Physiological types of substantia gelatinosa neurons in the rat spinal cord

Electrophysiological properties of neurons in the substantia gelatinosa (SG, or lamina II) were studied in vitro in spinal cord slices from 3-to 5-week-old rats. Based on the type of action potentials (APs) firing in response to long depolarization (0.5 to 0.8 sec), neurons were categorized into three types: tonic (APs were generated over the whole duration of the stimulus, n = 26, or 41.2%), adapting (a few APs occurred only at the beginning of stimulation, n = 8, 12.7%), and delayed-firing neurons, DFNs (APs occurred at the end of stimulation, n = 22, 35.1%); 11% of the cells had intermediate properties. Neurons of each type expressed distinct ion currents that were subthreshold for AP generation (< −40 mV). Tonic and adapting neurons either had no subthreshold currents (n = 21, or 61.3%) or expressed T-type calcium currents (n = 13, or 38.7%). All DFNs had outward A-type potassium currents. Statistical analysis confirmed this classification scheme: neurons of each type were differentially distributed in a 3-D parametric space of the main cellular properties. Distributions of tonic and adapting neurons partially overlapped, while that of DFNs differed significantly from both the above groups. It is suggested that DFNs perform a special function in the processing of sensory information; the functions of tonic and adapting neurons might be rather similar to each other. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 191–198, May–June, 2008.     

Effect of nifedipine on the ion channels formed by α1 subunits of the cardiac and vascular muscle L-type Ca channels

We investigated the voltage dependence of nifedipine sensitivity of the ion channels formed by α₁ subunits of the cardiac and smooth muscles (CM and SM, respectively) L-type Ca²⁺ channels stably expressed in Chinese hamster ovary (CHO) cells. Equilibrium inhibition of the α₁ subunits, directing Ba²⁺ current (I _α1), by different concentrations of nifedipine was measured at the holding potentials (V _h ) of −100 mV and −50 mV. AtV _h =−100 mV, the SM α₁ subunit was found to be 6-fold more sensitive for nifedipine than the subunit (K ₋₁₀₀=8.3 and 50.4 nM, respectively). Depolarization to −50 mV resulted in about sevenfold increase in the nifedipine potency for both subunits (K ₋₅₀=1.25 and 6.95 nM, respectively). The voltage dependence of steady-state inactivation could be fitted by a sum of two Boltzmann’s equations with slope factors of about 12 and 5 mV. The midpoints of both components in the CM α₁ subunit (−75.6 and −42.8 mV) were more negative than those in the SM subunit (−63.7 and −37.7 mV). The relative contribution of the less sloped component in the control was rather low, being less pronounced in the CM (0.15) than in the SM (0.34) subunits. Nifedipine shifted the midpoints of inactivation curves to more negative potentials. The shift was more pronounced for the SM α₁ subunit (−24.8 mV compared with −11.8 mV for the CM subunit in the presence of 10 nM nifedipine). Nifedipine differentially affected the two Boltzmann components of inactivation curves, more effectively inhibiting the steeper component. In the presence of 10 nM nifedipine, this component completely disappeared in the SM subunit, while its relative contribution in the CM subunit decreased from 0.85 to 0. 57, resulting in an apparent decrease in the steepness. These results are inconsistent with the receptor modulated hypothesis and suggest the existence of two mechanisms of inactivation characterized by different voltage dependence.     

Presteady-State Currents of the Rabbit Na+/Glucose Cotransporter (SGLT1)

