Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 m, a width of 21±5 , and a height of 10±4 m (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current =35±14 s is very short. Series resistancer _s, membrane resistancer _m, and membrane capacityc _m are calculated to be 192±48 k, 6.1±1.1 M, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 F/cm², a total membrane area of 1.86×10^–4 cm² is determined yielding a specific membrane resistanceR _m of 1,134 cm². Settling time of voltage clamp is 30 s. TTX-block of sodium current is described by 1:1 binding with aK _D value of 1.4×10^–6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between –40 and –30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m³h model. The time constant of activation decreases from 0.7 ms at –60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at –60 mV to 0.6 ms at +20 mV.Steady state inactivationh is characterized by the half maximum valueV _H=–76.1±4.3 mV and the slope parameters=–6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm and inactivationh overlap each other defining a maximum window current at –65 mV.     

A single non-L-, non-N-type Ca2+ channel in rat insulin-secreting RINm5F cells

Single high-voltage-activated (HVA) Ca²⁺ channel activity was recorded in rat insulinoma RINm5F cells using cell-attached and outside-out configurations. Single-channel recordings revealed three distinct Ca²⁺ channel subtypes: one sensitive to dihydropyridines (DHPs)-(L-type), another sensitive to -conotoxin (CTx)-GVIA (N-type) and a third type insensitive to DHPs and -CTx-GVIA (non-L-, non-N-type). The L-type channel was recorded in most patches between –30 and +30 mV The channel had pharmacological and biophysical features similar to the L-type channels described in other insulin-secreting cells (mean conductance 21 pS in control conditions and 24 pS in the presence of 5 M Bay K 8644). The non-L-, non-N-type channel was recorded in cells chronically treated with -CTx-GVIA in the presence of nifedipine to avoid the contribution of N- and L-type channels. Channel activity was hardly detectable below –10 mV and was recruited by negative holding potentials (< –90 mV). The channel open probability increased steeply from –10 to +40 mV Different unitary current sublevels could be detected and the current voltage relationship was calculated from the higher amplitude level with a slope conductance of 21 pS. Channel activity lasted throughout depolarizations of 300–800 ms with little sign of inactivation. Above 0 mV the channel showed a persistent flickering kinetics with brief openings (₀ 0.6 ms) and long bursts (_burst 60 ms) interrupted by short interburst intervals. The third HVA Ca²⁺ channel subtype, the N-type, had biophysical properties similar to the non-L-, non-N-type and was best identified in outside-out patches by its sensitivity to -CTx-GVIA. The channel was detectable only above –10 mV from a –90 mV holding potential, exhibited a fast flickering behaviour, persisted during prolonged depolarizations and had a slope conductance of about 19 pS. The present data provide direct evidence for a slowly inactivating non-L-, non-N-type channel in insulin-secreting RINm5F cells that activates at more positive voltages than the L-type channel and indicate the possibility of identifying unequivocally single HVA Ca²⁺ channels in cell-attached and excised membrane patches under controlled pharmacological conditions.     

Voltage-dependent noradrenergic modulation of ω-conotoxin-sensitive Ca2+ channels in human neuroblastoma IMR32 cells

High-threshold (HVA) Ca²⁺ channels of human neuroblastoma IMR32 cells were effectively inhibited by noradrenaline. At potentials between –20 mV and +10 mV, micromolar concentrations of noradrenaline induced a 50%–70% depression of HVA Ba²⁺ currents and a prolongation of their activation kinetics. Both effects were relieved at more positive voltages or by applying strong conditioning pre-pulses (facilitation). Facilitation restored the rapid activation of HVA channels and recruited about 80% of the noradrenaline-inhibited channels at rest. Re-inhibition of Ca²⁺ channels after facilitation was slow ( _r 36–45 ms) and voltage-independent between –30 mV and –90 mV. The inhibitory action of noradrenaline was dose-dependent (IC₅₀=84 nM), mediated by ₂-drenergic receptors and selective for -conotoxin-sensitive Ca²⁺ channels, which represent the majority of HVA channels expressed by IMR32 cells. The action of noradrenaline was mimicked by intracellular applications of GTP[S] and prevented by GDP[S] or by pre-incubation with pertussis toxin. The time course of noradrenaline inhibition measured during fast application (onset) and wash-out (offset) of the drug were independent of saturating agonist concentrations (10–50 M) and developed with mean time constants of 0.56 s ( _on) and 3.6 s ( _off) respectively. The data could be simulated by a kinetic model in which a G protein is assumed to modify directly the voltage-dependent gating of Ca²⁺ channels. Noradrenaline-modified channels are mostly inhibited at rest and can be recruited in a steep voltage-dependent manner with increasing voltages.     

