Pannexin1 Single Nucleotide Polymorphism and Platelet Reactivity in a Cohort of Cardiovascular Patients

Pannexin1 (Panx1), a membrane channel-forming protein permitting the passage of small-sized molecules, such as ATP, is expressed in human platelets. Recently, we showed that inhibiting Panx1 affects collagen-induced platelet aggregation but not aggregation triggered by other agonists. We also found that a single nucleotide polymorphism (SNP; rs1138800) in the Panx1 gene encoded for a gain-of-function channel (Panx1-400C) and was associated with enhanced collagen-induced platelet reactivity. Here, we assessed the association of this SNP with platelet reactivity in a cohort of 758 stable cardiovascular patients from the ADRIE study treated with aspirin and/or clopidogrel. We found that presence of the Panx1-400C allele was not associated with platelet reactivity in stable cardiovascular patients, irrespective of the platelet aggregation agonist used (collagen, ADP or arachidonic acid) or the anti-platelet drug regimen. Moreover, the Panx1-400A?>?C SNP did also not affect the re-occurrence of cardiac ischemic events in the same stable cardiovascular patient cohort.     

Pannexin-1 as a potentiator of ligand-gated receptor signaling

Pannexins are a class of plasma membrane spanning proteins that presumably form a hexameric, non-selective ion channel. Although similar in secondary structure to the connexins, pannexins notably do not form endogenous gap junctions and act as bona fide ion channels. The pannexins have been primarily studied as ATP-release channels, but the overall diversity of their functions is still being elucidated. There is an intriguing theme with pannexins that has begun to develop. In this review we analyze several recent reports that converge on the idea that pannexin channels (namely Panx1) can potentiate ligand-gated receptor signaling. Although the literature remains sparse, this emerging concept appears consistent between both ionotropic and metabotropic receptors of several ligand families.     

Pannexin-1 as a potentiator of ligand-gated receptor signaling

Pannexins are a class of plasma membrane spanning proteins that presumably form a hexameric, non-selective ion channel. Although similar in secondary structure to the connexins, pannexins notably do not form endogenous gap junctions and act as bona fide ion channels. The pannexins have been primarily studied as ATP-release channels, but the overall diversity of their functions is still being elucidated. There is an intriguing theme with pannexins that has begun to develop. In this review we analyze several recent reports that converge on the idea that pannexin channels (namely Panx1) can potentiate ligand-gated receptor signaling. Although the literature remains sparse, this emerging concept appears consistent between both ionotropic and metabotropic receptors of several ligand families.     

Electrophysiological and Theoretical Analysis of Depolarization-Dependent Outward Currents in the Dendritic Membrane of an Identified Nonspiking Interneuron in Crayfish

Depolarization-dependent outward currents were analyzed using the single-electrode voltage clamp technique in the dendritic membrane of an identified nonspiking interneuron (LDS interneuron) in situ in the terminal abdominal ganglion of crayfish. When the membrane was depolarized by more than 20 mV from the resting potential (65.0 ± 5.7 mV), a transient outward current was observed to be followed by a sustained outward current. Pharmacological experiments revealed that these outward currents were composed of 3 distinct components. A sustained component (I _s) was activated slowly (half rise time > 5 msec) and blocked by 20 mM TEA. A transient component (I _t1) that was activated and inactivated very rapidly (peak time < 2.5 msec, half decay time < 1.2 msec) was also blocked by 20 mM TEA. Another transient component (I _t2) was blocked by 100 M 4AP, activated rapidly (peak time < 10.0 msec) and inactivated slowly (half decay time > 131.8 msec). Two-step pulse experiments have revealed that both sustained and transient components are not inactivated at the resting potential: the half-maximal inactivation was attained at –21.0 mV in I _t1, and –38.0 mV in I _t2. I _s showed no noticeable inactivation. When the membrane was initially held at the resting potential level and clamped to varying potential levels, the half-maximal activation was attained at –36.0 mV in I _s, –31.0 mV in I _t1 and –40.0 mV in I _t2. The activation and inactivation time constants were both voltage dependent. A mathematical model of the LDS interneuron was constructed based on the present electrophysiological records to simulate the dynamic interaction of outward currents during membrane depolarization. The results suggest that those membrane conductances found in this study underlie the outward rectification of the interneuron membrane as well as depolarization-dependent shaping of the excitatory synaptic potential observed in current-clamp experiments.     