The rabbit Na⁺/glucose cotransporter (SGLT1) exhibits a presteady-state current after step changes in membrane voltage in the absence of sugar. These currents reflect voltage-dependent processes involved in cotransport, and provide insight on the partial reactions of the transport cycle. SGLT1 presteady-state currents were studied as a function of external Na⁺, membrane voltage V _m , phlorizin and temperature. Step changes in membrane voltage—from the holding V _h to test values, elicited transient currents that rose rapidly to a peak (at 3–4 msec), before decaying to the steady state, with time constants τ≈4–20 msec, and were blocked by phlorizin (K _i ≈30 μm). The total charge Q was equal for the application of the voltage pulse and the subsequent removal, and was a function of V _m . The Q-V curves obeyed the Boltzmann relation: the maximal charge Q _max was 4–120 nC; V _0.5, the voltage for 50% Q _max was −5 to +30 mV; and z, the apparent valence of the moveable charge, was 1. Q _max and z were independent of V _h (between 0 and −100 mV) and temperature (20–30°C), while increasing temperature shifted V _0.5 towards more negative values. Decreasing [Na⁺] _o decreased Q _max, and shifted V _0.5 to more negative voltages 9by −100 mV per 10-fold decrease in [Na⁺] _o ). The time constant τ was voltage dependent: the τ-V relations were bell-shaped, with maximal τ_max 8–20 msec. Decreasing [Na⁺] _o decreased τ_max, and shifted the τ-V curves towards more negative voltages. Increasing temperature also shifted the τ-V curves, but did not affect τ_max. The maximum temperature coefficient Q ₁₀ for τ was 3–4, and corresponds to an activation energy of 25 kcal/mole. Simulations of a 6-state ordered kinetic model for rabbit Na⁺/glucose cotransport indicate that charge-movements are due to Na⁺-binding/dissociation and a conformational change of the empty transporter. The model predicts that (i) transient currents rise to a peak before decay to steady-state; (ii) the τ-V relations are bell-shaped, and shift towards more negative voltages as [Na⁺] _o is reduced; (iii) τ_max is decreased with decreasing [Na⁺] _o ; and (iv) the Q-V relations are shifted towards negative voltages as [Na⁺] _o is reduced. In general, the kinetic properties of the presteady-state currents are qualitatively predicted by the model. Received: 12 August 1996/Revised: 30 September 1996     

Flow-Dependent Activation of Maxi K+ Channels in Apical Membrane of Rabbit Connecting Tubule

The Ca²⁺-activated maxi K⁺ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean number of open channels (NP _o ) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the stretch-activation of Ca²⁺-permeable cation channels, because the maxi K⁺ channel was not stretch-activated in both the cell-attached configuration using Ca²⁺-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K⁺ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K⁺ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K⁺ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV _t ) when luminal K⁺ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV _t was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced ΔV _t to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV _t to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV _t was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV _t =−7.2 ± 0.5 mV in control vs.ΔV _t =−6.7 ± 0.6 mV in AA). These results suggest that the Ca²⁺-activated maxi K⁺ channel is responsible for flow-dependent K⁺ secretion by coupling with the stretch-activated Ca²⁺-permeable cation channel in the rabbit CNT. Received: 21 August 1997/Revised: 20 March 1998     

K<Superscript>+</Superscript> Channels of Squid Giant Axons Open by an Osmotic Stress in Hypertonic Solutions Containing Nonelectrolytes

In hypertonic solutions made by adding nonelectrolytes, K⁺ channels of squid giant axons opened at usual asymmetrical K⁺ concentrations in two different time courses; an initial instantaneous activation (I _IN) and a sigmoidal activation typical of a delayed rectifier K⁺ channel (I _D). The current–voltage relation curve for I _IN was fitted well with Goldman equation described with a periaxonal K⁺ concentration at the membrane potential above −10 mV. Using the activation–voltage curve obtained from tail currents, K⁺ channels for I _IN are confirmed to activate at the membrane potential that is lower by 50 mV than those for I _D. Both I _IN and I _D closed similarly at the holding potential below −100 mV. The logarithm of I _IN/I _D was linearly related with the osmolarity for various nonelectrolytes. Solute inaccessible volumes obtained from the slope increased with the nonelectrolyte size from 15 to 85 water molecules. K⁺ channels representing I _D were blocked by open channel blocker tetra-butyl ammonium (TBA) more efficiently than in the absence of I _IN, which was explained by the mechanism that K⁺ channels for I _D were first converted to those for I _IN by the osmotic pressure and then blocked. So K⁺ channels for I _IN were suggested to be derived from the delayed rectifier K⁺ channels. Therefore, the osmotic pressure is suggested to exert delayed-rectifier K⁺ channels to open in shrinking rather hydrophilic flexible parts outside the pore than the pore itself, which is compatible with the recent structure of open K⁺ channel pore.     