Arginine-vasopressin induces mode-2 gating in L-type Ca2+ channels (smooth muscle cells of the urinary bladder of the guinea-pig)

The effect of arginine-vasopressin (AVP, 0.1 M) on elementary Ca²⁺ channel currents (L-type) was studied in cell-attached patches with 10 mM BaCl₂ as the charge carrier. At a constant potential of –30 mV, bath applied AVP increased the channel openness (NP _o) by a factor of 4.7±3.0 (mean±SD, n=9), the effect resulted from an increase in the frequency of opening (factor 2.5±0.8) and from a longer mean open time. Under control, openings longer than 5 ms contributed only 4% of the total, however, with the application of AVP this contribution increased to 29%. Under control, the open times were distributed along a single exponential (_o1=0.8±0.4 ms), a double exponential distribution was obtained during AVP (_o1=0.8±0.5 ms, _o2=7.5±0.7 ms). The Ca²⁺ agonist BAYk8644 (1 M) changed the open time distribution similarly to AVP (_o1=1.0±0.5 ms, _o2=9±2.8 ms). With 1 M BAYk8644 in the bath, AVP did not significantly increase the relative contribution of long openings, however, AVP increased the frequency of openings by a factor of 2.0±1 (n=6). The results are compatible with the idea that AVP can change the gating of L-type Ca²⁺ channels from mode 1 to mode 2.     

Glucose dependent K+-channels in pancreaticβ-cells are regulated by intracellular ATP

The resting conductance of cultured-cells from murine pancreases was investigated using the whole-cell, cell-attached and isolated patch modes of the patch-clamp technique. Whole-cell experiments revealed a high input resistance of the cells (>20 G per cell or>100 k·cm²), if the medium dialysing the cell interior contained 3 mM ATP. The absence of ATP evoked a large additional K⁺ conductance. In cell-attached patches single K⁺-channels were observed in the absence of glucose. Adition of glucose (20 mM) to the bath suppressed the channel activity and initiated action potentials. Similar single-channel currents were recorded from isolated patches. In this case the channels were reversibly blocked by adding ATP (3 mM) to the solution at the intracellular side of the membrane. The conductances (51 pS and 56 pS for [K⁺]₀=145 mM, T=21° C) and kinetics (at –70 mV: _open=2.2 ms and _closed=0.38 ms and 0.33 ms) of the glucose- and ATP-dependent channels were found to be very similar. It is concluded that both channels are identical. The result suggests that glucose could depolarize the-cell by increasing the cytoplasmic concentration of ATP.     

Cyclic AMP-dependent phosphorylation modifies the gating properties of L-type Ca2+ channels in bovine adrenal chromaffin cells

