GABA increases both the conductance and mean open time of recombinant GABAA channels co-expressed with GABARAP

The single channel properties of recombinant gamma-aminobutyric acid type A (GABA(A))alphabetagamma receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing alphabetagamma receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA(A) receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (> or =40 pS), and GABA (> or =10 microM) was able to increase their conductance, up to a maximum of 60 pS. The mean open time of GABA-activated channels in control cells expressing alphabetagamma receptors alone was 2.3 +/- 0.1 ms for the main 30-pS channel and shorter for the subconductance state (20 pS, 0.8 +/- 0.1 ms). Similar values were measured for the 30- and 20-pS channels active in patches from cells co-expressing GABARAP. However higher conductance channels (> or =40 pS) remained open longer, irrespective of whether GABA or GABA plus diazepam activated them. Plotting mean open times against mean conductances revealed a linear relationship between these two parameters. Since high GABA concentrations increase both the maximum single channel conductance and mean open time of GABA(A) channels co-expressed with GABARAP, trafficking processes must influence ion channel properties. This suggests that the organization of extrasynaptic GABA(A) receptors may provide a range of distinct inhibitory currents in the brain and, further, provide differential drug responses.     

Changes in membrane ionic conductance, but not changes in slip, can account for the non-linear dependence of the electrochemical proton gradient upon the electron-transport rate in chromatophores

Decrease in the rate of cyclic electron transport (JE) measured from the absorbance changes associated with reaction centre bacteriochlorophyll led to a less than proportionate decrease in the membrane potential (delta psi) measured by electrochromism. In principle this result can be explained either by a delta psi-dependent slip in the H+/e- coupling ratio (nE) or by a delta psi-dependent change in the membrane ionic conductance. Simultaneous measurement of the membrane ionic current (JDIS) did not reveal any significant changes in the H+/e- ratio (JDIS/JE) and showed that conductance changes (JDIS/delta psi) account quantitatively for the curved dependence of delta psi on JE. Simultaneous recordings of JDIS and the extravesicular pH from cresol-red absorbance changes, suggest that protons are the main current-carrying species across the chromatophore membrane at high values of delta psi in the presence and absence of Fo-ATPase inhibitor. At reduced delta psi the flux of other ions outweighs the hydrogen ion current.     

Ion channels activated by L-glutamate and GABA in cultured cerebellar neurons of the rat

Glutamate and GABA-receptor channels were investigated in explants of rat cerebellum grown in cell culture. The patch-clamp technique was used to examine neurons under whole cell clamp and the properties of channels were derived by analysis of glutamate and GABA-evoked current noise. In addition, single channel currents activated by glutamate were recorded from isolated outside-out patches of membrane. We found evidence for at least two types of glutamate receptor-channels in cerebellar cells. Some neurons exhibited a channel of 50 pS conductance with a Lorentzian noise spectrum of 5.9 ms time constant. Single channels were readily resolved both in whole cell clamp and excised patches. Other neurons possessed low conductance channels which produced two component spectra. Estimates of the single channel conductance gave a value of about 140 fS. GABA channel noise obtained from these cells was also fitted by two component spectra which gave single channel conductance of 16 pS.     

Cell-attached patch clamp study of the electropermeabilization of amphibian cardiac cells.

Potential gradients imposed across cell or lipid membranes break down the insulating properties of these barriers if an intensity and time-dependent threshold is exceeded. Potential gradients of this magnitude may occur throughout the body, and in particular in cardiac tissue, during clinical defibrillation, ablation, and electrocution trauma. To study the dynamics of membrane electropermeabilization a cell-attached patch clamp technique was used to directly control the potential across membrane patches of single ventricular cells enzymatically isolated from frog (Rana pipiens) hearts. Ramp waveshapes were used to reveal rapid membrane conductance changes that may have otherwise been obscured using rectangular waveshapes. We observed a step increase (delta t less than 30 microseconds) or breakdown in membrane conductance at transmembrane potential thresholds of 0.6-1.1 V in response to 0.1-1.0 kV/s voltage ramps. Conductance kinetics on a sub-millisecond time scale indicate that breakdown is preceded by a period of instability during which the noise and amplitude of the membrane conductance begin to increase. In some cells membrane breakdown was observed to be fully reversible when using an intershock interval of 1 min (20-23 degrees C). These findings support energetic models of membrane electropermeabilization which describe the formation of membrane pores (or growth of existing pores) to a conducting state (instability), followed by a rapid expansion of these pores when the energy barrier for the formation of hydrophilic pores is overcome (breakdown).     

