Apparent Reversal Potential Research Articles

When complex neuronal structures may not matter.

Much work has explored animal-to-animal variability and compensation in ion channel expression. Yet, little is known regarding the physiological consequences of morphological variability. We quantify animal-to-animal variability in cable lengths (CV = 0.4) and branching patterns in the Gastric Mill (GM) neuron, an identified neuron type with highly-conserved physiological properties in the crustacean stomatogastric ganglion (STG) of Cancer borealis. We examined passive GM electrotonic structure by measuring the amplitudes and apparent reversal potentials (Erevs) of inhibitory responses evoked with focal glutamate photo-uncaging in the presence of TTX. Apparent Erevs were relatively invariant across sites (mean CV ± SD = 0.04 ± 0.01; 7-20 sites in each of 10 neurons), which ranged between 100-800 µm from the somatic recording site. Thus, GM neurons are remarkably electrotonically compact (estimated λ > 1.5 mm). Electrotonically compact structures, in consort with graded transmission, provide an elegant solution to observed morphological variability in the STG.

Late-onset episodic ataxia type 2 associated with a novel loss-of-function mutation in the CACNA1A gene

We report a patient with typical features of episodic ataxia type 2 (EA2) but with onset in the sixth decade and associated interictal hand dystonia. He was found to bear the novel heterozygous missense mutation p.Gly638Asp (c.1913G > A) in the CACNA1A gene. Functional analysis of the mutation on P/Q channels expressed in HEK 293 cells revealed a reduction of Ca 2+ current densities, a left-shift in the apparent reversal potential, the slowing of inactivation kinetics and the increase in the rate of current recovery from inactivation. These results are consistent with a decrease in Ca 2+ permeability through mutant P/Q channels. To our knowledge, this is just the second patient with late onset EA2 linked to a CACNA1A mutation and the first to carry a loss-of-function missense mutation.

Caldendrin, a Neuron-specific Modulator of Cav/1.2 (L-type) Ca2+ Channels

EF-hand Ca2+-binding proteins such as calmodulin and CaBP1 have emerged as important regulatory subunits of voltage-gated Ca2+ channels. Here, we show that caldendrin, a variant of CaBP1 enriched in the brain, interacts with and distinctly modulates Cav1.2 (L-type) voltage-gated Ca2+ channels relative to other Ca2+-binding proteins. Caldendrin binds to the C-terminal IQ-domain of the pore-forming alpha1-subunit of Cav1.2 (alpha(1)1.2) and competitively displaces calmodulin and CaBP1 from this site. Compared with CaBP1, caldendrin causes a more modest suppression of Ca2+-dependent inactivation of Cav1.2 through a different subset of molecular determinants. Caldendrin does not bind to the N-terminal domain of alpha11.2, a site that is critical for functional interactions of the channel with CaBP1. Deletion of the N-terminal domain inhibits CaBP1, but spares caldendrin modulation of Cav1.2 inactivation. In contrast, mutations of the IQ-domain abolish physical and functional interactions of caldendrin and Cav1.2, but do not prevent channel modulation by CaBP1. Using antibodies specific for caldendrin and Cav1.2, we show that caldendrin coimmunoprecipitates with Cav1.2 from the brain and colocalizes with Cav1.2 in somatodendritic puncta of cortical neurons in culture. Our findings reveal functional diversity within related Ca2+-binding proteins, which may enhance the specificity of Ca2+ signaling by Cav1.2 channels in different cellular contexts.

