Voltage Clamp

Abstruct-Multilevel voltage source converters are emerging as a new breed of power converter options for high-power applications. The multilevel voltage source converters typically synthesize the staircase voltage wave from several levels of dc capacitor voltages. One of the major limitations of the multilevel converters is the voltage unbalance between different levels. The techniques to balance the voltage between different levels normally involve voltage clamping or capacitor charge control. There are several ways of implementing voltage balance in multilevel converters. Without considering the traditional magnetic coupled converters, this paper presents three recently developed multilevel voltage source converters: 1) diode-clamp, 2) flyingcapacitors, and 3) cascaded-inverters with separate dc sources. The operating principle, features, constraints, and potential applications of these converters will be discussed.

We describe methods for obtaining stable, whole-cell recordings from neurons in brain hermspheres from turtles and m brain shces from rats and turtles Synaptlc currents and membrane properties of central neurons can be stud~ed m voltage and current clamp m cells maintained wlttun their endogenous synapnc circuits. The methods described here are compatible wtth unmodified dissecting microscopes and recording chambers, and with brain shces of standard thickness (400-500 ~tm).

Andersen's syndrome is characterized by periodic paralysis, cardiac arrhythmias, and dysmorphic features. We have mapped an Andersen's locus to chromosome tified in unrelated patients. Expression of two of these mutations in Xenopus oocytes revealed loss of funcdevelopmental signaling in addition to its previously recognized function in controlling cell excitability in Salt Lake City, Utah 84112 5 Neuromuscular Pathology skeletal muscle and heart.

Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human excitatory amino acid transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in N-methyl-D-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel clustering. Functional properties of EAAT5 were examined in the Xenopus oocyte expression system by measuring radiolabeled glutamate flux and two-electrode voltage clamp recording. EAAT5-mediated L-glutamate uptake is sodium- and voltage-dependent and chloride-independent. Transporter currents elicited by glutamate are also sodium- and voltage-dependent, but ion substitution experiments suggest that this current is largely carried by chloride ions. These properties of EAAT5 are similar to the glutamate-elicited chloride conductances previously described in retinal neurons, suggesting that the EAAT5-associated chloride conductance may participate in visual processing.

Ca 2ϩ transients measured in failing human ventricular myocytes exhibit reduced amplitude, slowed relaxation, and blunted frequency dependence. In the companion article (O'Rourke B, Kass DA, Tomaselli GF, Kääb S, Tunin R, Marbán E. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart, I: experimental studies. Circ Res. 1999;84:562-570), O'Rourke et al show that Ca 2ϩ transients recorded in myocytes isolated from canine hearts subjected to the tachycardia pacing protocol exhibit similar responses. Analyses of protein levels in these failing hearts reveal that both SR Ca 2ϩ ATPase and phospholamban are decreased on average by 28% and that Na ϩ /Ca 2ϩ exchanger (NCX) protein is increased on average by 104%. In this article, we present a model of the canine midmyocardial ventricular action potential and Ca 2ϩ transient. The model is used to estimate the degree of functional upregulation and downregulation of NCX and SR Ca 2ϩ ATPase in heart failure using data obtained from 2 different experimental protocols. Model estimates of average SR Ca 2ϩ ATPase functional downregulation obtained using these experimental protocols are 49% and 62%. Model estimates of average NCX functional upregulation range are 38% and 75%. Simulation of voltage-clamp Ca 2ϩ transients indicates that such changes are sufficient to account for the reduced amplitude, altered shape, and slowed relaxation of Ca 2ϩ transients in the failing canine heart. Model analyses also suggest that altered expression of Ca 2ϩ handling proteins plays a significant role in prolongation of action potential duration in failing canine myocytes. (Circ Res. 1999;84:571-586.)

AbstractöTo investigate the basis of the £uctuating activity present in neocortical neurons in vivo, we have combined computational models with whole-cell recordings using the dynamic-clamp technique. A simpli¢ed`point-conductance' model was used to represent the currents generated by thousands of stochastically releasing synapses. Synaptic activity was represented by two independent fast glutamatergic and GABAergic conductances described by stochastic randomwalk processes. An advantage of this approach is that all the model parameters can be determined from voltage-clamp experiments. We show that the point-conductance model captures the amplitude and spectral characteristics of the synaptic conductances during background activity. To determine if it can recreate in vivo-like activity, we injected this point-conductance model into a single-compartment model, or in rat prefrontal cortical neurons in vitro using dynamic clamp. This procedure successfully recreated several properties of neurons intracellularly recorded in vivo, such as a depolarized membrane potential, the presence of high-amplitude membrane potential £uctuations, a low-input resistance and irregular spontaneous ¢ring activity. In addition, the point-conductance model could simulate the enhancement of responsiveness due to background activity.