成年大鼠海马CA1区锥体神经元外向整流氯离子单通道特性

采用膜片钳内面向外式技术,在急性分离成年大鼠海马CAl区锥体细胞上记录到了外向整流氯离子通道（outwardly rectifying chloride channel,ORCC）.长时间去极化（≥60 mV）刺激后,在30%的游离膜片上记录到有外向整流特性的单通道氯电流,膜电位在－60 mV到0 mV之间的单通道电导为(16.58±1.54) pS（n=10）,而在0 mV到+60 mV之间电导为（40.92±3.17） pS.通道开放概率有明显的电压依赖性(膜电位－60 mV时,P_o=0.44±0.12;膜电位为+60 mV时,P_o=0.86±0.06, n=10).在对称Cl^－浓度（150 mmol/L）时,通道翻转电位为(－4.17±1.84) mV.当溶液中部分NaCl被葡萄糖酸钠替代后,翻转电位为：(－34.23±4.86) mV (［Cl^－］_i/［Cl^－］_o=(30 mmol/L)/(150 mmol/L)),接近氯离子通道的理论值,这表明通道具有氯离子选择性.浴槽液中分别加入氯通道阻断剂DIDS和SITS可以使+40 mV的通道开放概率从(0.83±0.06)和(0.86±0.06)分别降低到(0.12±0.05)和(0.13±0.04)（n=5）,冲洗后可使开放概率基本恢复.上述研究结果显示,在成年大鼠海马CA1神经元上存在外向整流氯离子通道.     

p-CMBS Modifies Extrafacial Sulfhydryl Groups at the Chara Plasma Membrane: Activation of Ca2+ Influx and Inhibition of Two Different K+ Currents

The effect of the membrane impermeant sulfhydryl group (SH) reagent, p-chloromercuribenzenesulfonic acid (p-CMBS), on electrical membrane transport properties of the giant alga, Chara corallina, was determined. In an external medium with a high K⁺ concentration (5 mM) cells typically exhibited stable membrane potentials close to the K⁺equilibrium potential. The steady-state current-voltage (I-V) relation could be dissected into two distinct components: an almost linear ohmic leak current and a voltage-dependent K⁺ current. Adding 0.5 mM p-CMBS to the external medium resulted in an immediate, short depolarization transient (resembling the time course of an action potential) and was associated with a slow down of the cytoplasmic streaming velocity. The depolarization, as well as the streaming inhibition, could be abolished by pretreating cells with the Ca²⁺ channel inhibitor, LaCl₃. This suggests that the depolarization transient reflected a p-CMBS induced Ca²⁺ influx, a scenario known to trigger membrane excitation and slow down of cytoplasmic streaming. From the I-V analysis it appeared that p-CMBS also caused a reversible inhibition of two additional transmembrane currents: (1) a reduction of a leak current and (2) a modification of the deactivation kinetics of the voltage-dependent K⁺ channels. From the I-V difference analysis, the inhibited leak current was identified as a K⁺ current, because the reversal potential was close to the estimated K⁺ equilibrium potential. Control experiments have furthermore shown that the mercapto reagent, dithiothreitol, partly reversed the effect of p-CMBS. This strengthens the view that the action of the mercurial is related to a specific and direct modification of SH groups. The p-CMBS-evoked inhibition of K⁺ currents was not abolished by the LaCl₃ pretreatment, which suggests that the effect of the SH reagent is not induced indirectly by p-CMBS-triggered Ca²⁺ influx. Therefore, it is suggested that the mercurial interacts direcly with the K⁺ transport protein.     

琥珀酸对大鼠海马CA1区神经元电压依赖性钙电流的影响

目的：观察不同浓度的琥珀酸对大鼠海马CA1区神经元电压依赖性钙通道（voltage—dependent calcium channels,VDCC）电流的作用,初步探讨琥珀酸对神经元保护的电生理学基础。方法：采用传统全细胞膜片钳技术和制霉菌素（nystatin）穿孔膜片钳技术观察琥珀酸对海马CA1区神经元VDCC电流的影响。结果：不同浓度的琥珀酸（10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1）在海马CA1区对低电压激活（low—voltage activated,LVA）钙通道电流未见任何影响,而对高电压激活（high—voltage activated,HVA）钙通道电流的抑制呈浓度依赖性。对照组HVA钙电流为580．05±17．32pA,分别给予10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1。的琥珀酸后,HVA钙电流依次为563．74±16．65,517．99±15．24,444．66±13．26,405．32±19．11,269．03±9．96和86．41±3．25pA,同对照组相比差异有统计学意义（n=8,P〈0．01）。结论：琥珀酸能浓度依赖性地抑制HVA钙电流,而对LVA钙电流无影响。由此推测琥珀酸可能通过抑制HVA钙电流减少Ca^2＋内流而影响海马CA1区神经元的兴奋性,从而抑制癫痫的形成,其脑保护作用可能与此有关。     