We investigated the effects of cAMP-dependent phosphorylation on the voltage- and time-dependent gating properties of Ca²⁺ channel currents recorded from bovine adrenal chromaffin cells under whole-cell voltage clamp. Extracellular perfusion with the membrane-permeant activator of cAMP-dependent protein kinase, 8-bromo(8-Br)-cAMP (1 mM), caused a 49%, 29%, and 21% increase in Ca²⁺ current (I _Ca) amplitudes evoked by voltage steps to 0, +10, and +20 mV respectively (mean values from eight cells, p0.05). Analysis of voltage-dependent steady-state activation (m ) curves revealed a 0.70±0.27 charge increase in the activation-gate valency (z _m) following 8-Br-cAMP perfusion. Similar responses were observed when Ba²⁺ was the charge carrier, where z _m was increased by 1.33±0.34 charges (n=8). The membrane potential for half activation (V _1/2) was also significantly shifted 6 mV more negative for I _Ba (mean, n=8). The time course for I _Ba (and I _Ca) activation was well described by second-order m ² kinetics. The derived time constant for activation (_m) was voltage-dependent, and the _m/V relation shifted negatively after 8-Br-cAMP treatment. Ca²⁺ channel gating rates were derived from the (_m) and m ² values according to a Hodgkin-Huxley type m ² activation process. The forward rate ( _m) for channel activation was increased by 8-Br-cAMP at membrane potentials 0 mV, and the backward rate (_m) decreased at potentials +10 mV. Time-dependent inactivation of I _Ca consisted of a slowly decaying component (_h 300 ms) and a non-inactivating steady-state component. The currents contributed by the two inactivation processes displayed different voltage dependences, the effects of 8-Br-cAMP being exclusively on the slowly inactivating L-type component.     

Two types of transient outward currents in cardiac ventricular cells of mice

Ventricular cells of adult mice were prepared by an enzyme digestion procedure. Single channel currents were recorded by a conventional patch clamp technique from cell attached patches. Voltage steps from the holding potential of –80 mV to test potentials between –35 and +50 mV caused openings of two types of outward currents through single channels with the conductances of 27 and 12 pS, respectively. The averaged currents reveal transient time courses for both channel types. The current-voltage relations of both single channel currents were linear over the tested voltage range and intersected the voltage axis at –70 mV. This indicates that both single channel currents are mainly carried by potassium ions. All open and closed times were found to be voltage independent. The 27 pS channel had a mean open time of 3.9±1.0 ms (n=8). The closed time consisted of two components with ₁ = 2.1 ± 0.2 ms and ₂ = 50 ± 19 ms (n=8). The 12 pS channel had a mean open time of 34.0±5.2 ms (n=3) and the two components of the mean closed time have been calculated as ₁ = 8.3 ± 2.1 ms and ₂ = 120 ± 50 ms (n=3; all mean ±SD).     

Analysing ion channels with hidden Markov models

Ion channel current amplitudes () and open probabilities (P _o) have been analysed so far by defining a 50% threshold to distinguish between open and closed states of the channels. With this standard method (SM) it is very difficult or even impossible to analyse channels of different size in one membrane patch correctly. A stochastical model, named the hidden Markov model (HMM), separates between observation noise and the stochastic process of opening and closing of ion channels. The HMM allows the independent analysis of , P _o, and mean dwell times () of different channels in one membrane patch, without defining threshold levels. Using this method errors in the analysis are not summarized like in the SM because all different analysing procedures (e. g. filtering, setting of threshold, fitting processes) are done in one step. Two different K⁺ channels in excised basolateral membranes of the cortical collecting duct of rat (CCD) were analysed by the SM and the HMM. The value of the intermediate-conductance K⁺ channel (i-K⁺) was 3.9±0.1 pA (SM) and 3.8±0.2 pA (HMM) for 11 observations. The P _o value of this channel was 10.2±4.2% (SM) and 10.1±4.0% (HMM). The mean values were 5.4±0.6 ms for the open state and 9.6±2.2 ms and 145±21 ms for the closed states (SM) and 7.8±1.1 ms, 7.7±0.9 ms and 148±24 ms (HMM), respectively. For seven small-conductance K⁺ (s-K⁺) channels, which were found in the same membrane patches as the i-K⁺, an accurate analysis of P _o and was not possible with the SM. The value was 1.0±0.1 (SM), 0.9±0.1 (HMM) pA. P _o was 16.6±4.6%, the open value was 11.1±2.8 ms, and the closed value was 34.9±8.5 ms. The HMM allows the analysis of single-channel currents, P _o, and mean values when different or more than one ion channel(s) are colocalized in one membrane patch. Where analysis with the SM was possible results did not significantly differ from those obtained with the HMM. Thus for this kind of analysis the method of setting a 50% threshold appears justified.     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.     

