Hydrogen currents through aconitine-modified sodium channels in nerve fiber membranes

Ionic currents in nodal membrane treated with aconitine were measured under voltage clamp conditions when nodes were bathed in Na-free solutions. At pH lower than 4.6 inward ionic currents were detected which had kinetics and voltage range of activation analogous to those of aconitine-modified sodium channels at low pH. These currents were blocked by benzocaine (2 mM). Experiments with various concentrations of Ca2+, tris+, TEA+, choline+ ions showed that these ions are essentially impermeable both at normal and acidic pH. It is concluded that the inward currents observed are carried by H+ (or H3O+) ions through aconitine-modified sodium channels. From reversal potential measurements relative permeability (PH/PNa) of sodium channels is estimated to be 1059 +/- 88. The results suggest that the aconitine-modified channel is a rather wide water-filled pore and the rate of H+ passing through the channel is limited by its binding to an acidic group.     

Calcium-activated cationic channel in rat sensory neurons

Ion channels in sensory neurons are molecular sensors that detect external stimuli and transduce them to neuronal signals. Although Ca2+-activated nonselective cation (CAN) channels were found in many cell types, CAN channels in mammalian sensory neurons are not yet identified. In the present study, we describe an ion channel that is activated by intracellular Ca2+ in cultured rat sensory neurons. Half-maximal concentration of Ca2+ in activating the CAN channel was approximately 780 micro m. The current-voltage relationship of this channel was linear with a unit conductance of 28.8 +/- 0.4 pS at -60 mV in symmetrical 140 mm Na+ solution. The CAN channel was permeable to monovalent cations such as Na+, K+, Cs+, and Li+, but poorly permeable to Ca2+. The CAN channel in mammalian sensory neurons was reversibly blocked by intracellular adenine nucleotides, such as ATP, ADP, and AMP. Interestingly, single-channel currents activated by Ca2+ were blocked by fenamates, such as flufenamic acid, a class of nonsteroidal anti-inflammatory drugs. Thus, these results suggest that CAN channels in mammalian sensory neurons would participate in modulating nociceptive neural transmission in response to ever-changing intracellular Ca2+ in the local microenvironment.     

Single inward rectifier channels in horizontal cells

The ion channels responsible for inward rectification in horizontal cells were studied using the patch clamp technique applied to isolated cells from goldfish retina. Inward currents recorded from these cells were identified as due to the opening of inward rectifier channels based on their ion selectivity, channel gating behavior, and the effects of external blocking ions. The single channel conductance was 20 pS in 125 mM external K+. The null current potential shifted with changes in the K+ concentration as expected for a channel permeable to K+, and the channel appeared to have little permeability to Na+. The probability of a channel being in an open state increased as the membrane was hyperpolarized from the K+ equilibrium potential (0 to -10 mV) over potentials ranging to -80 mV, in the presence of external Na+. The closing rate was insensitive to membrane potential in the presence of external Na+. The opening rate of the channel increased as the membrane was hyperpolarized. The increase in the probability of a channel being open at negative potentials was therefore caused by the voltage sensitivity of the rate of channel opening.     

Electroregulated sodium channels of the somatic membrane of sympathetic neurons

Electrically-operated sodium channels in the somatic membrane of isolated neurons from the rat superior cervical ganglion have been studied by means of intracellular dialysis technique under voltage clamp conditions. It was shown that in this preparation sodium currents can be carried by two independent systems of sodium channels. The mathematical analysis of voltage-dependent TTX-sensitive fast sodium currents was performed by the Hodgkin-Huxley formalism; their kinetic properties were compared with those described in other objects. TTX-sensitive sodium channels in the somatic membrane of sympathetic neurons were found to be highly selective for Na+ ions. Kinetic and voltage-dependent characteristics of slow TTX-resistant sodium current were also described. This component of the sodium current was observed only in a few neurons (not more than 2%).     

Cobalt-dependent potentiation of net inward current density in Helix aspersa neurons.