A Ca2+-channel blocker derived from funnelweb spider toxin (FIX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na'-dependent plateau potential. In the squid giant synapse, FIX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2l current and of transmitter release. FIX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10-to 12-pS conductance in 80 mM Ba2l and 5-8 pS in 100 mM Ca2+ with voltagedependent open probabilities and kinetics. High Ba2l concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic

Cell surface receptors for gibbon ape leukemia virus (Glvr-1) and marine amphotropic retrovirus (Ram-i) are distinct but related proteins having multiple membranenning regions. Distant homology with a putative phosphate permease of Neurospora crussa suggested that these receptors might serve transport functions. By expression in Xenopus laevis oocytes and in mam l cells, we have identified Glvr-1 and Ram-i as sodium-dependent phosphate symporters. Two-electrode voltage-camp analysis indicates net cation influx, gestng that phosphate is transported with excess sodium ions. Phosphate uptake was reduced by >50% in mouse fibroblasts expressing amphotropic envelope glycoprotein, which binds to Ram-i, indicating that Ram-i Is a major phosphate transporter in these cells. RNA analysis shows wide but distinct tissue distributions, with Glvr-1 expression being highest in bone marrow and Ram-i in heart. Overexpression of Ram-i severely reprsd Glvr-i synthesis in fibroblasts, suggesting that transporter expression may be controlled by net phosphate accumulation. Accordingly, depletion of extracellular phosphate increased Ram-i and Glvr-i expression 3to 5-fold. These results suggest simple methods to modulate retroviral receptor expression, with possible applications to human gene therapy. Key issues in virology have been identification of cell-surface virus receptors, determination of receptor expression patterns, and elucidation ofthe effects of infection on the normal functions of these molecules. These issues are critical in the case of retroviruses because cells can become chronically infected, leading to receptor down-modulation and subsequent derangements in cellular functions. For example, the CD4 receptor for human immunodeficiency virus (HIV) is important for helper T-cell function, and interference with this normal function is an important aspect of HIV-induced disease (1). Indeed, three genes of HIV (env, nef, and vpu) can each mediate CD4 down-modulation (1-4). While several retroviral receptor cDNAs have been isolated (5-11), the only functionally characterized receptors are the above-mentioned CD4 for HIV and a sodium-independent cationic amino acid transporter for ecotropic murine leukemia viruses (MLVs) (12, 13). Among the cloned receptors with unknown functions are those that mediate infections of gibbon ape leukemia virus (GALV) and of mouse amphotropic retrovirus (these receptors are termed Glvr-1 and Ram-1, respectively) (7, 10, 11). Glvr-1 and Ram-1 are distinct proteins with multiple hydrophobic potential membrane-spanning sequences. Although they contain large un

In response to membrane depolarization, voltage-gated ion channels undergo a structural rearrangement that moves charges or dipoles in the membrane electric field and opens the channel-conducting pathway. By combination of site-specific fluorescent labeling of the Shaker potassium channel protein with voltage clamping, this gating conformational change was measured in real time. During channel activation, a stretch of at least seven amino acids of the putative transmembrane segment S4 moved from a buried position into the extracellular environment. This movement correlated with the displacement of the gating charge, providing physical evidence in support of the hypothesis that S4 is the voltage sensor of voltage-gated ion channels.

We have developed a mathematical model of the human atrial myocyte based on averaged voltage-clamp data recorded from isolated single myocytes. Our model consists of a Hodgkin-Huxley-type equivalent circuit for the sarcolemma, coupled with a fluid compartment model, which accounts for changes in ionic concentrations in the cytoplasm as well as in the sarcoplasmic reticulum. This formulation can reconstruct action

In rat neostriatal neurons, D1 dopamine receptors regulate the activity of cyclic AMP-dependent protein kinase (PKA) and protein phosphatase 1 (PP1). The influence of these signaling elements on high voltageactivated (HVA) calcium currents was studied using whole-cell voltage-clamp techniques. The application of D1 agonists or cyclic AMP analogs reversibly reduced N-and P-type Ca 2+ currents. Inhibition of PKA antagonized this modulation, as did inhibition of PP1, suggesting that the D~ effect was mediated by a PKA enhancement of PP1 activity directed toward Ca 2+ channels. In a subset of neurons, D~ receptor-mediated activation of PKA enhanced L-type currents. The differential regulation of HVA currents by the D1 pathway helps to explain the diversity of effects this pathway has on synaptic integration and plasticity in medium spiny neurons.