Excitable properties of adult skeletal muscle fibres from the honeybeeApis mellifera

In the hive, a wide range of honeybees tasks such as cell cleaning, nursing, thermogenesis, flight, foraging and inter-individual communication (waggle dance, antennal contact and trophallaxy) depend on proper muscle activity. However, whereas extensive electrophysiological studies have been undertaken over the past ten years to characterize ionic currents underlying the physiological neuronal activity in honeybee, ionic currents underlying skeletal muscle fibre activity in this insect remain, so far, unexplored. Here, we show that, in contrast to many other insect species, action potentials in muscle fibres isolated from adult honeybee metathoracic tibia, are not graded but actual all-or-none responses. Action potentials are blocked by Cd(2+) and La(3+) but not by tetrodotoxin (TTX) in current-clamp mode of the patch-clamp technique, and as assessed under voltage-clamp, both Ca(2+) and K(+) currents are involved in shaping action potentials in single muscle fibres. The activation threshold potential for the voltage-dependent Ca(2+) current is close to -40 mV, its mean maximal amplitude is -8.5+/-1.9 A/F and the mean apparent reversal potential is near +40 mV. In honeybees, GABA does not activate any ionic membrane currents in muscle fibres from the tibia, but L-glutamate, an excitatory neurotransmitter at the neuromuscular synapse induces fast activation of an inward current when the membrane potential is voltage clamped close to its resting value. Instead of undergoing desensitization as is the case in many other preparations, a component of this glutamate-activated current has a sustained component, the reversal potential of which is close to 0 mV, as demonstrated with voltage ramps. Future investigations will allow extensive pharmacological characterization of membrane ionic currents and excitation-contraction coupling in skeletal muscle from honeybee, a useful insect that became a model to study many physiological phenomena and which plays a major role in plant pollination and in stability of environmental vegetal biodiversity.

Effects of oleic acid on the high threshold barium current in seabassDicentrarchus labraxventricular myocytes

The present study employed a patch clamp technique in isolated seabass ventricular myocytes to investigate the hypothesis that oleic acid (OA), a mono-unsaturated fatty acid, can exert direct effects upon whole-cell barium currents. Acute application of free OA caused a dose-dependent depression of the whole-cell barium current that was evoked by a voltage step to 0 mV from a holding potential of -80 mV. The derived 50% inhibitory concentration (IC50) was 12.49+/-0.27 micromol l(-1). At a concentration of 30 micromol l(-1), OA significantly reduced the current density to about 45% of control values, but did not modify either the shape of the current-density voltage relationship or the apparent reversal potential. In addition, OA did not modify the voltage dependence of either steady state inactivation or activation curves. Taken together, these results indicate that physiological concentrations of free OA decrease the conductance of the L-type inward current, without altering its properties of selectivity and its voltage dependence. The inhibitory effect of OA upon the L-type calcium channel may translate, in vivo, into a protective effect against arrhythmias induced by Ca2+ overload.

Structure and Function of the Voltage Sensor of Sodium Channels Probed by a β-Scorpion Toxin

Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-scorpion toxin binding but alter voltage dependence of activation and enhance beta-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.

Transient rhythmic network activity in the somatosensory cortex evoked by distributed input in vitro

The initiation and maintenance of physiological and pathophysiological oscillatory activity depends on the synaptic interactions within neuronal networks. We studied the mechanisms underlying evoked transient network oscillation in acute slices of the adolescent rat somatosensory cortex and modeled its underpinning mechanisms. Oscillations were evoked by brief spatially distributed noisy extracellular stimulation, delivered via bipolar electrodes. Evoked transient network oscillation was detected with multi-neuron patch-clamp recordings under different pharmacological conditions. The observed oscillations are in the frequency range of 2–5 Hz and consist of 4–12 mV large, 40–150 ms wide compound synaptic events with rare overlying action potentials. This evoked transient network oscillation is only weakly expressed in the somatosensory cortex and requires increased [K +] o of 6.25 mM and decreased [Ca 2+] o of 1.5 mM and [Mg 2+] o of 0.5 mM. A peak in the cross-correlation among membrane potential in layers II/III, IV and V neurons reflects the underlying network-driven basis of the evoked transient network oscillation. The initiation of the evoked transient network oscillation is accompanied by an increased [K +] o and can be prevented by the K + channel blocker quinidine. In addition, a shift of the chloride reversal potential takes place during stimulation, resulting in a depolarizing type A GABA (GABA A) receptor response. Blockade of α-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA), N-methyl- d-aspartate (NMDA), or GABA A receptors as well as gap junctions prevents evoked transient network oscillation while a reduction of AMPA or GABA A receptor desensitization increases its duration and amplitude. The apparent reversal potential of −27 mV of the evoked transient network oscillation, its pharmacological profile, as well as the modeling results suggest a mixed contribution of glutamatergic, excitatory GABAergic, and gap junctional conductances in initiation and maintenance of this oscillatory activity. With these properties, evoked transient network oscillation resembles epileptic afterdischarges more than any other form of physiological or pathophysiological neocortical oscillatory activity.