Downregulation of ClC-2 by JAK2

JAK2 (Janus kinase-2) is activated by cell shrinkage and may thus participate in cell volume regulation. Cell volume regulatory ion channels include the small conductance Cl(-) channels ClC-2. The present study thus explored whether JAK2 influences ClC-2 activity. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild type JAK2, active (V617F)JAK2 or inactive (K882E)JAK2 and the Cl(-) channel activity determined by dual electrode voltage clamp. Expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Exposure of the oocytes expressing ClC-2 together with (V617F)JAK2 to the JAK2 inhibitor AG490 (40 μM) resulted in a gradual increase of the conductance. According to chemiluminescence JAK2 decreased the channel protein abundance in the cell membrane. The decline of conductance in ClC-2 and (V617F)JAK2 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with (V617F)JAK2 and oocytes expressing ClC-2 alone, indicating that (V617F)JAK2 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK2 down-regulates ClC-2 activity and thus counteracts Cl(-) exit, an effect which may impact on cell volume regulation.     

Effects of physostigmine on the voltage dependent ionic conductances of skeletal muscle fibres

The voltage dependent ionic conductances were studied by analysing the phase plane trajectories of action potentials evoked by electrical stimulation of the sartorius muscles of the frog (Rana esculenta). The delayed outward potassium current was measured also under voltage clamp conditions on muscle fibres of either the frog (Rana esculenta) or Xenopus laevis. On analysing the effect of physostigmine decreasing the peak amplitude, the rate of both the rising and falling phases of the action potentials, it was revealed that the alkaloid at a concentration of 1 mmol/l reduced significantly both the delayed potassium conductance and the outward ionic current values during the action potentials. The inhibition of sodium conductance and inward ionic current was less expressed. The maximum value of delayed potassium conductance measured under voltage clamp conditions was decreased by 1 mmol/l physostigmine. The time constant determined from the development of delayed potassium conductance was increased at a given membrane potential. The voltage vs. n relationship describing the membrane potential dependence of the delayed rectifier was not influenced by physostigmine. It has been concluded that physostigmine changes the time course of the action potentials by decreasing the value of both voltage dependent ionic conductances and by slowing down their kinetics. It is discussed that results obtained from the phase plane analysis of complex pharmacological effects can only be accepted with some restrictions.     

Identification and functional characterization of a dual GABA/taurine transporter in the bullfrog retinal pigment epithelium