1. End-plate currents have been studied in glycerol-treated frog sartorius nerve-muscle preparations with the voltage clamp technique. 2. The effects of temperature on the decay rate of end-plate currents were investigated over a temperature range from 10 to 30.50 C. The Q10 for the decay constant of end-plate currents depends somewhat on membrane potential; at-100 mV the decay constant has a Q10 of 2-7. 3. Peak end-plate current depends non-linearly on membrane potential with a decreasing slope conductance associated with hyperpolarization. 4. The 'instantaneous' voltage-current relationship for end-plate channels was determined by causing step changes in membrane potential during end-plate current flow. This relationship appears to be linear. 5. The interaction of acetylcholine with its receptor is viewed as being analogous to the first step in enzymic catalysis. On this view, acetylcholine binds to its receptor and induces a conformational change which is responsible for opening end-plate channels. By analogy to the first steps in the catalytic sequence of enzymes, the binding step is very rapid, almost diffusion-limited, and the conformational change is rate-limiting. 6. Equations describing this process have been derived. Expressions for the rate constants have also been derived by considering changing dipole moments of the transmitter-receptor complex associated with the conformational change. As the transmitter-receptor complex is in the membrane field, different conformational states have different energies, and the rate of conformational change thus depends on membrane potential. The equations thus derived are shown to account adequately for the time course of end-plate conductance change.

A slow muscarinic EPSP, accompanied by an increase in membrane input resistance, can be elicited in hippocampal CA1 pyramidal cells in vitro by electrical stimulation of cholinergic afferents in the slice preparation. Associated with the slow EPSP is a blockade of calcium-activated potassium afterhyperpolarizations (AHPs) (Cole and Nicoll, 1984a). In this study a single-electrode voltage clamp was used to examine the currents affected by activation of muscarinic receptors, using either bath application of carbachol or electrical stimulation of the cholinergic afferents. The 3 main findings of this study are that (1) of the 2 calcium-activated potassium currents (termed IAHP and IC) in hippocampal pyramidal cells, only IAHP is sensitive to carbachol; (2) IAHP is approximately 10-fold more sensitive to carbachol than is another muscarine-sensitive current, IM; and (3) neither blockade of IAHP nor of IM can account for the production of the slow EPSP. Rather, the slow EPSP appears to b...

We analyzed the synaptic physiology of unc-13 mutants in the nematode C. elegans. Mutants of unc-13 had normal nervous system architecture, and the densities of synapses and postsynaptic receptors were normal at the neuromuscular junction. However, the number of synaptic vesicles at neuromuscular junctions was two- to threefold greater in unc-13 mutants than in wild-type animals. Most importantly, evoked release at both GABAergic and cholinergic synapses was almost absent in unc-13 null alleles, as determined by whole-cell, voltage-clamp techniques. Although mutant synapses had morphologically docked vesicles, these vesicles were not competent for release as assayed by spontaneous release in calcium-free solution or by the application of hyperosmotic saline. These experiments support models in which UNC-13 mediates either fusion of vesicles during exocytosis or priming of vesicles for fusion.

We have studied the effects of the proteolytic enzyme Pronase on the membrane currents of voltage-clamped squid axons. Internal perfusion of the axons with Pronase rather selectively destroys inactivation of the Na conductance (g.).-At the level of a single channel, Pronase probably acts in an allor-none manner: each channel inactivates normally until its inactivation gate is destroyed, and then it no longer inactivates. Pronase reduces gN., possibly by destroying some of the channels, but after removal of its inactivation gate a Na channel seems no longer vulnerable to Pronase. The turn-off kinetics and the voltage dependence of the Na channel activation gates are not affected by Pronase, and it is probable that the enzyme does not affect these gates in any way. Neither the K channels nor their activation gates are affected in a specific way by Pronase. Tetrodotoxin does not protect the inactivation gates from Pronase, nor does maintained inactivation of the Na channels during exposure to Pronase. Our results suggest that the inactivation gate is a readily accessible protein attached to the inner end of each Na channel. It is shown clearly that activation and inactivation of Na channels are separable processes, and that Na channels are distinct from K channels.

