, stimulation of the P2Y₂ receptor promotes natriuresis and a decrease in blood pressure. Such observations identify purinergic signalling in the distal nephron as possibly causative, when dysfunctional, for certain forms of elevated blood pressure, and as a possible therapeutic target for the treatment of elevated blood pressure particularly that associated with salt sensitivity.     

Expanding the spectrum of Grik2 mutations: intellectual disability,behavioural disorder,epilepsy and dystonia

Isolated congenital anosmia (ICA) is a rare disorder, where otherwise healthy individuals present with an inability to smell since birth. A list of studies have described the genes involved in syndromic anosmia; however, the genetics of ICA is still in its infancy. Studies in mice show that the cyclic nucleotide‐gated channel subunit CNGA2, expressed in the olfactory epithelium has a crucial role in olfactory signal transduction. We have identified a novel X‐linked stop mutation in CNGA2 (c.634C>T, p.R212*) in two brothers with ICA using exome sequencing. No additional mutations in CNGA2 were identified in a cohort of 31 non‐related ICA individuals. Magnetic resonance brain imaging revealed diminished olfactory bulbs and flattened olfactory sulci. This is the first report of a mutation in the cyclic nucleotide‐gated gene CNGA2 and supports the critical role of this gene in human olfaction.     

Single pore translocation of folded,double-stranded,and tetra-stranded DNA through channel of bacteriophage phi29 DNA packaging motor

The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ∼35-fold slower speed of translocation per base-pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ∼32% of channel current blockage and with ∼64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine.     

KATP通道在心血管保护及糖尿病伴发心血管疾病发生发展中的作用

近年来诸多临床研究表明,当糖尿病患者伴发心脏病时,其死亡率、心力衰竭和心律失常的发生率和心血管事件风险均高于非糖尿病人群。糖尿病患者应用降糖药后心血管事件发生率的改变尚存争议,这一现象背后的分子机制较为复杂。 ATP敏感钾通道（ KATP通道）作为糖尿病和心血管疾病的共同发病机制,可以在一定程度上解释这一临床现象的原因。文章总结以下几方面的研究进展：① KATP通道构造、分布和作用机制的研究进展;② KATP通道的心血管保护作用的;③糖尿病状态下胰岛素、高血糖和异常代谢产物是如何通过影响KATP通道的表达和活性从而影响心血管系统;④ KATP通道阻滞剂（磺胺类降糖药）和KATP通道开放剂的研发进展。提出mitoKATP通道的相关机制研究应该是未来这一领域的研究重点,相关药物研发应该更加注重高选择性以及对血糖控制和心血管保护作用的兼顾。     

Molecular basis for pharmacological differences between brain and cardiac sodium channels

Sodium channels from brain and heart, whose primary structures are known, differ in their sensitivity to block by the guadinium toxins tetrodotoxin and saxitoxin and to block by external Zn²⁺ and Cd²⁺. Studies using site-directed mutagenesis have identified the SS2 and adjacent regions of all four repeats as critical determinants for toxin sensitivity. Within and in the immediate vicinities of the SS2 segments, there are only two amino-acid differences between rat brain sodium channel II and rat heart I sodium channel, both located in repeat I. Here we show that replacement of phenylalanine 385 of brain sodium channel by cysteine that is present at the equivalent position in heart channel (F385C) not only reduces sensitivity to the guadinium toxins but also increases sensitivity to Zn²⁺ and Cd²⁺, thus conferring properties of heart sodium channel on brain sodium channel. Replacement of asparagine at the second non-conserved position by arginine (N388R) only marginally affects sensitivity to the toxins, Zn²⁺ or Cd²⁺, but this mutation markedly reduces sensitivity to block by Ca²⁺ and Co²⁺. The double mutant channel (F385CN388R) shows combined properties of the two mutant channels. These results give a structural insight into the different properties of the two channel proteins.     

Voltage-gated Ca entry in isolated bovine capillary endothelial cells: evidence of a new type of BAY K 8644-sensitive channel

Isolated bovine capillary endothelial cells have been examined for voltage-dependent Ca entry. All cells displayed a low threshold activity, with the main characteristics of a T-type transient current, when examined using whole-cell recording for activation and inactivation and cell-attached conditions or inside-out patches for the elementary conductance (8 pS). 25% of the cells displayed an additional sustained current in 5 mM CaCl₂ above –40 mV, which was enhanced by application of BAY K 8644, but almost insensitive to superfusion with nicardipine. Two types of channels (2.8 and 21 pS, in 110 mM BaCl₂) were shown to have a BAY K 8644 sensitivity. The large conductance channels were L-type channels. The smaller events were elicited at more hyperpolarized potentials (by some 30 mV). Their mean open time was 16 ms in control conditions. In presence of BAY K 8644, additional long open times were observed (up to 100 ms as compared to 7.8 ms for the time constants of the slow mode of the L-type channel). We refer to these channels as SB channels: of small conductance and sensitive to BAY K 8644. In the presence of nicardipine, SB channels are not noticeably modified, in contrast to the L-type openings which are abolished. Also, SB open times are close to control values when nicardipine is added after a BAY K 8644 application. We suggest that, at physiological concentrations of divalent ions, an SB-type activity is elicited above –40 mV which generates the low threshold sustained current.