Intracellular microelectrodes, fluorescence imaging, and radiotracer flux techniques were used to investigate the physiological response of the retinal pigment epithelium (RPE) to the major retinal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). GABA is released tonically in the dark by amphibian horizontal cells, but is not taken up by the nearby Muller cells. Addition of GABA to the apical bath produced voltage responses in the bullfrog RPE that were not blocked nor mimicked by any of the major GABA-receptor antagonists or agonists. Nipecotic acid, a substrate for GABA transport, inhibited the voltage effects of GABA. GABA and nipecotic acid also inhibited the voltage effects of taurine, suggesting that the previously characterized beta- alanine sensitive taurine carrier also takes up GABA. The voltage responses of GABA, taurine, nipecotic acid, and beta-alanine all showed first-order saturable kinetics with the following Km's: GABA (Km = 160 microM), beta-alanine (Km = 250 microM), nipecotic acid (Km = 420 microM), and taurine (Km = 850 microM). This low affinity GABA transporter is dependent on external Na, partially dependent on external Cl, and is stimulated in low [K]o, which approximates subretinal space [K]o during light onset. Apical GABA also produced a significant conductance increase at the basolateral membrane. These GABA-induced conductance changes were blocked by basal Ba2+, suggesting that GABA decreased basolateral membrane K conductance. In addition, the apical membrane Na/K ATPase was stimulated in the presence of GABA. A model for the interaction between the GABA transporter, the Na/K ATPase, and the basolateral membrane K conductance accounts for the electrical effects of GABA. Net apical-to-basal flux of [3H]-GABA was also observed in radioactive flux experiments. The present study shows that a high capacity GABA uptake mechanism with unique pharmacological properties is located at the RPE apical membrane and could play an important role in the removal of GABA from the subretinal space (SRS). This transporter could also coordinate the activities of GABA and taurine in the SRS after transitions between light and dark.     

Inhibiton and gamma-aminobutyric acid-induced conductance on locust (Schistocerca gregaria) extensor tibiae muscle fibres

• 1.1. Increases in membrane conductance (g_m) were induced by GABA in distal bundles 32, 33 and 34 of extensor tibiae muscles of the locust (Schistocerca gregaria).
• 2.2. Bath application of GABA (10⁻⁵−5 × 10⁻³ M) induced reductions in muscle fibre space constant (λ).
• 3.3. GABA (5 × 10⁻³ M) induced additional membrane conductance of 2.21 ± 0.03 × 10⁻⁶ S/mm, 0.38 ± 0.03 × 10⁻⁶ S/mm and 0.29 ± 0.06 × 10⁻⁶ S/mm on muscle bundles 34, 33 and 32 respectively. The greater sensitivity of muscle fibres in bundle 34 to GABA is due at least in part to a larger number of GABA receptors on bundle 34 muscle fibres.
• 4.4. The decrement of electrotonic potentials in the presence of GABA were measured over distances of both half fibre length and whole fibre length. Good agreement was obtained between changes in space constant produced by GABA using half fibre length and whole fibre length data.
• 5.5. By taking into account changes in space constant induced by GABA it was possible to demonstrate that presynaptic GABA receptors were involved in the inhibition of slow excitatory postsynaptic potentials by GABA.
• 6.6. “Slow” excitatory postsynaptic potentials recorded under current clamp were inhibited in a dose-dependent manner by GABA. This inhibition was not dependent on muscle-fibre GABA sensitivity and could not be completely accounted for by GABA-induced changes in the cable properties of the muscle fibres.

     

Influence of intercellular clefts on potential and current distribution in a multifiber preparation.

A theoretical model is presented for current and voltage clamp of multifiber bundles in a double sucrose gap. Attention is focused on methodological errors introduced by the intercellular cleft resistance. The bundle is approximated by a continuous geometry. Voltage distribution, as a function of radial distance and time, is defined by a parabolic partial differential equation which is specified for different membrane characteristics. Assuming a linear membrane, analytical solutions are given for current step and voltage step conditions. The theoretical relations (based on Bessel functions) may be used to calculate membrane conductance and capacity from experimental clamp data. The case of a nonlinear membrane with standard Hodgkin-Huxley kinetics for excitatory Na current is treated assuming maximum Na conductances (gNa) of 120, 10, and 1 mmho/cm2. Numerical simulations are presented for potential and current distribution in a bundle of 60 microns diameter during depolarizing voltage steps. Adequate voltage control is restricted to the peripheral fibers of the bundle whereas the membrane potential of the inner fibers deviates from the command level during early inward current, tending to the Na equilibrium potential. In the peak current-voltage diagram the loss of voltage control is reflected by an increased steepness of the negative region and a decreased slope conductance of the positive region. With gNa = 120 mmho/cm2, the positive slope conductance is approximately 25% of the slope expected from ideal space clamping. With the lower values of gNa, the slope conductance ratio is in the order of 50%. Implications of the results for an experimental voltage clamp analysis of early inward current on multifiber preparations are discussed.     