Varenicline, a new nicotinic ligand based on the structure of cytisine, has recently been approved by the U.S. Food and Drug Administration for use as a smoking cessation aid. Varenicline has been shown to be a partial agonist of ␣4␤2 receptors, and in equilibrium binding assays, it is highly selective for the ␣4␤2 receptor. In this study, we have examined the functional activity of varenicline at a variety of rat neuronal nicotinic receptors expressed in Xenopus laevis oocytes and assayed under two-electrode voltage clamp. We also find that varenicline is a potent, partial agonist at ␣4␤2 receptors, with an EC 50 of 2.3 Ϯ 0.3 M and an efficacy (relative to acetylcholine) of ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine.

Two novel mutations (R85C and R85H) on the extracellular immunoglobulin-like domain of the sodium channel ␤1 subunit have been identified in individuals from two families with generalized epilepsy with febrile seizures plus (GEFS؉). The functional consequences of these two mutations were determined by co-expression of the human brain NaV1.2 ␣ subunit with wild type or mutant ␤1 subunits in human embryonic kidney (HEK)-293T cells. Patch clamp studies confirmed the regulatory role of ␤1 in that relative to NaV1.2 alone the NaV1.2؉␤1 currents had right-shifted voltage dependence of activation, fast and slow inactivation and reduced use dependence. In addition, the NaV1.2؉␤1 current entered fast inactivation slightly faster than NaV1.2 channels alone. The ␤1(R85C) subunit appears to be a complete loss of function in that none of the modulating effects of the wild type ␤1 were observed when it was co-expressed with NaV1.2. Interestingly, the ␤1(R85H) subunit also failed to modulate fast kinetics, however, it shifted the voltage dependence of steady state slow inactivation in the same way as the wild type ␤1 subunit. Immunohistochemical studies revealed cell surface expression of the wild type ␤1 subunit and undetectable levels of cell surface expression for both mutants. The functional studies suggest association of the ␤1(R85H) subunit with the ␣ subunit where its influence is limited to modulating steady state slow inactivation. In sum-mary, the mutant ␤1 subunits essentially fail to modulate ␣ subunits which could increase neuronal excitability and underlie GEFS؉ pathogenesis. (S. Petrou). Abbreviations: EGFP, enhanced green fluorescent protein; GEFSϩ, generalized epilepsy with febrile seizures plus; HEK, human embryonic kidney; PBS, phosphate-buffered saline; SCN1A, sodium channel ␣1 subunit; SCN1B, sodium channel ␤1 subunit; WT, wild type.

It is often assumed that all neurons of the same cell type have identical intrinsic properties, both within an animal and between animals. We exploited the large size and small number of unambiguously identifiable neurons in the crab stomatogastric ganglion to test this assumption at the level of channel mRNA expression and membrane currents (measured in voltage-clamp experiments). In lateral pyloric (LP) neurons, we saw strong correlations between measured current and the abundance of Shal and BK-KCa mRNAs (encoding the Shal-family voltage-gated potassium channel and large-conductance calcium-activated potassium channel, respectively). We also saw two-to fourfold interanimal variability for three potassium currents and their mRNA expression. Measurements of channel expression in the two electrically coupled pyloric dilator (PD) neurons showed significant interanimal variability, but copy numbers for IH (encoding the hyperpolarization-activated, inward-current channel) and Shal mRNA in the two PD neurons from the same crab were similar, suggesting that the regulation of some currents may be shared in electrically coupled neurons.

In several neuronal types of the CNS, glutamate and GABA receptors mediate a persistent current which reflects the presence of a low concentration of transmitters in the extracellular space. Here, we further characterize the tonic current mediated by ambient glutamate in rat hippocampal slices. A tonic current of small amplitude (53.99 ± 6.48 pA at +40 mV) with the voltage dependency and the pharmacology of NMDA receptors (NMDARs) was detected in virtually all pyramidal cells of the CA1 and subiculum areas. Manipulations aiming at increasing D-serine or glycine extracellular concentrations failed to modify this current indicating that the glycine binding sites of the NMDARs mediating the tonic current were saturated. In contrast, non-transportable inhibitors of glutamate transporters increased the amplitude of this tonic current, indicating that the extracellular concentration of glutamate primarily regulates its magnitude. Neither AMPA/kainate receptors nor metabotropic glutamate receptors contributed significantly to this tonic excitation of pyramidal neurons. In the presence of glutamate transporter inhibitors, however, a significant proportion of the tonic conductance was mediated by AMPA receptors. The tonic current was unaffected when inhibiting vesicular release of transmitters from neurons but was increased upon inhibition of the enzyme converting glutamate in glutamine in glial cells. These observations indicate that ambient glutamate is mainly of glial origin. Finally, experiments with the use-dependent antagonist MK801 indicated that NMDARs mediating the tonic conductance are probably extra-synaptic NMDARs.