Glutathione accelerates sodium channel inactivation in excised rat axonal membrane patches

The effects of glutathione were studied on the gating behaviour of sodium channels in membrane patches of rat axons. Depolarizing pulses from –120 to –40 mV elicited sodium currents of up to 500 pA, indicating the simultaneous activation of up to 250 sodium channels. Inactivation of these channels in the excised, inside-out configuration was fitted by two time constants ( _h1=0.81 ms; _h2= 5.03 ms) and open time histograms at 0 mV revealed a biexponential distribution of channel openings ( _short=0.28 ms; _long=3.68 ms). Both, the slow time constant of inactivation and the long lasting single channel openings disappeared after addition of the reducing agent glutathione (2–5 mM) to the bathing solution. Sodium channels of excised patches with glutathione present on the cytoplasmatic face of the membrane had inactivation kinetics similar to channels recorded in the cell-attached configuration. These observations indicate that redox processes may contribute to the gating of axonal sodium channels.     

Ca currents in human neuroblastoma IMR32 cells: kinetics,permeability and pharmacology

We have investigated the kinetics, permeability and pharmacological properties of Ca channels in in vitro differentiated IMR32 human neuroblastoma cells. The lowthreshold (LVA, T) Ca current activated positive to –50 mV and inactivated fully within 100 ms in a voltage-dependent manner. This current persisted in the presence of 3.2 M -conotoxin (-CgTx) or 40 M Cd and showed a weaker sensitivity to Ni and amiloride than in other neurons. The high-threshold Ca currents (HVA,L and N) turned on positive to –30 mV, and inactivated slowly and incompletely during pulses of 200 ms duration. The amplitude of the HVA currents and the number of ¹²⁵I--CgTx binding sites increased markedly during cell differentiation. In agreement with recent reports, 6.4 M -CgTx blocked only about 85% of the Ba currents through HVA channels in 50% of the cells. Residual -CgTx-resistant currents proved to be more sensitive to dihydropyridines (DHP) than total HVA currents. Bay K 8644 (1 M) had a clear agonistic action on -CgTx-resistant currents and was preferred to other Ca antagonists for identifying HVA DHP-sensitive channels. Compared to the -CgTx-sensitive, the DHP-sensitive currents turned on at slightly more negative potentials and showed a weaker sensitivity to voltage. The two HVA currents were otherwise hardly distinguishable in terms of activation/inactivation kinetics, Ca/Ba permeability and sensitivity to holding potentials. This suggests that currently used criteria for identifying multiple types of neuronal Ca channels (T,L,N) may be widely misleading if not supported by pharmacological assays.     

Modification of electrophysiological and pharmacological properties of K channels in neuroblastoma cells induced by the oxidant chloramine-T

The effects of chloramine-T (CL-T) on voltage-dependent potassium channels in neuroblastoma cells were analysed using the whole-cell current recording technique. CL-T irreversibly decreased the peak whole — cell K current, considerably slowed its inactivation and shifted its activation-voltage curve towards positive voltages by 6 mV. Under control conditions, the inactivation of the whole-cell K current could be described by the sum of two exponentials, F and S, whose time constants at +50 mV were _F=1.00±0.15 s and _S=5.72±0.47 s respectively. After CL-T, it could be described by the sum of two (S₁ and S₂) or three (F, S₁ and S₂) exponentials whose time constants at +50 mV were: _F=0.81±0.22 s, _S1=6.46±0.60 s and _S2=48.56±3.64 s. Under control conditions, F and S inactivating components of the whole-cell K current were blocked by 4-aminopyridine, with a Hill coefficient of 1 and apparent dissociation constants of 0.04 and 0.7 mM respectively. After CL-T, both S₁ and S₂ components were equally blocked by 4-aminopyridine with a Hill coefficient of 0.25, being reduced to 64% of their control values by 10 mM. CL-T is known to slow the inactivation of sodium channels and to oxidize sulphydryl amino acids and unsaturated lipids. It is concluded that the inactivation gates of voltage-dependent sodium and potassium channels are either constituted of the same amino acid residues or are controlled by unsaturated lipids surrounding or bound to the channel proteins.     