K-Aggravated Myotonia Mutations at Residue G1306 Differentially Alter Deactivation Gating of Human Skeletal Muscle Sodium Channels

Summary Fast inactivation and deactivation gating were compared between wild-type human voltage-gated skeletal muscle sodium channel (hNa_V1.4) and potassium-aggravated myotonia (PAM) mutations G1306A, G1306E, and G1306V. Cell-attached macropatches were used to compare wild-type and PAM-gating properties in normal extracellular K⁺ (4 mM), decreased K⁺ (1 mM), and increased K⁺ (10 mM). G1306E/A increased the apparent valence of the conductance (g(V)) curve. Compared to hNa_V1.4, the steady-state inactivation (h_∞) curve was depolarized for G1306E/A but hyperpolarized by G1306V, and this mutation increased apparent valence. G1306A/E slowed the rate of current rise towards peak activation. G1306V slowed open-state deactivation, inactivated-state deactivation, and recovery from fast inactivation. G1306A/E abbreviated open-state deactivation at negative commands. These mutants slowed open-state deactivation at more positive commands, at voltages for which fast inactivation might influence tail current decay. G1306E abbreviated recovery delay without affecting recovery rate. Low K⁺ increased peak current in hNa_V1.4 and in G1306V. For G1306E, low K⁺ increased the rate of entry into fast inactivation, hyperpolarized the g(V) and h_∞ curves, and increased recovery delay. Biophysical underpinnings of PAM caused by mutations of G1306 thus vary with the specific mutation, and hyperkalemic exacerbation of effects of mutations at this residue are not direct.     

Inhibitory Effects of Honokiol on the Voltage-Gated Potassium Channels in Freshly Isolated Mouse Dorsal Root Ganglion Neurons

Voltage-gated potassium (K_V) currents, subdivided into rapidly inactivating A-type currents (I _A) and slowly inactivating delayed rectifier currents (I _K), play a fundamental role in modulating pain by controlling neuronal excitability. The effects of Honokiol (Hon), a natural biphenolic compound derived from Magnolia officinalis, on K_V currents were investigated in freshly isolated mouse dorsal root ganglion neurons using the whole-cell patch clamp technique. Results showed that Hon inhibited I _A and I _K in concentration-dependent manner. The IC₅₀ values for block of I _A and I _K were 30.5 and 25.7 µM, respectively. Hon (30 µM) shifted the steady-state activation curves of I _A and I _K to positive potentials by 17.6 and 16.7 mV, whereas inactivation and recovery from the inactivated state of I _A were unaffected. These results suggest that Hon preferentially interacts with the active states of the I _A and I _K channels, and has no effect on the resting state and inactivated state of the I _A channel. Blockade on K⁺ channels by Hon may contribute to its antinociceptive effect, especially anti-inflammatory pain.

     

Membrane responses to changes in the extracellular potassium concentration in isolated hippocampal pyramidal neurons

The responses of freshly isolated hippocampal pyramidal neurons to rapid, elevations of the external potassium concentration ([K⁺] _out ) were investigated using the whole-cell variation of a patch-clamp technique. An elevation of [K⁺] _out induced a two-phase inward current at the membrane potentials more negative than the reversal potential for K ions. This current consisted of a leakage, current and a time-dependent current (τ=40–50 msec at 21°C), the latter designated below asI _ΔK. It displayed first-order activation kinetics that showed neither voltage, nor concentration dependence. The amplitude of this current was determined by the external K⁺ concentration and increased with hyperpolarization. Voltage dependence ofI _ΔK measured within the range from −20 to −120 mV was similar to that for inward rectifier. Activation ofI _ΔK was utterly dependent on Na⁺; substitution of extracellular Na⁺ with choline chloride almost completely depressedI _ΔK.I _ΔK was absent in the cells freshly dissociated from the nodosal and dorsal root ganglia. This suggests that this earlier unrecognized current is instrumental in preserving densely packed hippocampal pyramidal neurons from sudden increases in [K⁺] _out and following spontaneous over-excitation. It prevents the neurons from responding to K⁺-induced depolarizations by slowing down potassium influx.     

Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K⁺ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (E_m) was not sensitive to the external K⁺ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K⁺-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (E_rev) of the s-ORC conductance. The s-ORC was blocked by Ba²⁺ (K_1/2 = 0.3–1.3mM) and verapamil (K_1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba²⁺. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K⁺ concentration. The slow activation of the IRC (t_1/2 ≈ 0.5 s) and its negative activation potential (V_threshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes. Received: 28 March 1996 / Accepted: 11 November 1996     

Simulation of GABA B -receptor-mediated K+ current in thalamocortical relay neurons: tonic firing,bursting, and oscillations

Until recently, the presence of γ-aminobutyric acid (GABA) in the thalamus has usually been associated with the "classical" GABA _A Cl⁻-dependent receptor. However, the discovery of a slower, long-lasting, K⁺-dependent inhibitory postsynaptic potential (IPSP) mediated by GABA _B receptors in projection cells of the dorsal lateral geniculate nucleus has led researchers to reconsider its role in modulating the behavior of these cell groups (Crunelli et al. 1988; Crunelli and Leresche 1991). Of particular interest is the role of this K⁺ current in the activation of the low-threshold Ca²⁺ current, I _T , of thalamocortical relay (TCR) neurons responsible for bursting activity (Jahnsen and Llinás 1984 a, b). Considering the time scale on which the GABA _B -receptor-activated K⁺ current operates, it is ideally suited to foster sustained rhythmicity in TCR cells reciprocally connected to neurons of the nucleus reticularis thalami (NRT) as well as interneurons at frequencies observed in vivo (Steriade and Llinás 1988). In this study we show that small changes in the duration and amplitude of the K⁺-dependent IPSPs can have marked effects on TCR cell groups including a shift from single-spike firing (tonic) to bursting behavior. We further show that a single GABA _B -mediated IPSP is sufficient to activate the low-threshold Ca²⁺ response and that sustained oscillations are possible given the presence of excitatory TCR connections to GABAergic NRT cells or interneurons of the dorsal lateral thalamus. These combined effects are examined with regard to their role in generating the well known 7 – 14 Hz spindle rhythm as well as slower 6 – 8 Hz oscillations observed in TCR cells in vivo (Steriade and Llinás 1988). Received: 13 October 1993 / Accepted in revised form: 24 February 1994     

Effects of Copper on T-Type Ca<Superscript>2+</Superscript> Channels in Mouse Spermatogenic Cells

Low voltage-activated, rapidly inactivating T-type Ca²⁺ channels are found in a variety of cells, where they regulate electrical activity and Ca²⁺ entry. In whole-cell patch-clamp recordings from mouse spermatogenic cells, trace element copper (Cu²⁺) inhibited T-type Ca²⁺ current (I _T-Ca) with IC₅₀ of 12.06 μM. Inhibition of I _T-Ca by Cu²⁺ was concentration-dependent and mildly voltage-dependent. When voltage stepped to −20 mV, Cu²⁺ (10 μM) inhibited I _T-Ca by 49.6 ± 4.1%. Inhibition of I _T-Ca by Cu²⁺ was accompanied by a shift of −2.23 mV in the voltage dependence of steady-state inactivation. Cu²⁺ upshifted the current–voltage (I-V) curve. To know the change of the gating kinetics of T-type Ca²⁺ channels, we analyzed the effect of Cu²⁺ on activation, inactivation, deactivation and reactivation of T-type Ca²⁺ channels. Since T-type Ca²⁺ channels are a key component in capacitation and the acrosome reaction, our data suggest that Cu²⁺ can affect male reproductive function through T-type Ca²⁺ channels as a preconception contraceptive material.     