A low concentration of transition metal ions Co2+ and Ni2+ increases the inward current density in neurons from the land snail Helix aspersa. The currents were measured using a single electrode voltage-clamp/internal perfusion method under conditions in which the external Na+ was replaced by Tris+, the predominant external current carrying cation was Ca2+, and the internal perfusate contained 120 mM Cs+/0 K+; 30 mM tetraethylammonium (TEA) was added externally to block K+ current. In the presence of Co2+ (3 mM) or Ni2+ (0.5 mM) inward Ca2+ currents were stimulated normally by voltage-dependent activation of Ca2+ channels. There was a 5-10% decrease in the rate of rise of the inward current. The principal effect of Co2+ and Ni2+ in increasing the current density seems to be a decrease in the rate at which the inward currents decline during a depolarizing voltage pulse. The results may be due to a decrease in a voltage-dependent or Ca(2+)-dependent outward current and/or an inhibition of Ca2+ channel inactivation. Outward current under these conditions (zero internal K+) was significant and most likely due to Cs+ efflux through the voltage-activated or Ca(2+)-activated nonspecific cation channels. Co2+ is an extremely effective blocker of this outward current. These results are not an artifact of internal perfusion or the special ionic conditions. Intracellular recording of unperfused neurons in normal Helix Ringer's solution showed that the Ca(2+)-dependent action potential duration was increased significantly by low concentrations of Co2+. This result is consistant with the Co(2+)-dependent increase in inward (depolarizing) current seen in voltage-clamp experiments.     

Brevetoxin modulates neuronal sodium channels in two cell lines derived from rat brain

Single Na+ channel currents were recorded from cell-attached membrane patches from two neuronal cell lines derived from rat brain, B50 and B104, and compared before and after exposure of the cells to purified brevetoxin, PbTx-3. B50 and B104 Na+ channels usually exhibited fast activation and inactivation as is typical of TTX-sensitive Na+ channels. PbTx-3 modified channel gating in both cell lines. PbTx-3 caused (1) significant increases in the frequency of channel reopening, indicating a slowing of channel inactivation, (2) a change in the voltage dependence of the channels, promoting channel opening during steady-state voltage clamp of the membrane at voltages throughout the activation range of Na+ currents, but notably near the resting potential of these cells (-60 - -50 mV), and (3) a significant, 6.7 mV hyperpolarized shift in the threshold potential for channel opening. Na+ channel slope conductance did not change in PbTx-3-exposed B50 and B104 neurons. These effects of Pbx-3 may cause hyperexcitability as well as inhibitory effects in intact brain.     

Cations differentially affect subpopulations of L-glutamate receptors in rat synaptic plasma membranes

Several cations were examined for their ability to specifically affect one of the 3 L-glutamate (L-Glu) binding sites in rat forebrain synaptic plasma membranes (i.e. Na+-dependent, Cl--dependent and Cl--independent). Na+-dependent binding was potently inhibited by K+ and NH4+ ions. Other monovalent cations tested (Cs+, Li+, triethylammonium) had no effect on this binding site. Polyvalent cations (Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Cr3+) also had little effect on the Na+-dependent L-Glu binding site. Cl--dependent L-Glu binding was potently inhibited by Na+ ions but was not affected by other monovalent ions. All of the divalent cations were potent inhibitors of both Cl--dependent and -independent binding. The results show that these binding sites of L-Glu can be distinguished by their response to cations and suggest possible novel modes of regulation in vivo.     

Changes in ionic conductances induced by cAMP in Helix neurons

Adenosine 3',5'-cyclic monophosphate (cAMP) was injected by a fast and quantitative pressure injection method into voltage-clamped identified Helix neurons. The intracellular elevation of cAMP caused an inward current which was not accompanied by a significant change in membrane conductance in a negative potential range with little activation of voltage-dependent membrane conductances. Near resting potential Na+ ions were the main carrier of the cAMP-induced inward current as measured with ion-selective microelectrodes. TTX did not affect the Na+ influx. K+ and less effective Ca2+ could substitute for Na+ in carrying the inward current. In the presence of Na+, divalent cations such as Ca2+ and Mg2+, and also La3+ exerted an inhibitory influence on the cAMP-induced inward current, and Ca2+ as measured with ion-selective microelectrodes did not contribute significantly to the current. Thus, the inward current was of a non-specific nature. Simultaneously to this cAMP action, the membrane permeability for K+ ions was decreased by cAMP. This effect became particularly obvious when K+ currents were activated by long-lasting, depolarizing voltage steps. In this situation a reduced K+ efflux following cAMP injection was observed by means of K+-selective microelectrodes located near the external membrane surface. Outward K+ currents were less reduced by cAMP if external Ca2+ was replaced by Ni2+. The nearly compensatory increase and decrease of two membrane conductances in the same neuron explained the lack of change in the cell input resistance despite the considerable depolarizing action of intracellularly elevated cAMP.     