Neuronal mechanisms for pitch analysis in the time domain

Summary Many units in the auditory midbrain nucleus (MLD) of the Guinea fowl are found to be tuned to amplitude modulated tones (AM). For a given response maximum the relationship of the period _m of the modulation frequency f_m and the period _c of the carrier frequency f_c may be given by an empirical equation: m · _m + n · _c = 1 · ₁, where m, n and 1 are small integers typical for a unit. ₁ is a time constant of 0.4 ms. The temporal pattern of the neuronal response support these findings. The averages of spike trains oscillate with periods multiple to ₁. These oscillations are elicited by stimulus onsets and zero crossings of f_m and may be coupled strongly to f_m depending on f_c. Variation of f_m or f_c shifts the mean delay of the phase coupled activity proportional to m · _m and n · _c, respectively. These effects may be explained with activity phase coupled to f_c which coincides at the level of the recorded units with oscillations coupled to f_m. This is expressed by the above given periodicity equation. Psychophysical results with AM-stimuli indicate that the mechanisms described and the periodicity equation are adequate for the explanation of the analysis of periodicity pitch in humans. Hence the period corresponding to pitch is defined by m · _m + n · _c = 1 · ₁, where n and 1 are integers and ₁ = 0.4 ms. Plots of _p as a function of _c reveal steps at 0.4 ms intervals indicating that the neuronal time constant is the same in both species.Supported by the DFG, SFB 45     

Ca2+ not cyclic AMP mediates the fluid secretory response to isoproterenol in the rat mandibular salivary gland: Whole-cell patch-clamp studies

We have performed whole-cell patch-clamp studies on dispersed seccretory cells of the rat mandibular gland to determine how -adrenergic stimulation causes fluid secretion. When the pipette contained a high K⁺ solution, the resting membrane potential averaged –33 mV±1.1 (SEM,n=34) and the clamped cell showed strong outward rectification. We monitored K⁺ and Cl^– currents for periods of 15 min by recording the currents needed to clamp the cell potential at 0 and –80 mV, respectively. Isoproterenol (1–2 mol/l) caused increases in the clamp current at 0 mV (the K⁺ current) and at –80 mV (the Cl^– current) in about 80% of cases, although the responses were variable in size and time-course; the responses were indistinguishable from those induced by acetylcholine or the Ca²⁺ ionophore, A23187. The -adrenergic antagonist, phentolamine (1–2 mol/l), had no effect on the response, but the -adrenergic antagonist, propranolol (10 mol/l), blocked it completely. The isoproterenol response could not be mimicked by application to either surface of the cell membrane, of cyclic AMP (100 mol/l), forskolin (1 or 20 mol/l) or cholera toxin (2.5 g/ml). However, increasing the Ca²⁺-chelating capacity of the pipette solution by raising its EGTA concentration from the customary 0.5 to 20 mmol/l, blocked the response to isoproterenol, suggesting that -adrenergic agonists activate Cl^– and K⁺ channels by raising cytosolic Ca²⁺. Since neomycin, which blocks phospholipase C, blocked the action of isoproterenol without impairing the cell responsiveness to A23187, it appears that isoproterenol, like muscarinic agonists, increased cytosolic Ca²⁺ via the phosphatidylinositol cycle.This project was supported by the National Health and Medical Research Council of Australia     

Inhibitory synaptic channels activated by γ-aminobutyric acid (GABA) in crayfish muscle