Modelling the Electrical Activity of Pancreatic α-cells Based on Experimental Data from Intact Mouse Islets

Detailed experimental data from patch clamp experiments on pancreatic α-cells in intact mouse islets are used to model the electrical activity associated with glucagon secretion. Our model incorporates L- and T-type Ca²⁺ currents, delayed rectifying and A-type K⁺ currents, a voltage-gated Na⁺ current, a KATP conductance, and an unspecific leak current. Tolbutamide closes KATP channels in the α-cell, leading to a reduction of the resting conductance from 1.1 nS to 0.4 nS. This causes the α-cell to depolarise from −76 mV to 33 mV. When the basal membrane potential passes the range between −60 and −35 mV, the α-cell generates action potentials. At higher voltages, the α-cell enters a stable depolarised state and the electrical activity ceases. The effects of tolbutamide are simulated by gradually reducing the KATP conductance (g _K,ATP ) from 500 pS to 0 pS. When g _K,ATP is between 72 nS and 303 nS, the model generates action potentials in the same voltage range as the α-cell. When g _K,ATP is lower than 72 nS, the model enters a stable depolarised state, and firing of action potentials is inhibited due to voltage-dependent inactivation of the Na⁺ and T-type Ca²⁺ currents. This is in accordance with experimental results. Changing the inactivation parameters to those observed in somatostatin-secreting δ-cells abolishes the depolarised inactive state, and leads to β-cell like electrical activity with action potentials generated even after complete closure of the KATP channels.     

Analysis of GABA-induced inhibition of spontaneous firing in chick accessory lobe neurons

It has been hypothesized that chick accessory lobes (ALs) contain functional neurons and act as a sensory organ of equilibrium. It was reported that neurons located in an outer layer of ALs showed γ-aminobutyric acid (GABA)- and glutamic acid decarboxylase (GAD)-like immunoreactivity more strongly than centrally located neurons, which were surrounded by the GAD-immunoreactive terminals. We investigated effects of GABA on the electrical activity of AL neurons. About 50% of embryonic AL neurons exhibited spontaneous firing. In the on-cell recording, GABA, muscimol, and GABA in combination with CGP35348 inhibited this firing. In whole-cell voltage clamp recordings, GABA and muscimol evoked a transient current. The mean reversal potential of GABA-evoked currents was close to the theoretical reversal potential of Cl⁻. These results indicate that GABA exerts the inhibitory effect on the firing through the activation of GABA_A receptors. In addition, the intracellular concentration of Cl⁻ was estimated to be about 16 mM in measurements with the gramicidin-perforated configuration, indicating the physiological reversal potential of the GABA current was about −60 mV. In conclusion, AL neurons have an intrinsic mechanism to evoke the spontaneous firing, which can be arrested by the inhibitory mechanism through the activation of the GABA_A receptors.     

The effects of amyloid peptides on A-type K+ currents of Drosophila larval cholinergic neurons: modeled actions on firing properties

In a previous paper we described the actions of beta-amyloid on an A-type K⁺ current from Drosophila 3^rd Instar larval neurons. The results were a depolarizing shift in the steady-state voltage dependence of inactivation and an increase in the rate of recovery from inactivation of the current. In this work we have used the simulation program NEURON to construct a model cell. We then use the model to predict the effects of changing the A-type K⁺ current as was observed in the amyloid treated neurons on the firing properties of the cell. We show that changing the steady-state voltage dependence of inactivation of the current to a more depolarized level as observed in experiments in beta-amyloid treated neurons causes an increase in the threshold for the initiation of repetitive firing. However, increasing the rate of recovery from inactivation had no effect. Changing both properties simultaneously had no additional effect over changing the voltage dependence of inactivation alone. Thus, a change in the steady-state properties of the A-type K⁺ current as seen in the amyloid-treated Drosophila cholinergic neurons is sufficient to alter the firing properties of the modeled cell.     