Molecular Mechanism for Divergent Regulation of Cav1.2 Ca2+ Channels by Calmodulin and Ca2+-binding Protein-1

Ca(2+)-binding protein-1 (CaBP1) and calmodulin (CaM) are highly related Ca(2+)-binding proteins that directly interact with, and yet differentially regulate, voltage-gated Ca(2+) channels. Whereas CaM enhances inactivation of Ca(2+) currents through Ca(v)1.2 (L-type) Ca(2+) channels, CaBP1 completely prevents this process. How CaBP1 and CaM mediate such opposing effects on Ca(v)1.2 inactivation is unknown. Here, we identified molecular determinants in the alpha(1)-subunit of Ca(v)1.2 (alpha(1)1.2) that distinguish the effects of CaBP1 and CaM on inactivation. Although both proteins bind to a well characterized IQ-domain in the cytoplasmic C-terminal domain of alpha(1)1.2, mutations of the IQ-domain that significantly weakened CaM and CaBP1 binding abolished the functional effects of CaM, but not CaBP1. Pulldown binding assays revealed Ca(2+)-independent binding of CaBP1 to the N-terminal domain (NT) of alpha(1)1.2, which was in contrast to Ca(2+)-dependent binding of CaM to this region. Deletion of the NT abolished the effects of CaBP1 in prolonging Ca(v)1.2 Ca(2+) currents, but spared Ca(2+)-dependent inactivation due to CaM. We conclude that the NT and IQ-domains of alpha(1)1.2 mediate functionally distinct interactions with CaBP1 and CaM that promote conformational alterations that either stabilize or inhibit inactivation of Ca(v)1.2.

Differential Role of the α1C Subunit Tails in Regulation of the Cav1.2 Channel by Membrane Potential, β Subunits, and Ca2+ Ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.

K+ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.

Multiple Structural Elements Contribute to the Slow Kinetics of the Cav3.3 T-type Channel

Molecular cloning and expression studies established the existence of three T-type Ca(2+) channel (Ca(v)3) alpha(1) subunits: Ca(v)3.1 (alpha(1G)), Ca(v)3.2 (alpha(1H)), and Ca(v)3.3 (alpha(1I)). Although all three channels are low voltage-activated, they display considerable differences in their kinetics, with Ca(v)3.1 and Ca(v)3.2 channels activating and inactivating much faster than Ca(v)3.3 channels. The goal of the present study was to determine the structural elements that confer the distinctively slow kinetics of Ca(v)3.3 channels. To address this question, a series of chimeric channels between Ca(v)3.1 and Ca(v)3.3 channels were constructed and expressed in Xenopus oocytes. Kinetic analysis showed that the slow activation and inactivation kinetics of the Ca(v)3.3 channel were not completely abolished by substitution with any one portion of the Ca(v)3.1 channel. Likewise, the Ca(v)3.1 channel failed to acquire the slow kinetics by simply adopting one portion of the Ca(v)3.3 channel. These findings suggest that multiple structural elements contribute to the slow kinetics of Ca(v)3.3 channels.

Loperamide mobilizes intracellular Ca2+ stores in insulin-secreting HIT-T15 cells.