Small crayfish muscle fibres were voltage clamped and synaptic current elicited by superfused GABA solutions was measured. Analysis of the fluctuations of synaptic current and of relaxations of the current after voltage steps yielded analogous results. The current has two components. The first component is characterized by the opening of synaptic channels with a single channel conductance =9 pS and an average open time =5 ms, measured at 23°C and-100 mV. depends on the membrane potential, _E = ₀ · e ^E/, and was about +100 mV in the average. The channel open time agrees with the time constant of decay of the inhibitory postsynaptic current (IPSC) elicited by a nerve stimulus. The current is carried by chloride ions. The second current component is much slower, the average channel open time was _s = 33 ms at 23°C and-60 mV. The open time _s of the slow component also was shortened on hyperpolarization. The reversal potential for the current component was more positive than-50 mV. This slow component also seems to be a synaptic one.This investigation was supported by a grant of the Deutsche Forschungsgemeinschaft     

GM-CSF counteracts hemorrhage-induced suppression of cytokine-producing capacity

Objective and design: To study whether a treatment with the hematopoietic growth factor GM-CSF restores the attenuated ex-vivo cytokine-producing capacity of macrophages after sublethal hemorrhagic shock.Subjects: Male Sprague-Dawley rats.Treatment: 20 g/animal of recombinant murine GM-CSF after shock via arterial line.Methods: Hemorrhagic shock was established by pressure-controlled bleeding to a mean arterial pressure of 50 mm Hg for 35–40 min and consecutive resuscitation. 24 h after hemorrhage, lipopolysaccharide (LPS)-induced cytokine production of isolated macrophages derived from different compartments was measured.Results: A significant reduction of LPS-induced TNF production was found in whole blood cultures (1.0 ± 0.7 ng/ml after sham vs. 0.23 ± 0.08 ng/ml after shock operation), macrophages derived from spleen (0.88 ± 0.23 ng/ml after sham vs. 0.03 ± 0.1 ng/ml after shock operation), peritoneum (2.2 ± 0.7 ng/ml after sham vs. 0.29 ± 0.4 ng/ml after shock operation) and bronchoalveolar fluid (0.65 ± 0.13 ng/ml after sham vs. 0.003 ± 0.027 ng/ml after shock operation, mean ± S.D.). In cells from animals treated with GM-CSF a significantly enhanced LPS-induced TNF production in splenic, alveolar and peritoneal macrophages was found after shock compared to the cells derived from untreated animals (peritoneum: 289 ± 366 ng/ml TNF after shock vs. 2066 ± 94 ng/ml TNF after shock and GM-CSF; lung: 9 ± 12 ng/ml TNF after shock vs. 64 ± 17 ng/ml TNF after shock and GM-CSF; spleen: 58 ± 96 ng/ml TNF after shock vs. 548 ± 47 ng/ml TNF after shock and GM-CSF). Blood cultures collected from rats after hemorrhagic shock did not show a significant increase of TNF-production after GM-CSF treatment.Conclusion: Hemorrhagic shock caused a depression of the TNFa response to LPS which was partly counteracted by treatment with GM-CSF. Therefore, GM-CSF represents a promising approach to normalise trauma- and shock-induced immune dysfunction.Received 4 April 2003; returned for revision 3 July 2003; accepted by A. Falus 25 August 2003     

Characterization of two components of the N-like,high-threshold-activated calcium channel current in differentiated SH-SY5Y cells

Retinoic acid differentiated SH-SY5Y cells exhibit only a high-threshold-activated (–30 to –20 mV) whole cell calcium channel current. When barium was used as the charge carrier, the high-threshold-activated current showed bi-exponential inactivation kinetics during a 500 ms voltage step from –90 to +10mV. The time constants of inactivation were approximately 75 and 750 ms. The fast inactivating component was more sensitive than the slow inactivating component to steady-state inactivation at depolarized holding potentials. The calcium channel current was inhibited by externally applied cadmium (10–300 M) and gadolinium (10–30 M) as well as by high concentrations of nickel and cobalt, Conus toxin (1 M) irreversibly blocked the calcium channel current. However, the dihydropyridine agonist, BAY K 8644 (3–10 M) and antagonists, nifedipine (3–10 M) and nimodipine (10 M) did not affect either component of the calcium channel current. Agents which blocked the calcium channel current did not exhibit any selectivity for the fast inactivating over the slow inactivating component of the current. These results indicate that whilst the calcium channel current recorded in differentiated SH-SY5Y cells can be classified on the basis of the blocking agents as being of the N type, the current shows more than one form of inactivation.     