Antidepressant- and cocaine-sensitive serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) dictate clearance of extracellular 5-HT after release. To explore protein kinase C-mediated SERT regulation, we generated a stable human SERT (hSERT)-expressing cell line (293-hSERT) and evaluated modulation of 5-HT activity via studies of 5-HT flux, hSERT-mediated currents under voltage clamp, and surface distribution of SERT protein. 293-hSERT cells exhibit saturable, high-affinity, and antidepressant-sensitive 5-HT uptake as well as hSERT-dependent whole-cell currents. In these cells, the protein kinase C activator beta-PMA caused a time-dependent reduction in 5-HT uptake capacity (Vmax) after acute application and a reduction in SERT-mediated currents. Effects of beta-PMA were mimicked by the phorbol ester beta-PDBu, were not observed with the inactive alpha-isomers, and could be blocked by treatment of cells with the protein kinase C inhibitor staurosporine. Biotinylation/immunoblot...

Neonicotinoid insecticides are compounds acting agonistically on insect nicotinic acetylcholine receptors (nAChR). They are especially active on hemipteran pest species such as aphids, whiteflies, and planthoppers, but also commercialized to control many coleopteran and some lepidopteran pest species. The most prominent member of this class of insecticides is imidacloprid. All neonicotinoid insecticides bind with high affinity (I 50 -values around 1 nM) to [ 3 H]imidacloprid binding sites on insect nAChRs. One notable ommission is the neonicotinoid thiamethoxam, showing binding affinities up to 10,000-fold less potent than the others, using housefly head membrane preparations. Electrophysiological whole cell voltage clamp studies using neurons isolated from Heliothis virescens ventral nerve cord showed no response to thiamethoxam when applied at concentrations of 0.3 mM, although the symptomology of poisoning in orally and topically treated noctuid larvae suggested strong neurotoxicity. Other neonicotinoids, such as clothianidin, exhibited high activity as agonists on isolated neurons at concentrations as low as 30 nM. There was no obvious correlation between biological efficacy of thiamethoxam against aphids and lepidopterans and receptor affinity in electrophysiological and binding assays. Pharmacokinetic studies using an LC-MS/MS approach to analyze haemolymph samples taken from lepidopteran larvae revealed that thiamethoxam orally applied to 5th instar Spodoptera frugiperda larvae was rapidly metabolized to clothianidin, an open-chain neonicotinoid. Clothianidin shows high affinity to nAChRs in both binding assays and whole cell voltage clamp studies. When applied to cotton plants, thiamethoxam was also quickly metabolized, with clothianidin being the predominant neonicotinoid in planta briefly after application, as indicated by LC-MS/MS analyses. Interestingly, the N-desmethylated derivative of thiamethoxam, N-desmethyl thiamethoxam, was not significantly produced in either lepidopteran larvae or in cotton plants, although it was often mentioned as a possible metabolite, being nearly as active as imidacloprid. In conclusion, our investigations show that thiamethoxam is likely to be a neonicotinoid precursor for clothianidin.

Exogenous application of acetylcholine elicits inward currents in hippocampal interneurons that are mediated via a-bungarotoxin-sensitive nicotinic acetylcholine receptors, but synaptic responses mediated via such receptors have never been reported in mammalian brain. In the present study, EPSCs were evoked in hippocampal interneurons in rat brain slices by electrical stimulation and were recorded by using whole-cell voltage-clamp techniques. Nicotinic EPSCs were isolated pharmacologically, using antagonists to block other known types of ligand-gated ion channels, and then were tested with either a-bungarotoxin or methyllycaconitine, which are selective antagonists for nicotinic acetylcholine receptors that contain the a7 receptor subunit. Each antagonist proved highly effective at reducing the remaining synaptic current. Evoked a7-mediated nicotinic EPSCs also were desensitized by superfusion with 1 mM nicotine, had extrapolated reversal potentials near 0 mV, and showed strong inwa...