1 We have investigated the effects of loperamide on intracellular Ca(2+) stores and membrane K(+) channels in insulin-secreting hamster insulinoma (HIT-T15) cells. 2 In cell-attached patch-clamp mode, loperamide (3-250 micro M) activated large single-channel currents. The loperamide-activated currents were tentatively identified as Ca(2+)-activated K(+) channel (K(Ca)) currents based on their single-channel conductance (145 pS), apparent reversal potential, and insensitivity to tolbutamide. Smaller single-channel currents with a conductance (32 pS) indicative of adenosine triphosphate-sensitive K(+) channels (K(ATP) channels) were also recorded, but were insensitive to loperamide. 3 Surprisingly, the loperamide-activated currents persisted in the absence of extracellular Ca(2+). Yet under these conditions, we still measured loperamide-induced Ca(2+) increases. These effects are dose dependent. Loperamide had no effects in the inside-out patch configuration, suggesting that loperamide does not directly activate the channels with large conductance, but does so secondarily to release of Ca(2+) from intracellular stores. 4 Carbachol (100 micro M), an agonist of muscarinic receptors, which mediates IP(3)-dependent intracellular Ca(2+) release, enhanced the effects of loperamide on K(Ca) channels. 5 Both the putative K(Ca) currents and Ca(2+) signals induced by loperamide (with '0' [Ca(2+)](o)) were abolished when the intracellular Ca(2+) stores had been emptied by pretreating the cells with either carbachol or thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor that blocks reuptake of calcium. 6 These data indicate that loperamide in insulin-secreting beta-cells evokes intracellular Ca(2+) release from IP(3)-gated stores and activates membrane currents that appear to be carried by K(Ca), rather than K(ATP) channels.

Effects of prostaglandin F2alpha on membrane currents in rabbit middle cerebral arterial smooth muscle cells.

Although PGF(2alpha) affects contractility of vascular smooth muscles, no studies to date have addressed the electrophysiological mechanism of this effect. The purpose of our investigation was to examine the direct effects of PGF(2alpha) on membrane potentials, Ca(2+)-activated K(+) (K(Ca)) channels, delayed rectifier K(+) (K(V)) channels, and L-type Ca(2+) channels with the patch-clamp technique in single rabbit middle cerebral arterial smooth muscle cells (SMCs). PGF(2alpha) significantly hyperpolarized membrane potentials and increased the amplitudes of total K(+) currents. PGF(2alpha) increased open-state probability but had little effect on the open and closed kinetics of K(Ca) channels. PGF(2alpha) increased the amplitudes of K(V) currents with a leftward shift of the activation and inactivation curves and a decrease in the activation time constant. PGF(2alpha) decreased the amplitudes of L-type Ca(2+) currents without any significant change in threshold or apparent reversal potentials. This study provides the first finding that the direct effects of PGF(2alpha) on middle cerebral arterial SMCs, at least in part, could attenuate vasoconstriction.

Intramembrane Charge Movement and L-Type Calcium Current in Skeletal Muscle Fibers Isolated from Control and mdx Mice

Dystrophin-deficient muscle fibers from mdx mice are believed to suffer from increased calcium entry and elevated submembranous calcium level, the actual source and functional consequences of which remain obscure. Here we compare the properties of the dihydropyridine receptor as voltage sensor and calcium channel in control and mdx muscle fibers, using the silicone-voltage clamp technique. In control fibers charge movement followed a two-state Boltzmann distribution with values for maximal charge, midpoint voltage, and steepness of 23 ± 2 nC/ μF, −37 ± 3 mV, and 13 ± 1 mV ( n = 7). Essentially identical values were obtained in mdx fibers and the time course of charge recovery from inactivation was also similar in the two populations ( τ ≈ 6 s). In control fibers the voltage dependence of the slow calcium current elicited by 100-ms–long pulses gave values for maximal conductance, apparent reversal potential, half-activation potential, and steepness factor of 156 ± 15 S/F, 65.5 ± 2.9 mV, −0.76 ± 1.2 mV, and 6.2 ± 0.5 mV ( n = 17). In mdx fibers, the half-activation potential of the calcium current was slightly more negative (−6.2 ± 1.2 mV, n = 16). Also, when using longer pulses, the time constant of calcium current decay was found to be significantly larger (by a factor of 1.5–2) in mdx than in control fibers. These changes in calcium current properties are unlikely to be primarily responsible for a dramatic alteration of intracellular calcium homeostasis. They may be speculated to result, at least in part, from remodeling of the submembranous cytoskeleton network due to the absence of dystrophin.