Neural nicotinic acetylcholine responses in solitary mammalian retinal ganglion cells

Using the patch-clamp technique,whole-cell recordings from solitary rat retinal ganglion cells in culture have established the nicotinic nature of the acetylcholine responses in these central neurons. Currents produced by acetylcholine (5–20 mol/l) or nicotine (5–20 mol/l) reversed in polarity near –5 mV and were unaffected by atropine (10 mol/l). Agonist-induced currents were blocked by low doses(2–10 mol/l) of the classical ganglionic antagonists hexamethonium and mecamylamine, as well as by d-tubocurarine and dihydro--erythroidine (the latter two do not discriminate clearly between ganglionic and neuromuscular junction receptors). Treatment with the potent neuromuscular blocking agent -bungarotoxin (10 mol/l) did not affect the cholinergic responses of these cells, while toxin F (0.2 mol/l), a neural nicotinic receptor antagonist, readily abolished acetylcholine-induced currents. Thus, the experiments performed to date show that the nicotinic responses of retinal ganglion cells in the central nervous system share the pharmacology of autonomic ganglion cells in the peripheral nervous system. The ionic current carried by the nicotinic channels was selective for cations, similar to that described for nicotinic channels in other tissues. In addition, single-channel currents elicited by acetylcholine were observed in whole-cell recordings with seals > 5 G as well as in occasional outside-out patches of membrane. These acetylcholine-activated events, which had a unitary conductance of 48 pS and a reversal potential of 0 mV, represent the ion channels that mediate the neural nicotinic responses observed in these experiments on retinal ganglion cells.     

TTX-sensitive Na+ channels and Ca2+ channels of the Land N-type underlie the inward current in acutely dispersed coeliac-mesenteric ganglia neurons of adult rats

Inward membrane currents of sympathetic neurons acutely dispersed from coeliac-superior mesenteric ganglia (C-SMG) of adult rats were characterized using the whole-cell variant of the patch-clamp technique. Current-clamp studies indicated that C-SMG neurons retained electrical properties similar to intact ganglia. Voltage-clamp studies designed to isolate Na⁺ currents revealed that tetrodotoxin (TTX, 1 M) completely inhibited the large transient inward current. Half activation potential (V _h) and slope factor (K) were –26.8 mV and 6.1 mV, respectively. Inactivation parameters for V _h and K were –65 mV and 8.2 mV, respectively. Voltage-clamp studies also revealed a high-voltage-activated sustained inward Ca²⁺ current which was blocked by the removal of external Ca²⁺ or the presence of Cd²⁺ (0.1 mM). The dihydropyridine agonist, (+)202–791 (1 M), caused a small increase (20%) in the amplitude of the Ca²⁺ current at more negative potentials and markedly prolonged the tail currents. -Conotoxin GIVA (, CgTX, 15 M) caused a 66% inhibition of the high-voltage-activated Ca²⁺ current amplitude. Norepinephrine (1 M) caused a 49% reduction in the peak Ca²⁺ current. This study is the first demonstration that dispersed C-SMG neurons from adult rats retain electrical characteristics similar to intact ganglia. A TTX-sensitive Na⁺ current as well as a high voltage-activated sustained Ca²⁺ current underlie the inward current in C-SMG neurons. The macroscopic Ca²⁺ current is composed of a small dihydropyridinesensitive (L-type current) and a large -CgTx-sensitive (N-type current) component. Thus, acutely dispersed CSMG neurons are suitable for examining the biophysical properties and modulation of membrane currents of adult prevertebral sympathetic neurons in normal and diseased states.     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.