Motor pattern switching in the heartbeat pattern generator of the medicinal leech: membrane properties and lack of synaptic interaction in switch interneurons

The motor program for heartbeat in the medicinal leech is produced by a central pattern generator that regularly switches between two alternative coordination states. A pair of switch heart interneurons reciprocally alternate between rhythmically active and inactive states to effect these switches. During spontaneous switches in the activity state of switch interneurons, there was no correlation between the duration of a particular activity state and beat period, indicating that the timing networks for the switch cycle and the beat cycle are relatively independent. Simultaneous recordings from two switch heart interneurons showed that a perturbation in the electrical activity of one does not influence switching of the other and that there is no synaptic interaction between them. Using voltage clamp, we characterized an L-like Ca²⁺ current (measured as Ba²⁺ currents), inactivating and non-inactivating K⁺ currents, a persistent Na⁺ current, and a hyperpolarization-activated inward current in switch interneurons. Dynamic clamp experiments show that “subtraction” of an artificial switch leak conductance (described previously by Gramoll et al. 1994) from a switch interneuron when it is in the inactive state causes it to display activity associated with the active state. We discuss how the switch leak conductance may interact with the intrinsic currents of switch interneurons to control their activity state. Accepted: 1 December 1998     

Blockade of a KCa channel with synthetic peptides from noxiustoxin: A K+ channel blocker

Using the outside-out configuration of the patch-clamp method, we studied the effect of several synthetic peptides corresponding to various segments from the N-terminal region of noxiustoxin (NTX) on single Ca²⁺-activated K⁺ (K_Ca) channels of small conductance obtained from cultured bovine aortic endothelial cells. These peptides induced diverse degrees of fast blockade in the endothelial K_Ca channel. The most effective blockers were the peptides NTX_1–39 (IC₅₀=0.5 m) and NTX_1–20 comprising the first 20 amino acids from the native toxin (IC₅₀ 5 m), while less effective was the hexapeptide NTX_1–6, from the first six amino acid residues of NTX (IC₅₀ = 500 m). This was the minimum sequence required to block the channel.By testing overlapping sequences from the entire molecule, specially those corresponding to the N-terminal region of NTX, we have been able to determine their different apparent affinities for the K_Ca channel. Synthetic peptides from the C-terminal region produced no effect on the K_Ca channel at the concentrations tested (up to 1 mm). These results confirm that in the N-terminal region of the NTX is located part of the sequence that may recognize K⁺ channels, as we have suggested previously from in vivo experiments. The blockade induced by native NTX was poorly affected by changes in membrane potential; however, the blockage induced by synthetic peptides lacking the C-terminal region was partially released by depolarization.This study was supported by grant HL-45880 from the National Institutes of Health, and by grant 900946 from the American Heart Association to D.L.K. and Howard Hughes Medical Institute No. 75191-527104, CONACyT-Mexico No. 0018-N9105, and DGAPA-UNAM No. IN 202689 to L.D.P. This work was partially supported by a Grant-in-aid No. 92014250 from the American Heart Association to L.V.     

Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

大鼠全脑缺血后海马CA1区锥体神经元L型钙通道电流的改变(英)

已有研究表明在脑缺血期间及再灌流后早期,海马CA1锥体神经元细胞内钙浓度明显升高,这一钙超载被认为是缺血性脑损伤的重要机制之一.电压依赖性钙通道是介导正常CA1神经元钙内流的主要途径.实验观察了脑缺血再灌流后早期海马CA1锥体神经元电压依赖性L型钙通道的变化.以改良的四血管闭塞法制作大鼠15 min前脑缺血模型,在急性分离的海马CA1神经元上,采用膜片钳细胞贴附式记录L型电压依赖性钙通道电流.脑缺血后CA1神经元L型钙通道的总体平均电流明显增大,这是由于通道的开放概率增加所致.进一步分析单通道动力学显示,脑缺血后通道的开放时间变长,通道的开放频率增大.研究结果提示L型钙通道功能活动增强可能参与了缺血后海马CA1锥体神经元的细胞内钙浓度升高.     