T-tubule localization of the inward-rectifier K(+) channel in mouse ventricular myocytes: a role in K(+) accumulation.

1. The properties of the slow inward 'tail currents' (I(tail)) that followed depolarizing steps in voltage-clamped, isolated mouse ventricular myocytes were examined. Depolarizing steps that produced large outward K(+) currents in these myocytes were followed by a slowly decaying inward I(tail) on repolarization to the holding potential. These currents were produced only by depolarizations: inwardly rectifying K(+) currents, I(K1), produced by steps to potentials negative to the holding potential, were not followed by I(tail). 2. For depolarizations of equal duration, the magnitude of I(tail) increased as the magnitude of outward current at the end of the depolarizing step increased. The apparent reversal potential of I(tail) was dependent upon the duration of the depolarizing step, and the reversal potential shifted to more depolarized potentials as the duration of the depolarization was increased. 3. Removal of external Na(+) and Ca(2+) had no significant effect on the magnitude or time course of I(tail). BaCl(2) (0.25 mM), which had no effect on the magnitude of outward currents, abolished I(tail) and I(K1) simultaneously. 4. Accordingly, I(tail) in mouse ventricular myocytes probably results from K(+) accumulation in a restricted extracellular space such as the transverse tubule system (t-tubules). The efflux of K(+) into the t-tubules during outward currents produced by depolarization shifts the K(+) Nernst potential (E(K)) from its 'resting' value (close to -80 mV) to more depolarized potentials. This suggests that I(tail) is produced by I(K1) in the t-tubules and is inward because of the transiently elevated K(+) concentration and depolarized value of E(K) in the t-tubules. 5. Additional evidence for the localization of I(K1) channels in the t-tubules was provided by confocal microscopy using a specific antibody against Kir2.1 in mouse ventricular myocytes.

The Voltage-Dependent Proton Pumping in Bacteriorhodopsin Is Characterized by Optoelectric Behavior

The light-driven proton pump bacteriorhodopsin (bR) was functionally expressed in Xenopus laevis oocytes and in HEK-293 cells. The latter expression system allowed high time resolution of light-induced current signals. A detailed voltage clamp and patch clamp study was performed to investigate the ΔpH versus Δ ψ dependence of the pump current. The following results were obtained. The current voltage behavior of bR is linear in the measurable range between −160 mV and +60 mV. The pH dependence is less than expected from thermodynamic principles, i.e., one ΔpH unit produces a shift of the apparent reversal potential of 34 mV (and not 58 mV). The M 2–BR decay shows a significant voltage dependence with time constants changing from 20 ms at +60 mV to 80 ms at −160 mV. The linear I-V curve can be reconstructed by this behavior. However, the slope of the decay rate shows a weaker voltage dependence than the stationary photocurrent, indicating that an additional process must be involved in the voltage dependence of the pump. A slowly decaying M intermediate (decay time > 100 ms) could already be detected at zero voltage by electrical and spectroscopic means. In effect, bR shows optoelectric behavior. The long-lived M can be transferred into the active photocycle by depolarizing voltage pulses. This is experimentally demonstrated by a distinct charge displacement. From the results we conclude that the transport cycle of bR branches via a long-lived M 1* in a voltage-dependent manner into a nontransporting cycle, where the proton release and uptake occur on the extracellular side.

Role of GABA(A)-mediated inhibition in controlling the responses of regular spiking cells in turtle visual cortex.