Effects of ApC, a sea anemone toxin, on sodium currents of mammalian neurons

We have characterized the actions of ApC, a sea anemone polypeptide toxin isolated from Anthopleura elegantissima, on neuronal sodium currents (I(Na)) using current and voltage-clamp techniques. Neurons of the dorsal root ganglia of Wistar rats (P5-9) in primary culture were used for this study. These cells express tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) I(Na). In current-clamp experiments, application of ApC increased the average duration of the action potential. Under voltage-clamp conditions, the main effect of ApC was a concentration-dependent increase in the TTX-S I(Na) inactivation time course. No significant effects were observed on the activation time course or on the current peak-amplitude. ApC also produced a hyperpolarizing shift in the voltage at which 50% of the channels are inactivated and caused a significant decrease in the voltage dependence of Na+ channel inactivation. No effects were observed on TTX-R I(Na). Our results suggest that ApC slows the conformational changes required for fast inactivation of the mammalian Na+ channels in a form similar to other site-3 toxins, although with a greater potency than ATX-II, a highly homologous anemone toxin.     

Inhibition of serotonin uptake into mouse brain synaptosomes by ionophores and ion-channel agents.

[3H]Serotonin uptake into mouse cerebrocortical synaptosomes was decreased by the K+ ionophore valinomycin, the K+ and Na+ ionophore gramicidin, and the proton ionophore carbonylcyanide m-chlorophenylhydrazone. The Na+/H+ exchanger monensin reduced uptake at non-depolarizing concentrations. Uptake was also decreased by inhibition of the Na+, K(+)-ATPase with ouabain and by tetrodotoxin-sensitive activation of voltage-dependent sodium channels with veratridine, batrachotoxin and scorpion venom. In contrast, the Ca2+ channel agents BAY K8644 and nimodipine were ineffective. The effect of reducing the Na+ gradient depended upon whether the internal Na+ concentration was raised (i.e. by scorpion venom, monensin) or the external Na+ concentration was lowered (37 mM NaCl in the medium).     

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.     

Steady growth cone currents revealed by a novel circularly vibrating probe: a possible mechanism underlying neurite growth

The rate and direction of neurite growth have been shown in a number of studies to be determined by the distribution of adhesive sites on the growth cone. Recent evidence showing that the application of extrinsic electric fields can redistribute membrane molecules and alter both the rate and direction of neurite growth have raised the question whether endogenous electric fields might be produced by steady currents in growth cones. To investigate this question, we have devised a novel circularly vibrating microprobe capable of measuring current densities in the range of 5 nA/cm2 (near the theorectical limit of sensitivity), with a spatial resolution of 2 micron. The design of this device and the development of a novel algorithm for computing current vectors on-line is described. Using this probe we have found that cultured goldfish retinal ganglion cell growth cones generate steady inward currents at their tips. The measured currents, in the range of 10-100 nA/cm2, appear to flow into the filopodia at their tips and back outward near the junctures of the filopodia and the growth cone. The currents appear to be produced only during active growth. Ion substitution experiments support the conclusion that the majority of this current is carried by Ca2+ ions, which we postulate flow through a population of activated voltage-sensitive Ca2+ channels located on the filopodial tips. Calculation of the transmembrane current density (4 X 10(-6) nA/cm2) leads to an estimate of channel density (10 channels/micron2) in close agreement with the measured density of Ca2+ channels in other systems. The assumption that calcium channel proteins are conveyed to nerve terminals by active transport, whereas sodium channel proteins are conveyed passively by a slower somatofugal diffusion process [Strichartz et al, 1984], would explain why developing neurons tend to display Ca2+-sensitive electrogenesis at their growing tips, and Na+-sensitive action potentials later in development. In order to gain some insight into the possible role of these steady growth currents, we estimated the membrane depolarization and axial voltage gradient they produce. It is likely that the currents produce sufficient membrane depolarization (approximately equal to 4 mV) to cause autogenous activation of ion channel permeabilities. Similarly, the axial voltage gradient (approximately equal to 4 mV/cm) would be expected to move intracytoplasmic vesicles by electrophoresis at a rate (20-40 microns/hr) very close to that at which the filopodia are observed to grow.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ethanol inhibits voltage-gated sodium channels in cultured superior cervical ganglion neurons

To determine whether actions on sodium channels contribute to ethanol's depressant effects on the autonomic nervous system, the acute effects of ethanol on Na+ currents in primary cultured superior cervical ganglion were examined by whole-cell patch clamp recordings. Ethanol inhibited Na+ currents concentration dependently, and decreased action potential firing. Ethanol (100 mM) did not affect activation curve, but resulted in a left shift of the inactivation curve and prolonged the recovery from inactivation. This finding indicates that the channels in the inactivated state are more susceptible to ethanol than those in the resting state. For the first time, this study demonstrates acute inhibitory effects of ethanol on sodium channel gating in sympathetic neurons.     