Hysteresis in the voltage dependence of HCN channels: conversion between two modes affects pacemaker properties

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are important for rhythmic activity in the brain and in the heart. In this study, using ionic and gating current measurements, we show that cloned spHCN channels undergo a hysteresis in their voltage dependence during normal gating. For example, both the gating charge versus voltage curve, Q(V), and the conductance versus voltage curve, G(V), are shifted by about +60 mV when measured from a hyperpolarized holding potential compared with a depolarized holding potential. In addition, the kinetics of the tail current and the activation current change in parallel to the voltage shifts of the Q(V) and G(V) curves. Mammalian HCN1 channels display similar effects in their ionic currents, suggesting that the mammalian HCN channels also undergo voltage hysteresis. We propose a model in which HCN channels transit between two modes. The voltage dependence in the two modes is shifted relative to each other, and the occupancy of the two modes depends on the previous activation of the channel. The shifts in the voltage dependence are fast (tau approximately 100 ms) and are not accompanied by any apparent inactivation. In HCN1 channels, the shift in voltage dependence is slower in a 100 mM K extracellular solution compared with a 1 mM K solution. Based on these findings, we suggest that molecular conformations similar to slow (C-type) inactivation of K channels underlie voltage hysteresis in HCN channels. The voltage hysteresis results in HCN channels displaying different voltage dependences during different phases in the pacemaker cycle. Computer simulations suggest that voltage hysteresis in HCN channels decreases the risk of arrhythmia in pacemaker cells.     

大鼠全脑缺血后海马CA1区锥体神经元L型钙通道电流的改变(英文)

已有研究表明在脑缺血期间及再灌流后早期,海马CA1锥体神经元细胞内钙浓度明显升高,这一钙超载被认为是缺血性脑损伤的重要机制之一.电压依赖性钙通道是介导正常CA1神经元钙内流的主要途径.实验观察了脑缺血再灌流后早期海马CA1锥体神经元电压依赖性L型钙通道的变化.以改良的四血管闭塞法制作大鼠 15min前脑缺血模型,在急性分离的海马CA1神经元上,采用膜片钳细胞贴附式记录L型电压依赖性钙通道电流.脑缺血后CA1神经元L型钙通道的总体平均电流明显增大,这是由于通道的开放概率增加所致.进一步分析单通道动力学显示,脑缺血后通道的开放时间变长,通道的开放频率增大.研究结果提示L型钙通道功能活动增强可能参与了缺血后海马CA1锥体神经元的细胞内钙浓度升高     

GAT1 (GABA:Na+:Cl-) cotransport function. Steady state studies in giant Xenopus oocyte membrane patches.

Neurotransmitter transporters are reported to mediate transmembrane ion movements that are poorly coupled to neurotransmitter transport and to exhibit complex "channel-like" behaviors that challenge the classical "alternating access" transport model. To test alternative models, and to develop an improved model for the Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporter, GAT1, we expressed GAT1 in Xenopus oocytes and analyzed its function in detail in giant membrane patches. We detected no Na+- or Cl--dependent currents in the absence of GABA, nor did we detect activating effects of substrates added to the trans side. Outward GAT1 current ("reverse" transport mode) requires the presence of all three substrates on the cytoplasmic side. Inward GAT1 current ("forward" transport mode) can be partially activated by GABA and Na+ on the extracellular (pipette) side in the nominal absence of Cl-. With all three substrates on both membrane sides, reversal potentials defined with specific GAT1 inhibitors are consistent with the proposed stoichiometry of 1GABA:2Na+:1Cl-. As predicted for the "alternating access" model, addition of a substrate to the trans side (120 mM extracellular Na+) decreases the half-maximal concentration for activation of current by a substrate on the cis side (cytoplasmic GABA). In the presence of extracellular Na+, the half-maximal cytoplasmic GABA concentration is increased by decreasing cytoplasmic Cl-. In the absence of extracellular Na+, half-maximal cytoplasmic substrate concentrations (8 mM Cl-, 2 mM GABA, 60 mM Na+) do not change when cosubstrate concentrations are reduced, with the exception that reducing cytoplasmic Cl- increases the half-maximal cytoplasmic Na+ concentration. The forward GAT1 current (i.e., inward current with all extracellular substrates present) is inhibited monotonically by cytoplasmic Cl- (Ki, 8 mM); cytoplasmic Na+ and cytoplasmic GABA are without effect in the absence of cytoplasmic Cl-. In the absence of extracellular Na+, current-voltage relations for reverse transport current (i.e., outward current with all cytoplasmic substrates present) can be approximated by shallow exponential functions whose slopes are consistent with rate-limiting steps moving 0.15-0.3 equivalent charges. The slopes of current-voltage relations change only little when current is reduced four- to eightfold by lowering each cosubstrate concentration; they increase twofold upon addition of 100 mM Na+ to the extracellular (pipette) side.     