Regulation of intrinsic excitability in hippocampal neurons by activity-dependent modulation of the KV2.1 potassium channel

K_V2.1 is the prominent somatodendritic sustained or delayed rectifier voltage-gated potassium (Kv) channel in mammalian central neurons, and is a target for activity-dependent modulation via calcineurin-dependent dephosphorylation. Using hanatoxin-mediated block of K_V2.1 we show that, in cultured rat hippocampal neurons, glutamate stimulation leads to significant hyperpolarizing shifts in the voltage-dependent activation and inactivation gating properties of the K_V2.1-component of delayed rectifier K⁺ (IK) currents. In computer models of hippocampal neurons, these glutamate-stimulated shifts in the gating of the K_V2.1-component of IK lead to a dramatic suppression of action potential firing frequency. Current-clamp experiments in cultured rat hippocampal neurons showed glutamate-stimulation induced a similar suppression of neuronal firing frequency. Membrane depolarization also resulted in similar hyperpolarizing shifts in the voltage-dependent gating properties of neuronal IK currents, and suppression of neuronal firing. The glutamate-induced effects on neuronal firing were eliminated by hanatoxin, but not by dendrotoxin-K, a blocker of K_V1.1-containing channels. These studies together demonstrate a specific contribution of modulation of K_V2.1 channels in the activity-dependent regulation of intrinsic neuronal excitability.     

Action potentials in primary osteoblasts and in the MG-63 osteoblast-like cell line

Whole-cell patch-clamp analysis revealed a resting membrane potential of −60 mV in primary osteoblasts and in the MG-63 osteoblast-like cells. Depolarization-induced action potentials were characterized by duration of 60 ms, a minimal peak-to-peak distance of 180 ms, a threshold value of −20 mV and a repolarization between the spikes to −45 mV. Expressed channels were characterized by application of voltage pulses between −150 mV and 90 mV in 10 mV steps, from a holding potential of −40 mV. Voltages below −60 mV induced an inward current. Depolarizing voltages above −30 mV evoked two currents: (a) a fast activated and inactivated inward current at voltages between −30 and 30 mV, and (b) a delayed-activated outward current that was induced by voltages above −30 mV. Electrophysiological and pharmacological parameters indicated that hyperpolarization activated strongly rectifying K⁺ (K_ir) channels, whereas depolarization activated tetrodotoxin sensitive voltage gated Na⁺ (Na_v) channels as well as delayed, slowly activated, non-inactivating, and tetraethylammonium sensitive voltage gated K⁺ (K_v) channels. In addition, RT-PCR showed expression of Na_v1.3, Na_v1.4, Na_v1.5, Na_v1.6, Na_v1.7, and K_ir2.1, K_ir2.3, and K_ir2.4 as well as K_v2.1. We conclude that osteoblasts express channels that allow firing of action potentials.     

A highly stable Cu/Zn superoxide dismutase from <Emphasis Type="Italic">Withania somnifera</Emphasis> plant: gene cloning,expression and characterization of the recombinant protein

A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg⁻¹) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95°C, respectively. At 95°C, thermal inactivation rate constant (K _d) of the enzyme was 2.46 × 10⁻³ min⁻¹ and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5–11.0). It also showed a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have useful applications.     