The specific membrane capacitance (C m ) of a neuron influences synaptic efficacy and determines the speed with which electrical signals propagate along dendrites and unmyelinated axons. The value of this important parameter remains controversial. In this study, C m was estimated for the somatic membrane of cortical pyramidal neurons, spinal cord neurons, and hippocampal neurons. A nucleated patch was pulled and a voltage-clamp step was applied. The exponential decay of the capacitative charging current was analyzed to give the total membrane capacitance, which was then divided by the observed surface area of the patch. C m was 0.9 F/cm 2 for each class of neuron. To test the possibility that membrane proteins may alter C m , embryonic kidney cells (HEK-293) were studied before and after transfection with a plasmid coding for glycine receptor/channels. The value of C m was indistinguishable in untransfected cells and in transfected cells expressing a high level of glycine channels, indicating that differences in transmembrane protein content do not significantly affect C m . Thus, to a first approximation, C m may be treated as a "biological constant" across many classes of neuron.

A B S r R A C T The relative permeability of endplate channels to many organic cations was determined by reversal-potential criteria. Endplate currents induced by iontophoretic "puffs" of acetylcholine were studied by a Vaseline gap, voltage clamp method in cut muscle fibers. Reversal potential changes were measured as the NaCl of the bathing medium was replaced by salts of organic cations, and permeability ratios relative to Na + ions were calculated from the Goldman-Hodgkin-Katz equation. 40 small monovalent organic cations had permeability ratios larger than 0.1. The most permeant including NH4 +, hydroxylamine, hydrazine, methylamine, guanidine, and several relatives of guanidine had permeability ratios in the range 1.3-2.0. However, even cations such as imidazole, choline, tris(hydroxymethyl)aminomethane, triethylamine, and glycine methylester were appreciably permeant with permeability ratios of 0.13-0.95. Four compounds with two charged nitrogen groups were also permeant. Molecular models of the permeant ions suggest that the smallest cross-section of the open pore must be at least as large as a square, 6.5 ,~ • 6.5/~. Specific chemical factors seem to be less important than access or friction in determining the ionic selectivity of the endplate channel.

ABSIRACT Behavioral sensitization of the gill-withdrawal reflex of Aplysia is caused by presynaptic facilitation at the synapses of the mechanoreceptor sensory neurons of the reflex onto the motor neurons and interneurons. The presynaptic facilitation has been shown to be simulated by serotonin (the putative presynaptic facilitatory transmitter) and by cyclic AMP and to be accompanied by an increase in the Ca2+ current of sensory neuron cell bodies exposed to tetraethylammonium. This increase in the Ca2+ current could result from either a direct action on the Ca2+ channel or an action on an opposing K+ current. Here we report voltage clamp experiments which indicate that the increase in Ca2+ current associated with presynaptic facilitation results from a decrease in a K+ current. Stimulation of the connective (the pathway that mediates sensitization) or application of serotonin causes a decrease in a voltage-sensitive, steady-state outward current measured under voltage clamp as well as an increase in the transientnetinward and a decrease in the transient outward currents elicited by brief depolarizing command steps. The reversal potential of the steady-state synaptic current is sensitive to extracellular K+ concentration, and both the steady-state synaptic current and the changes in the transient currents are blocked by K+ current blocking agents and by washout of KV. These results suggest that serotonin and the natural transmitter released by connective stimulation act to decrease a voltage-sensitive K+ current. The decrease in K+ current prolongs the action potential, and this in turn increases the duration of the inward Ca2+ current and thereby enhances transmitter release.

Although the physiological basis of erythermalgia, an autosomal dominant painful neuropathy characterized by redness of the skin and intermittent burning sensation of extremities, is not known, two mutations of Na v 1.7, a sodium channel that produces a tetrodotoxinsensitive, fast-inactivating current that is preferentially expressed in dorsal root ganglia (DRG) and sympathetic ganglia neurons, have recently been identified in patients with primary erythermalgia. Na v 1.7 is preferentially expressed in small-diameter DRG neurons, most of which are nociceptors, and is characterized by slow recovery from inactivation and by slow closed-state inactivation that results in relatively large responses to small, subthreshold depolarizations. Here we show that these mutations in Na v 1.7 produce a hyperpolarizing shift in activation and slow deactivation. We also show that these mutations cause an increase in amplitude of the current produced by Na v 1.7 in response to slow, small depolarizations. These observations provide the first demonstration of altered sodium channel function associated with an inherited painful neuropathy and suggest that these physiological changes, which confer hyperexcitability on peripheral sensory and sympathetic neurons, contribute to symptom production in hereditary erythermalgia.