Hydrogen ion block of single sodium channels in neuroblastoma cells

The effect of external pH on the amplitude of currents through single sodium channels in cultured mouse neuroblastoma cells C 1300, clone N18A-1 was studied. Currents through single sodium channels in outside-out membrane patches were measured at normal (7.2) and low (5.4) pH of the external solution. With a decrease of the external pH to 5.4, about two-fold reversible reduction of the amplitude of single sodium channel currents (at testing potentials of -10-30 mV) was observed. The data obtained confirm the suggestion that the inhibition of macroscopic sodium currents with lowering of pH of the extracellular solution is due to the decrease in the ionic current flowing through single open channels.     

Nav 1.1 dysfunction in genetic epilepsy with febrile seizures-plus or Dravet syndrome

Relatively few SCN1A mutations associated with genetic epilepsy with febrile seizures-plus (GEFS+) and Dravet syndrome (DS) have been functionally characterized. In contrast to GEFS+, many mutations detected in DS patients are predicted to have complete loss of function. However, functional consequences are not immediately apparent for DS missense mutations. Therefore, we performed a biophysical analysis of three SCN1A missense mutations (R865G, R946C and R946H) we detected in six patients with DS. Furthermore, we compared the functionality of the R865G DS mutation with that of a R859H mutation detected in a GEFS+ patient; the two mutations reside in the same voltage sensor domain of Na(v) 1.1. The four mutations were co-expressed with β1 and β2 subunits in tsA201 cells, and characterized using the whole-cell patch clamp technique. The two DS mutations, R946C and R946H, were nonfunctional. However, the novel voltage sensor mutants R859H (GEFS+) and R865G (DS) produced sodium current densities similar to those in wild-type channels. Both mutants had negative shifts in the voltage dependence of activation, slower recovery from inactivation, and increased persistent current. Only the GEFS+ mutant exhibited a loss of function in voltage-dependent channel availability. Our results suggest that the R859H mutation causes GEFS+ by a mixture of biophysical defects in Na(v) 1.1 gating. Interestingly, while loss of Na(v) 1.1 function is common in DS, the R865G mutation may cause DS by overall gain-of-function defects.     

Depolarization of nonmyelinated fibers of the rat vagus nerve produced by activation of protein kinase C

The effect of activation of protein kinase C by phorbol esters has been studied on the nonmyelinated (C) fibers of the rat vagus nerve. Grease-gap recording at room temperature was used to monitor changes in the resting and action potentials. Effects of phorbol esters on the rate of efflux of 86Rb and 14C-guanidinium were also measured. The active isomer beta-phorbol 12,13-dibutyrate (PDBu), applied for 10 min at concentrations of 10 nM to 3 microM, caused a slowly developing depolarization, which persisted after the drug was washed out. The action potential was concomitantly reduced. These effects did not occur with the inactive isomer alpha-phorbol 12,13-didecanoate. The PDBu-induced depolarization was reduced by about 75% if Na+ was replaced by the impermeant cation N-methyl-(+)-glucamine (NMG); the residual effect was almost abolished if the nerves were presoaked in a solution containing gluconate in place of Cl-. It was concluded that increases in conductance mainly to Na+ and Cl- were responsible for the depolarization. The response was unaffected by tetrodotoxin or calcium-channel blockers. Omission of Ca2+, surprisingly, enhanced the PDBu-induced depolarization 3-5-fold; furthermore, addition of 2 mM Ca2+ following a PDBu-induced depolarization recorded in Ca2+-free solution caused a pronounced repolarization. This effect of Ca2+ occurred also with Sr2+ and Ba2+, but not with other divalent cations or with La3+. Divalent cations known to block Ca channels inhibited the repolarizing action of Ca2+. These results suggested that Ca2+ acts intracellularly, either to block Na channels opened by PDBu or to activate protein phosphatases. The PDBu-induced response in Ca2+-free solution was increased 2-fold by a reduction in pH from 7.4 to 6.5. Under normal conditions the nerve was reversibly depolarized by this pH change; after PDBu this pH sensitivity was enhanced, and depolarization occurred at a less acidic pH. PDBu caused a 3-4-fold increase in the rate of efflux of 86Rb (a marker for K+ ions) and of 14C-guanidinium (a marker for Na+ ions) from preloaded nerves. These effects, in contrast to the depolarization, were transient.(ABSTRACT TRUNCATED AT 400 WORDS)     