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).     

Exponentiated exponential model (Gompertz kinetics) of Na+ and K+ conductance changes in squid giant axon

The conductance changes, gK(t) and gNa(t), of squid giant axon under voltage clamp (Hodgkin and Huxley, 1952) may be modeled by exponentiated exponential functions (Gompertz kinetics) from any holding potential VO to any membrane clamp potential V. The equation constants are set by the membrane potential V, and include, for any voltage step in the case of gK, the initial conductance, gO, the asymptote conductance g, and rate constant k: gK = g exp(-be-kt) where b = 1n g/gO. Equations of similar form relate g and k to the voltage V, and govern the corresponding parameters of the gNa system. For the gNa, the fast phase y = y exp (-be-kt) is cut down in proportion to a slow process p = (1 - p)e-k't + p, and thus gNa = py. The expo-exponential functions involve fewer constants than the Hodgkin-Huxley model. In particular, the role of the n, m, h parameters appears to be filled largely by 1n (g/gO) in the case of gK and by 1n (y/yO) in the case of gNa. Membrane action potentials during current clamp may be computed from the conductances generated by use of the appropriate differential forms of the equations; diverse other membrane behaviors may be predicted.     

Numerical analysis of the voltage-clamp technique applied to frog neuromuscular junctions.

The nonlinear cable equation was solved numerically by means of an implicit procedure. The correlation between end-plate length and fiber diameter was determined in frog (Rana pipiens) sartorius muscles stained with gold chloride (Löwit, 1875). The diameter of the fibers stained by the Löwit method was 80 (74-85) micron (median and its 95% confidence interval for 52 fibers), the length of the end plates in the same fibers was 382 (353-417) micron. The fibers simulated were 80 micron in diameter. To solve the equation the muscle fibers were represented by 500 segments 20 micron long, and the equation was solved in steps of 10 microseconds; a double exponential function was incorporated to the first seven segments to represent the neuromuscular junction. The potential of the first segment of the cable was set to the clamping level and the membrane potential of the remaining segments calculated. The current needed to hold the first segment was estimated by adding the current flowing through the first segment to the current flowing from it to the second segment. Our results indicate that the lack of space clamp in the point voltage-clamp studies of the frog neuromuscular junction introduces serious errors in the estimates of the end-plate conductance value, the kinetics of the conductance changes, and the reversal potential of the end-plate currents. The possibility of an efficient voltage-clamp technique is also explored. Our calculations suggest that the study of end-plate current and conductance is possible with little error if the end-plate potential is controlled at both ends of the synaptic area simultaneously.     

Synaptic inhibition and cell communication; impairment of cell-to-cell coupling produced by gamma-aminobutyric acid (GABA) in the somatic musculature of Ascaris lumbricoides

The influence of gamma-aminobutyric acid (GABA) (10(-5) M) on the electrical coupling of giant somatic muscle cells of Ascaris lumbricoides was investigated. GABA enhanced the resting potential of the cells and abolished the spike activity. The coupling coefficient (V2/V1) was reduced by 58.8% while the input resistance (Rin) was decreased by 38.8%. The decline in Rin was not related to unlinearity of the current-voltage relation. As the time constant of cell membrane was reduced by 28.4% by the addition of GABA the effect of the neurotransmitter on cell-to-cell coupling seems to be mainly related to a decrease in resistance of the non-junctional membrane due to an increase in chloride conductance.     

Energy dependence and functional reconstitution of the gamma-aminobutyric acid carrier from synaptic vesicles.