This paper describes the interaction of apamin, a bee venom neurotoxin, with the mouse neuroblastoma cell membrane. Voltage-clamp analyses have shown that apamin at low concentrations specifically blocks the Ca2+-dependent K+ channel in differentiated neuroblastoma cells. Binding experiments with highly radiolabeled toxin indicate that the dissociation constant of the apamin-receptor complex in differentiated neuroblastoma cells is 15-22 pM and the maximal binding capacity is 12 fmol/mg of protein. The receptor is destroyed by proteases, suggesting that it is a protein. The binding capacity of neuroblastoma cells for radiolabeled apamin dramatically increases during the transition from the nondifferentiated to the differentiated state. The number of Ca2+-dependent K+ channels appears to be at most 1/5th the number of fast Na+ channels in differentiated neuroblastoma. The binding of radiolabeled apamin to its receptor is antagonized by monovalent and divalent cations. Na+ inhibition of the binding of 125I-labeled apamin is of the competitive type (Kd(Na+) = 44 mM). Guanidinium and guanidinated compounds such as amiloride or neurotensin prevent binding of 125I-labeled apamin, the best antagonist being neurotensin.

Voltage-clamp analysis was applied to study the currents associated with the uptake of extracellular y-aminobutyric acid (GABA) by the cloned transporter GAT1 expressed at high efficiency in Xenopus oocytes. Steadystate GABA currents were increased at higher extracellular [GABA], [Na+], and [CI-] and at more negative potentials. The Hill coefficient for Na+ exceeded unity, suggesting the involvement of two Na+ ions. In the absence of CABA, voltage jumps produced transient currents that behaved like capacitive charge movements; these were suppressed by the uptake inhibitor SKF-89976A, were shifted to more negative potentials at lower external [Na'] and [CI-1, and had an effective valence of 1.1 elementary charge. A turnover rate per transporter of 6-131s at maximal [GABA] (-80 mV, 96 mM NaCI, 22OC) is given both by the kinetics of voltage jump relaxations and by the ratio between the maximal GABA currents and the charge movements. These quantitative data are necessary for evaluating the roles of GATl in synaptic function.

Tight-seal whole-cell recordings were made from cleaned somata of CA3 pyramidal cells deep in hippocampal slices from 19-21-d-old rats. The cells were filled with biocytin, and their voltage responses to short current pulses were recorded. After washout of initial sag, responses scaled linearly with injected current and were stable over time. The dendritic and axonal arbors of four cells were reconstructed and measured using light microscopy. Dendritic spines and axonal boutons were counted and the additional membrane area was incorporated into the relevant segments. The morphology of each neuron was converted into a detailed branching cable model by assuming values for specific membrane capacitance Cm and resistance Rm, and cytoplasmic resistivity Ri. These parameters were optimized for each cell by directly matching the model's response to that of the real cell by means of a modified weighted least-squares fitting procedure. By comparing the deviations between model and experi...

1. The involvement of N-methyl-D-aspartate (NMDA) receptors in the response to single-shock (0.033 Hz) stimulation of the Schaffer collateral-commissural pathway in hippocampal slices has been investigated using current- and voltage-clamp techniques. 2. In the presence of Mg2+ (1 or 2 mM) at membrane potentials near rest, the selective NMDA antagonist D-2-amino-5-phosphonovalerate (APV) had no effect on the excitatory postsynaptic potential (EPSP) and the biphasic inhibitory postsynaptic potential (IPSP) evoked by Schaffer collateral-commissural stimulation. The recurrent IPSP evoked by antidromic stimulation of alvear fibres was also unaffected by APV. 3. The introduction of a Mg2+-free perfusate led, at high stimulus intensity, to an orthodromically evoked epileptiform discharge but little change in the recurrent IPSP. APV suppressed a large proportion of the enhanced response in Mg2+-free perfusate. 4. EPSPs and excitatory postsynaptic currents (EPSCs) evoked in Mg2+-free perfusa...

The current contributions of individual ionic channels can be measured by electrically isolating a small patch of membrane. To do this, the tip of a small pipette is brought into close contact with an enzymatically cleaned membrane of a hypersensitive amphibian or mammalian muscle fiber. Current flowing through the pipette is measured. If the pipette contains cholinergic agonist at ft-molar concentrations, square pulse current waveforms can be observed which represent the activation of individual acetylcholine-receptor channels. The square pulses have amplitudes of 1 to 3 pA and durations of 10-lOOms.