The visual cortex of freshwater turtles contains pyramidal cells, which have a regular spiking (RS) firing pattern, and several categories of aspiny, inhibitory interneurons. The interneurons show diverse firing patterns, including the fast spiking (FS) pattern. Postsynaptic potentials (PSPs) evoked in FS cells by visual stimulation of the retina reach their peak amplitudes as much as 200 ms before PSPs in RS cells (Mancilla et al., 1998). FS cells could, consequently, control the amplitudes of light-evoked PSPs in RS cells by producing disynaptic, feedforward inhibitory postsynaptic potentials (IPSPs) that overlap in time with geniculocortical excitatory postsynaptic potentials (EPSPs). Since FS cells receive recurrent, excitatory inputs from RS cells, they could also control the amplitudes of light-evoked PSPs in RS cells via polysynaptic, feedback inhibition. The in vitro geniculocortical preparation of Pseudemys scripta was used to characterize the temporal relationships of EPSPs and IPSPs produced in RS cells by electrical activation of geniculate afferents and by diffuse light flashes presented to the retina. GABA(A) receptor-mediated inhibition was blocked using extracellular application of bicuculline (3.5 mM) or intracellular perfusion of picrotoxin (1 microM) in individual RS cells. Electrical stimulation of thalamic afferents produced compound PSPs. Blockade of GABA(A) receptor-mediated IPSPs with either bicuculline or picrotoxin provided evidence for both early and late IPSPs in RS cells. Analysis of the apparent reversal potentials of light-evoked PSPs indicated the existence of early IPSPs during the first 140-300 ms following light onset. Light responses of cells perfused with picrotoxin diverged from control light responses at about 300 ms after light onset and had maximum amplitudes that were significantly different from control light responses. These experiments indicate that the responses of RS cells to both electrical and natural stimulation of geniculate afferents are controlled by both early and late IPSPs, consistent with activation of both feedforward and feedback pathways.

Blue light activates potassium-efflux channels in flexor cells from Samanea saman motor organs via two mechanisms.

Light-induced leaflet movement of Samanea saman depends on the regulation of membrane transporters in motor cells. Blue light (BL) stimulates leaflet opening by inducing K(+) release from the flexor motor cells. To elucidate the mechanism of K(+)-efflux (K(D))-channel regulation by light, flexor motor cell protoplasts were patch-clamped in a cell-attached configuration during varying illumination. Depolarization elicited outward currents through single open K(D) channels. Changes in cell membrane potential (E(M)) were estimated by applying voltage ramps and tracking the change of the apparent reversal potential of K(D)-channel current. BL shifted E(M) in a positive direction (i.e. depolarized the cell) by about 10 mV. Subsequent red light pulse followed by darkness shifted E(M) oppositely (i.e. hyperpolarized the cell). The BL-induced shifts of E(M) were not observed in cells pretreated with a hydrogen-pump inhibitor, suggesting a contribution by hydrogen-pump to the shift. BL also increased K(D)-channel activity in a voltage-independent manner as reflected in the increase of the mean net steady-state patch conductance at a depolarization of 40 mV relative to the apparent reversal potential (G(@40)). G(@40) increased by approximately 12 pS without a change of the single-channel conductance, possibly by increasing the probability of channel opening. Subsequent red-light and darkness reversed the change in G(@40). Thus, K(+) efflux, a determining factor for the cell-volume decrease of flexor cells, is regulated by BL in a dual manner via membrane potential and by an independent signaling pathway.

Morphological and membrane properties of rat magnocellular basal forebrain neurons maintained in culture.