The energy dependence of gamma-aminobutyric acid (GABA) uptake was characterized in rat brain synaptic vesicles and in proteoliposomes reconstituted with a new procedure from vesicular detergent extracts. The proteoliposomes displayed high ATP-dependent GABA uptake activity with properties virtually identical to those of intact vesicles. GABA uptake was similar at chloride concentrations of 0 and 150 mM, i.e. conditions under which either the membrane potential (delta psi) or the pH difference (delta pH) predominates. Delta psi was gradually dissipated by increasing the concentration of SCN-. GABA uptake was reduced by 10 mM SCN-, showing less sensitivity to delta psi reduction than glutamate uptake but more than dopamine uptake. Dissipation of delta pH with NH+4 abolished GABA uptake at pH 7.3, whereas no significant inhibition occurred at pH 6.5. In contrast, dopamine uptake was inhibited more strongly, even at pH 6.5, and glutamate uptake was not reduced in either condition. We conclude that GABA uptake is driven by both components of the proton electrochemical gradient, delta pH and delta psi, and that this is different from the uptake of both dopamine and glutamate, which is more strongly dependent on delta pH and delta psi, respectively. Thus, our data suggest that GABA uptake is electrogenic and occurs in exchange for protons.     

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes.

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 micrograms/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd = 3.34 +/- 0.36 nM without tagetone and Kd,t = 5.86 +/- 0.86 nM with tagetone; p < 0.05, two tailed Student's t-test) without affecting maximal binding (Bmax = 488 +/- 24 fmoles/mg protein, and Bmax,t = 500 +/- 25 fmoles/mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 microM with and without tagetone respectively). GABA was able to decrease merocyanine delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 microM. Tagetone, at a concentration of 50 micrograms/ml and in the presence of GABA 30 microM or 60 microM, enhanced the ability of GABA alone on decreasing delta A570-610. Tagetone alone did not change delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine delta A570-610 value are mainly exerted at the membrane potential level (delta psi m). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding: on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.     

Axon voltage-clamp simulations. III. Postsynaptic region.

This is the third in a series of four papers in which we present the numerical simulations of the application of the voltage clamp technique to excitable cells. In this paper we discuss the problem of voltage clamping a region of a cylindrical cell using microelectrodes for current injection and voltage recording. A recently developed technique (Llinás et al., 1974) of internal application of oil drops to electrically insulate a short length of the postsynaptic region of the squid giant synapse is evaluated by simulation of the voltage clamp of an excitable cylindrical cell of finite length with variable placement of the current and voltage electrodes. Our results show that ENa can be determined quite accurately with feasible oil gap lengths but that the determination of the reversal potential for the synaptic conductance, ES, can be considerably in error. The error in the determination of ES dependp, and especially the membrane resistance at the time the synaptic conductance occurs. It is shown that the application of tetraethylammonium chloride to block the active potassium conductance very significantly reduces the error in the determination of ES. In addition we discuss the effects of cable length and electrode position on the apparent amplitude and time course of the syn aptic conductance change. These results are particularly relevant to the application of the voltage clamp technique to cells with nonsomatic synapses. The method of simulation presented here provides a tool for evaluation of voltage clamp analysis of synaptic transmission for any cell with known membrane parameters and geometry.     

Fusion of membranes during fertilization. Increases of the sea urchin egg''s membrane capacitance and membrane conductance at the site of contact with the sperm

The early events of fertilization that precede and cause activation of an egg have not been fully elucidated. The earliest electrophysiological change in the sea urchin egg is a sperm-evoked increase of the egg's membrane conductance. The resulting depolarization facilitates entry of the fertilizing sperm and precludes the entry of supernumerary sperm. The sequence of the increase in the egg's membrane conductance, gamete membrane fusion, egg activation, and sperm entry, including causal relationships between these events, are not known. This study reports the use of whole egg voltage clamp and loose patch clamp to monitor simultaneously changes of membrane conductance and capacitance at the site of sperm-egg contact. Measurements were made during sperm-egg interactions where sperm entry readily proceeded or was precluded by maintaining the egg's membrane potential either at large, negative values or at positive values. Whenever the sperm evoked an increase of the egg's membrane conductance, that increase initiated abruptly, was localized to the site of sperm attachment, and was accompanied by a simultaneous abrupt increase of the membrane capacitance. This increase of capacitance indicated the establishment of electrical continuity between gametes (possibly fusion of the gametes' plasma membranes). If sperm entry was blocked by large negative membrane potentials, the capacitance cut off rapidly and simultaneously with a decrease of the membrane conductance, indicating that electrical continuity between gametes was disrupted. When sperm entry was precluded by positive membrane potentials, neither conductance nor capacitance increased, indicating that sperm entry was halted before the fusion of membranes. A second, smooth increase of capacitance was associated with the exocytosis of cortical granules near the sperm in eggs that were activated. Electrical continuity between the gametes always preceded activation of the egg, but transient electrical continuity between the gametes alone was not always sufficient to induce activation.     

Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish

The kinetics of the membrane current during the anomalous or inward- going rectification of the K current in the egg cell membrane of the starfish Mediaster aequalis were analyzed by voltage clamp. The rectification has instantaneous and time-dependent components. The time- dependent increase in the K conductance for the negative voltage pulse as well as the decrease in the conductance for the positive pulse follows first-order kinetics. The steady-state conductance increases as the membrane potential becomes more negative and reaches the saturation value at about -40 mV more negative than the K equilibrium potential, V(K). The entire K conductance can be expressed by g(K).n; g g(K) represents the component for the time-independent conductance which depends on V-V(K) and [K+]o, and n is a dimensionless number (1 is greater than or equal to n is greater than or equal to 0) and determined by two rate constants which depend only on V-V(K). Cs+ does not carry any significant current through the K channel but blocks the channel at low concentration in the external medium. The blocking effect increases as the membrane potential is made more negative and the potential-dependent blocking by the external Cs+ also has instantaneous and time-dependent components.     

The cross desensitization and modulation of Cl currents activated by gamma-aminobutyric acid and L-glutamate in the isolated neurons of Aplysia]

Chlorine conductance gated by gamma-aminobutyric acid (GABA) and L-glutamate in the medial pleural neurons of aplysia was studied using voltage clamp technique and a continuous microperfusion system that allowed rapid agonist application. Both GABA and glutamate elicited current responses that rapidly activated and then decayed. Glutamate response could be blocked by perfusion of aspartate or taurine and the GABA current showed voltage dependence. Thus the currents exhibited cross desensitization. It has been found that very low concentrations of acetylcholine (10(-8) to 10(-14) M) which have no electrophysiologic responses of their own, modulate the response to a constant application of GABA. During cooling the preparation blocked this effect, it is possible to suggest that the small doses of acetylcholine effect the membrane chemosensitivity through the cell biochemical mechanism.     

Effect of toxin 1 from Androctonus australis Hector on sodium currents in giant axons of Loligo forbesi

The effects of external application of micromolar concentrations of toxin 1 of the scorpion, Androctonus australis Hector, on the sodium conductance of squid giant axons have been studied quantitatively using the voltage clamp technique. Toxin concentrations which induce long plateau action potentials under current clamp conditions were found to simultaneously decrease the peak conductance and increase the delayed sodium conductance. Return to holding potential level after step depolarizations was accompanied by large exponential tails of current. The toxin-induced maintained sodium conductance increased with membrane depolarization independently of the peak conductance. Depolarizing conditioning prepulses to - 30 mV were found to almost totally inactivate the peak sodium current but to leave the delayed conductance unaffected. This property was taken as an indication that the total current is made of the added contributions of two distinct populations on sodium channels : fast activating and inactivating channels and slow activating channels. These two channel populations were separated from each other and analysed. It was found that the fast channels were almost identical to normal channels whereas the slow channels had a much slower (nearly exponential) kinetics and activated for more positive values of membrane potential. These observations strongly support the second hypothesis of Gillespie and Meves (1980) that the peak conductance and maintained conductance reflect the existence of two separate populations of channels. They further indicate that slow channels probably originate from the modification by the toxin of normal voltage-sensitive channels.