The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors. In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M. sexta APN protein, or a cadherin Cry1Ab ecto-binding domain. In voltage clamping assays with dissected midgut from the susceptible insect, M. sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation. In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus). In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A. Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity. The mode of action of Vip3A supports its use as a novel insecticidal agent.

The absorption, fluorescence, dichroism, and birefringence of stained squid axons were measured during action potentials and voltage clamp steps in an effort to find large optical signals that could be used to monitor membrane potential. Changes in all four optical properties were found that were linearly related to membrane potential and, with several new dyes, the signal-to-noise ratios were larger than any obtained previously. The problem of photodynamic damage was greatly diminished; with a merocyaninerhodanine dye, the photodynamic damage associated with intense light and the presence of oxygen was negligible. The absorption change obtained with this dye was relatively large; it could be measured with a signal-to-noise ratio of 100:1 during a single action potential.

Membrane channels are subject to a wide variety of regulatory mechanisms that can be affected by activity. We present a model of a stomatogastric ganglion (STG) neuron in which several Ca2+-dependent pathways are used to regulate the maximal conductances of membrane currents in an activity-dependent manner. Unlike previous models of this type, the regulation and modification of maximal conductances by electrical activity is unconstrained. The model has seven voltage-dependent membrane currents and uses three Ca2+ sensors acting on different time scales. Starting from random initial conditions over a given range, the model sets the maximal conductances for its active membrane currents to values that produce a predefined target pattern of activity approximately 90% of the time. In these models, the same pattern of electrical activity can be produced by a range of maximal conductances, and this range is compared with voltage-clamp data from the lateral pyloric neuron of the STG. If the...

A technique is described which allows neurons to be voltage clamped with a single microelectrode, and the advantages of this circuit with respect to conventional bridge techniques are discussed. In this circuit, the single microelectrode is rapidly switched from a current passing to a recording mode. The circuitry consists of: (1) an electronic switch; (2) a high impedance, ultralow input capacity amplifier; (3) a sample-and-hold module; (4) conventional voltage clamping circuitry. The closed electronic switch allows current to flow through the electrode. The switch then opens, and the electrode is in a recording mode. The low input capacity of the preamplifier allows the artifact from the current pulse to rapidly abate, after which time the circuit samples the membrane potential. This cycle is repeated a t rates up to 10 kHz. The voltage clamping amplifier senses the output of the sample-and-hold module and adjusts the current pulse amplitude to maintain the desired membrane potential. The system was evaluated in Aplysia neurons by inserting two microelectrodes into a cell. One electrode was used to clamp the cell and the other to independently monitor membrane potential a t a remote location in the soma.

A significant percentage of confirmed severe acute respiratory syndrome (SARS) patients experienced gastrointestinal symptoms, and the viral sequence was detectable in the stool of most patients. At present, the knowledge of the pathology of the digestive system in SARS patients is limited. Because a resurgence of the SARS epidemic is constantly possible, there is an urgent need to understand the involvement of the digestive system in this new disease.

In higher plants changes and oscillations in cytosolic free Ca 2؉ concentration ([Ca 2؉ ] i ) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca 2؉ ] i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca 2؉ ] i to voltage and its interaction with ABA. We recorded [Ca 2؉ ] i by Fura2 f luorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a twoelectrode voltage clamp in intact Vicia guard cells. Freerunning oscillations between voltages near ؊50 mV and ؊200 mV were associated with oscillations in [Ca 2؉ ] i , and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca 2؉ ] i to increase, often in excess of 1 M, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca 2؉ ] i that spread centripetally from the peripheral cytoplasm within 5-10 s and relaxed over 40-60 s thereafter. The [Ca 2؉ ] i increases showed a voltage threshold near ؊120 mV and were sensitive to external Ca 2؉ concentration. Substituting Mn 2؉ for Ca 2؉ to quench Fura2 f luorescence showed that membrane hyperpolarization triggered a divalent inf lux. ABA affected the voltage threshold for the [Ca 2؉ ] i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca 2؉ ] i . These results demonstrate a capacity for voltage to evoke [Ca 2؉ ] i increases, they point to a dual interaction with ABA in triggering and propagating [Ca 2؉ ] i increases, and they implicate a role for voltage in ''conditioning'' [Ca 2؉ ] i signals that regulate ion channels for stomatal function.