Divalent metals differentially block cloned T-type calcium channels

We tested divalent metals including Cu2+, Pb2+, and Zn2+ to determine their pharmacological profiles for blockade of cloned T-type Ca2+ channels (alpha1G, alpha1 H, and alpha1I). Effects of the metals were also evaluated for native low and high voltage-activated Ca2+ channels in rat sympathetic pelvic neurons. Cu2+ and Zn2+ blocked three T-type channel isoforms in a concentration-dependent manner with a higher affinity for alpha1H currents (IC50 = 0.9 microM and 2.3 microM). In pelvic neurons, only Zn2+ showed strong selectivity for T-type Ca2+ currents over high voltage-activated Ca2+ currents. Conversely, Pb2+ block on Ca2+ channels did not show distinctive selectivity. Taken together, these results suggest that besides Ni2+, Cu2+ and Zn2+ can be used as selective blockers of alpha1 H at low concentrations.     

快速老化小鼠海马神经元电压门控离子通道特点

目的：观察快速老化小鼠（Senescence-accelerated mouse,SAM)海马神经元的基本离子通道特点，并对抗快速老化亚系（SAM－resistance/1,SAMR1)与快速老化亚系（SAM－prone/8,SMAP8)的基本离子通道特点进行了比较，探讨了离子通道变化在衰老中的可能角度，方法：应用全细胞记录方式，观察并比较原代培养SAMR1和SAMP8海马神经元的电压门控离子通道及膜参数。结果：原代培养SAMR1和SAMP8海马神经元电压门控Na^2 通道电流（INa)和电压门控延迟整流K^ 通道电流（IK）的电学特点和幅度基本一致。SAMP8的电压门控Ca^2 通道电流（ICa)和瞬时外向K^ 通道电流（IA）的幅值则大于相同培养天数的SAMR1。经膜电容校正所得的ICa电流密度也表现出增大的变化规律。结论：SAMP8与SAMR1神经元间IA和ICa的差异可能与其神经系统变异而产生的学习记忆功能下降有关。     

Effects of divalent cations on the frequency of spontaneous action potentials from the lateral line organ of Xenopus laevis

The effect of superfusion of the internal surface of the skin of Xenopus laevis with saline containing Co2+, Ca2+, Mg2+, or Ba2+, on the frequency of spontaneous action potentials of the lateral line nerve, was studied to investigate the role of extracellular Ca2+ in spontaneous neural activity. Addition of divalent cations to frog saline, either singly or as a mixture of two different ions, produced concentration-dependent suppression of spontaneous rate. The rank order of potency for suppression by each ion, perfused alone, was Co2+ greater than Ca2+ greater than Mg2+ greater than Ba2+. Suppression by combinations of Mg2+ and Co2+, or of Ca2+ and Co2+, was approximated by the sum of the suppressive effects of each cation. Ca2+ was more suppressive than Mg2+ when each of these ions was paired with the same amount of Co2+, while Ca2+ was approximately as suppressive as Co2+ when similarly paired with Mg2+. One interpretation of the suppression by Ca2+ invokes the hypothesis that divalent cations suppress spontaneous activity by charge screening of voltage-sensitive Na+ channels on afferent dendrites and that release of neurotransmitter by the influx of extracellular Ca2+ through voltage-sensitive Ca2+ channels of hair cells may not be the sole mechanism for generation of spontaneous activity in the lateral line. These results quantify the relative suppressive potency of common divalent cations in the lateral line, and serve as a caveat to investigators who interpret a blockade of action potentials by high concentrations of Co2+ or Mg2+ as sufficient evidence for dependence of neurotransmission upon extracellular Ca2+, particularly in acousticolateralis systems.     

Interaction of forskolin with voltage-gated K+ channels in PC12 cells

Forskolin (FSK) directly blocks a distinct class of voltage-dependent K+ channels in pheochromocytoma cells. We have studied the biophysical mechanism of FSK action on these channels. The mean open duration decreased linearly with [FSK], indicating that a single molecule of FSK interacts with a single open K+ channel. FSK did not alter the voltage dependence of activation or the latency to first opening. Whole-cell currents in the presence of FSK did not show a rising phase in tail currents, suggesting that FSK-bound channels can close. We used a kinetic scheme in which FSK binds preferentially to the open state of the channel to describe its interaction with the K+ channel. This scheme is analogous to the modulated receptor hypothesis used to describe the interaction of local anesthetics with voltage-dependent Na+ channels.