Enhancement of Lanthanum (III) on Sodium Currents in Acutely Isolated Hippocampal CA1 Neurons of Rat

The effects of lanthanum (III) (La³⁺) on voltage-gated sodium channel currents (I _Na) in freshly dissociated rat hippocampal CA1 neurons were studied using the whole-cell patch clamp techniques. La³⁺ reversibly enhanced I _Na in a concentration- and voltage-dependent manner. The 50% enhancement concentration (EC₅₀) of La³⁺ on I _Na was 9.93 μM. In addition, 10 μM La³⁺ shifted the steady state activation curve of I _Na towards positive potential and the steady state inactivation curve towards negative potential without changing the slope factor. These results indicated that La³⁺ could increase the amplitudes of I _Na and change the activation and inactivation courses of I _Na even in very low concentration.     

Pannexin 1 as a driver of inflammation and ischemia–reperfusion injury

Pannexin 1 (Panx1) is a ubiquitously expressed protein forming large conductance channels that are central to many distinct inflammation and injury responses. There is accumulating evidence showing ATP released from Panx1 channels, as well as metabolites, provide effective paracrine and autocrine signaling molecules that regulate different elements of the injury response. As channels with a broad range of permselectivity, Panx1 channels mediate the secretion and uptake of multiple solutes, ranging from calcium to bacterial derived molecules. In this review, we describe how Panx1 functions in response to different pro-inflammatory stimuli, focusing mainly on signaling coordinated by the vasculature. How Panx1 mediates ATP release by injured cells is also discussed. The ability of Panx1 to serve as a central component of many diverse physiologic responses has proven to be critically dependent on the context of expression, post-translational modification, interacting partners, and the mode of stimulation.     

The pharmacological activity of nicotine and nornicotine on nAChRs subtypes: relevance to nicotine dependence and drug discovery

Cigarette smoking and other forms of tobacco use deliver an array of pharmacologically active alkaloids, including nicotine and ultimately various metabolites of these substances. While nornicotine is a significant component in tobacco as well as a minor systemic metabolite of nicotine, nornicotine appears to be N-demethylated locally in the brain where it accumulates at relatively high levels after chronic nicotine administration. We have now examined the effects of nornicotine on specific combinations of neuronal nicotinic acetylcholine receptor (nAChR) subunits expressed in Xenopus oocytes and compared these responses to those evoked by acetylcholine and nicotine. Of the nAChR subtypes studied, we have found that alpha7 receptors are very responsive to nornicotine (EC50 approximately 17 micromol/L I(max) 50%, compared with acetylcholine (ACh)). nAChRs containing the ligand-binding domain of the alpha6 subunits (in the form of an alpha6/alpha3 chimera) are also strongly responsive to nornicotine (EC50 approximately 4 micromol/L I(max) 50%, compared with ACh). Alpha7-type nAChRs have been suggested to be potential therapeutic targets for Alzheimer's disease, schizophrenia and possibly other pathologies. nAChRs containing alpha6 subunits have been suggested to have a role in nicotine-evoked dopamine release. Thus, understanding the actions of nornicotine in the brain may have significance for both emerging therapeutics and the management of nicotine dependence.     

Characterisation of the Role of Calcium in AlF4-Induced Long-Term Potentiation in Area CA1 of Rat Hippocampus

We investigated calcium influx in the long lasting potentiation induced in area CA1 of rat hippocampus by brief bath application of the G-protein activator AlF₄ ^–(NaF/AlCl₃). Brief (10 min) bath application of AlF₄ ^– in standard saline (with 2mM Ca²⁺) consistently induced a long lasting potentiation which was not observed if AlF₄ ^– was bath-applied in nominally calcium free saline. Increasing the potential calcium influx, either by raising extracellular calcium concentration to 3.5 mM or by addition of the voltage operated calcium channel (VOCC) agonist BayK8644. failed to increase the number of slices exhibiting potentiation or the mean level of potentiation. Bath application of AlF₄ ^– in the presence of the VOCC antagonist failed to block the potentiation and AlF₄ ^– readily induced a long lasting potentiation under voltage clamp conditions, strongly suggesting that the calcium influx required for AlF₄-induced potentiation is not through NMDA receptors or VOCC channels. It is suggested that the calcium required may be provided by an ongoing recharging and emptying of IP3 sensitive intracellular Ca²⁺ stores.