Morphological and electrophysiological characteristics of magnocellular neurons from basal forebrain nuclei of postnatal rats (11-14 days old) were examined in dissociated cell culture. Neurons were maintained in culture for periods of 5-27 days, and 95% of magnocellular (>23 micron diam) neurons stained positive with acetylcholinesterase histochemistry. With the use of phase contrast microscopy, four morphological subtypes of magnocellular neurons could be distinguished according to the shape of their soma and pattern of dendritic branching. Corresponding passive and active membrane properties were investigated with the use of whole cell configuration of the patch-clamp technique. Neurons of all cell types displayed a prominent (6-39 mV; 6.7-50 ms duration) spike afterdepolarization (ADP), which in some cells reached firing threshold. The ADP was voltage dependent, increasing in amplitude and decreasing in duration with membrane hyperpolarization with an apparent reversal potential of -59 +/- 2.3 (SE) mV. Elevating [Ca2+]o (2.5-5.0 mM) or prolonging spike repolarization with 10 mM tetraethylammonium (TEA) or 1 mM 4-aminopyridine (4-AP), potentiated the ADP while it was inhibited by reducing [Ca2+]o (2.5-1 mM) or superfusion with Cd2+ (100 microM). The ADP was selectively inhibited by amiloride (0.1-0.3 mM or Ni2+ 10 microM) but unaffected by nifedipine (3 microM), omega-conotoxin GVIA (100 nM) or omega-agatoxin IVA (200 nM), indicating that Ca2+ entry was through T-type Ca2+ channels. After inhibition of the ADP with amiloride (300 microM), depolarization to less than -65 mV revealed a spike afterhyperpolarization (AHP) with both fast and slow components that could be inhibited by 4-AP (1 mM) and Cd2+ (100 microM), respectively. In all cell types, current-voltage relationships exhibited inward rectification at hyperpolarized potentials >/=EK (approximately -90 mV). Application of Cs+ (0.1-1 mM) or Ba2+ (1-10 microM) selectively inhibited inward rectification but had no effect on resting potential or cell excitability. At higher concentrations, Ba2+ (>10 microM) also inhibited an outward current tonically active at resting potential (VH -70 mV), which under current-clamp conditions resulted in small membrane depolarization (3-10 mV) and an increase in cell excitability. Depolarizing voltage commands from prepulse potential of -90 mV (VH -70 mV) in the presence of tetrodotoxin (0.5 microM) and Cd2+ (100 microM) to potentials between -40 and +40 mV cause voltage activation of both transient A-type and sustained delayed rectifier-type outward currents, which could be selectively inhibited by 4-AP (0.3-3 mM) and TEA (1-3 mM), respectively. These results show that, although acetylcholinesterase-positive magnocellular basal forebrain neurons exhibit considerable morphological heterogeneity, they have very similar and characteristic electrophysiological properties.

The Effect of the Oxidant Hypochlorous Acid on theL-Type Calcium Current in Isolated Ventricular Cardiomyocytes

Disturbances of cellular calcium homeostasis due to oxidative stress are involved in reperfusion associated phenomena like myocardial stunning and reperfusion induced arrhythmias. This study investigates the effect of the major neutrophil-derived oxidant hypochlorous acid (HOCl) on thel-type calcium current (ICa,L) of hamster ventricular cardiomyocytes. Using the whole-cell recording configuration of the patch-clamp technique, ICa,Lwas recorded over 12.5 min (0.1 Hz). Application of HOCl or buffer (for control) via a second micropipette in close proximity to the cell was started att=1 min. To study the influence of increased intracellular calcium buffer concentration and of ATP on HOCl-induced effects, internal solutions were composed as follows (EGTA/ATP in mmol/l): group I (standard) 0.5/0.0, group II 5.0/0.0, group III 0.5/1.0, and group IV 5.0/1.0. Application of 10, 20 and 40μmol/l HOCl (under group I-conditions) caused a dose-dependent decrease in peak ICa,Lto 82±3.2, 66±4.2 and 36±4.3% of baseline value (v94±4.8% in controls, mean±s.e.m.,P<0.05), and integrated ICa,Lwithout affecting apparent reversal potential, activation and inactivation kinetics. HOCl-induced (40μmol/l) decrease in ICa,Lwas partially inhibited in group II and III. Peak currents of these groups averaged 51±4.7 and 52±4.2% of baseline after 11.5 min administration of HOCl. Peak current in group IV cells decreased to 65±3.8% of baseline value (P<0.05 between group I–IV andvcontrols). Oxidative stress-induced decrease in ICa,Lmay be explained by energy depletion or calcium overload rather than by direct oxidative inactivation of channel proteins. A decrease in ICa,Lmay contribute to the shortening of action potential during reperfusion.