The contribution of cationic conductances to the potential of rod photoreceptors

The contribution of cationic conductances in shaping the rod photovoltage was studied in light adapted cells recorded under whole-cell voltage- or current-clamp conditions. Depolarising current steps (of size comparable to the light-regulated current) produced monotonic responses when the prepulse holding potential (V _h) was −40 mV (i.e. corresponding to the membrane potential in the dark). At V _h = −60 mV (simulating the steady-state response to an intense background of light) current injections <35 pA (mimicking a light decrement) produced instead an initial depolarisation that declined to a plateau, and voltage transiently overshot V _h at the stimulus offset. Current steps >40 pA produced a steady depolarisation to ≈−16 mV at both V _h. The difference between the responses at the two V _h was primarily generated by the slow delayed-rectifier-like K⁺ current (I _Kx), which therefore strongly affects both the photoresponse rising and falling phase. The steady voltage observed at both V _h in response to large current injections was instead generated by Ca-activated K⁺ channels (I _KCa), as previously found. Both I _Kx and I _KCa oppose the cation influx, occurring at the light stimulus offset through the cGMP-gated channels and the voltage-activated Ca²⁺ channels (I _Ca). This avoids that the cation influx could erratically depolarise the rod past its normal resting value, thus allowing a reliable dim stimuli detection, without slowing down the photovoltage recovery kinetics. The latter kinetics was instead accelerated by the hyperpolarisation-activated, non-selective current (I _h) and I _Ca. Blockade of all K⁺ currents with external TEA unmasked a I _Ca-dependent regenerative behaviour.     

Kinetic Analysis of Miniature Synaptic Currents in Rat Substantia Gelatinosa Neurons

Interneurons of the substantia gelatinosa (SG) form a complex synaptic network in the dorsal horn of the spinal cord. The properties of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively) were studied in spinal cord slices of 3- to 4-week-old rats. The reversal potentials of the currents were close to 0 mV for excitatory and –70 mV for inhibitory events. Under recording conditions close to physiological ones (holding potential –40 mV, temperature 32°C, low intracellular [Cl^–]), the mean rise times of these currents were, respectively, 1.0 and 1.8 msec. The decay of the currents was monoexponential in the majority of occurrences (94 and 91.4%), with a time constant (τ) of 2.7 msec for mEPSCs and 7.2 msec for mIPSCs. A part (8.6%) of mIPSCs had an additional slow component with τ = 30.1 msec. All mEPSCs were blocked by 10 mM CNQX, an antagonist of the AMPA/kainate subtype of glutamate receptors. Monoexponential mIPSCs were blocked by 1 mM strychnine, an antagonist of glycine receptors, while two-component mIPSCs required the additional presence of 10 mM bicuculline, a blocker of GABAA receptors. Only two cells of 23 (~9%) demonstrated pure GABA-ergic mIPSCs (τ = 26.2 msec). It is concluded that, under physiological conditions, AMPA/kainate but not NMDA receptors mediate excitatory synaptic transmission in SG neurons. Synaptic inhibition is mediated predominantly by glycine receptors, with mild fractions of IPSCs provided by GABA-ergic transmission and GABA/glycine co-release.     

An Inactivating Inward-rectifying K Current Present in Prolactin Cells from the Pituitary of Lactating Rats

Primary cultures containing a high percentage of lactotrophs were obtained by dissociating the pituitary of rats following 14–18 days of lactation. Lactotrophs with a distinctive appearance were recorded after 1–35 days in vitro and identified by immunocytochemical staining for prolactin. Whole-cell voltage clamp measurements in isotonic KCl solution from a holding potential of −40 mV revealed the presence of inward-rectifying K currents with a time-dependent, Na⁺-independent inactivation at potentials negative to −60 mV. The time for complete inactivation was strikingly different between lactotrophs, varying between 1 sec and more than 5 sec at −120 mV, and was not related to time in culture. The reversal potential shifted 59 mV (25°C) for a tenfold change in external K⁺ concentration, demonstrating the selectivity of the channel for K⁺ over Na⁺. The inward-rectifying K current was blocked by 5 mm Ba²⁺ and partially blocked by 10 mm TEA. Chloramine-T (1 and 2 mm) produced a total block of the inward-rectifying K current in lactotrophs. Thyrotropin-releasing hormone (500 nm) significantly reduced the inward-rectifying K current in about half of the lactotrophs. This current is similar to the inward-rectifying K current previously characterized in clonal somatomammotrophic pituitary cells (GH₃B₆). The variability of the rate of inactivation of this current in lactotrophs and its responsiveness to TRH is discussed. Received: 28 September 1995/Revised: 11 December 1995