Patch-clamp study on membrane properties and transmitter activated currents of rabbit area postrema neurons

Using the patch-clamp technique in combination with sliced tissue preparation the membrane properties of newborn rabbit area postrema neurons were investigated. The neurons responded upon depolarization with a fast Na ⁺-current followed by an inactivating and non-inactivating K ⁺-current. GABA-activated currents were investigated resulting in a large Cl^--conductance, indicating the expression of GABA_A-receptors. The expression of glutamate receptor mRNA was studied by in situ hybridization and electrophysiological measurements of these receptors by means of the patch-clamp technique. As a main result it was found that ionotropic glutamate receptors in the area postrema are composed of flop variants of the GluA-, GluB- and GluC-subunits.Abbreviations AP area postrema - GABA -aminobutyric acid - Glu glutamate - I–V current-voltage - SFO subfornical organ     

Is the Voltage Gate of Connexins CO<Subscript>2</Subscript>-sensitive? Cx45 Channels and Inhibition of Calmodulin Expression

The sensitivity of Cx45 channels to CO₂, transjunctional voltage (V _j) and inhibition of calmodulin (CaM) expression was tested in oocytes by dual voltage clamp. Cx45 channels are very sensitive to V _j and close with V _j preferentially by the slow gate, likely to be the same as the chemical gate. With a CO₂-induced drop in junctional conductance (G _j), both the speed of V _j-dependent inactivation of junctional current (I _j) and V _j sensitivity increased. With 40-mV V _j-pulses, the of single exponential I _j decay reversibly decreased by 40% during CO₂ application, and G_{j steady state}/G_{j peak} decreased multiphasically, indicating that both kinetics and V _j sensitivity of chemical/slow V _j gating are altered by changes in [H⁺]_i and/or [Ca²⁺]_i. CaM expression was inhibited with oligonucleotides antisense to CaM mRNA. With 15 min CO₂, relative junctional conductance (G _jt/G _jt0) dropped to 0% in controls, but only by 17% in CaM-antisense oocytes. Similarly, V _j sensitivity was significantly lessened in CaM-antisense oocytes. The data indicate that both the speed and sensitivity of V _j-dependent inactivation of the junctional current of Cx45 channels are affected by CO₂ application, and that CaM plays a key role in channel gating.     

Simulations of voltage clamping poorly space-clamped voltage-dependent conductances in a uniform cylindrical neurite

Significant error is made by using a point voltage clamp to measure active ionic current properties in poorly space-clamped cells. This can even occur when there are no obvious signs of poor spatial control. We evaluated this error for experiments that employ an isochronal I(V) approach to analyzing clamp currents. Simulated voltage clamp experiments were run on a model neuron having a uniform distribution of a single voltage-gated inactivating ionic current channel along an elongate, but electrotonically compact, process. Isochronal Boltzmann I(V) and kinetic parameter values obtained by fitting the Hodgkin-Huxley equations to the clamp currents were compared with the values originally set in the model. Good fits were obtained for both inward and outward currents for moderate channel densities. Most parameter errors increased with conductance density. The activation rate parameters were more sensitive to poor space clamp than the I(V) parameters. Large errors can occur despite normal-looking clamp curves.     

Crayfish sensory terminals and motor neurones exhibit two distinct types of GABA receptors

Motor neurones of the crayfish walking system display inhibitory responses evoked either by γ-amino butyric acid (GABA) or glutamate, possibly involving the same receptor (Pearlstein et al. 1994). In order to test if this sensibility to both GABA and glutamate was a specific property of crayfish GABA receptors, pharmacological characteristics of GABA-evoked responses in both sensory terminals from CB chordotonal organ and motor neurones of the walking system have been compared. Both receptors are GABA-gated Cl⁻ channels activated by specific GABA_A (muscimol, isoguvacine), GABA_B (3-aminopropyl phosphinic acid), and GABA_C (cis-4-amino crotonic acid) agonists, and blocked by competitive (β-guanidino propionic acid) and non-competitive (picrotoxin) antagonists. They were insensitive to specific GABA_A (bicuculline, SR-95531) and GABA_B (phaclofen) antagonists. Furthermore, in both cases, nipecotic acid and the modulatory drug diazepam had no effect. However, our results demonstrate that GABA receptors of sensory terminals are different from those of motor neurones. GABA-induced desensitisation only occurred in sensory terminals. Moreover, glutamate was shown to activate GABA-gated Cl⁻ channels in motor neurones, but not in sensory terminals. Therefore, GABA is likely to be the endogenous neurotransmitter of presynaptic inhibition in sensory terminals, whereas inhibition between antagonistic motor neurones would be achieved by glutamate. Accepted: 10 July 1996     

Ca2+-activated K+ conductance of the human red cell membrane: Voltage-dependent Na+ block of outward-going currents

Summary Human red cells were prepared with various cellular Na⁺ and K⁺ concentrations at a constant sum of 156mm. At maximal activation of the K⁺ conductance,g _K(Ca), the net efflux of K⁺ was determined as a function of the cellular Na⁺ and K⁺ concentrations and the membrane potential,V _m , at a fixed [K⁺]_ex of 3.5mm.V _m was only varied from (V _m –E _K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g _K(Ca) as a function of cellular Na⁺ and K⁺ concentrations atV _m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na⁺. Since the present Ca²⁺-activated K⁺ channels have been shown to be of the multi-ion type, the experimental data from each set of Na⁺ and K⁺ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na⁺ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na⁺ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca²⁺ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg _K(Ca) for outward currents at (V _m –E _K)>25 25 mV reflects a voltage-dependent Na⁺ block of the Ca²⁺-activated K⁺ channels.     

The GABAA Receptor Complex as a Target for Fluoxetine Action

The clinically important antidepressant fluoxetine is established as a selective serotonin reuptake inhibitor. This study demonstrates that fluoxetine also interacts with the GABA_A receptor complex. At concentrations above 10 M fluoxetine inhibited the binding of both [³H]GABA (IC₅₀ = 2 mM) and [³H]flunitrazepam (IC₅₀ = 132 M ) to the GABA_A receptor complex in brain cortical membranes. Low fluoxetine concentrations (1 nM) enhanced GABA-stimulated Cl^– uptake by a rat cerebral cortical vesicular preparation. At higher concentrations (100 M and 1 mM), however, fluoxetine inhibited GABA-stimulated Cl^– uptake, an effect related to a reduction in E_max. These observations might assist in an explanation of the basis of the antidepressant action of fluoxetine.     

Pretreatment of Rat Brain Synaptosomes with GABA Increases Subsequent GABA Uptake Via GABAB Receptor Activation

Pretreatment with 100 M GABA of synaptosomes purified from rat brain results in an increased uptake of the labelled neurotransmitter in subsequent incubations. The effect is blocked by a GABA_B receptor antagonist, 2-hydroxy-saclofen. The effect is mimicked by baclofen and the baclofen effect is blocked by saclofen too. Lower GABA concentrations (up to 50 M) do not result in an increase of subsequent GABA uptake. Treatment of synaptosomes with 8-Br-cAMP results in a decreased GABA uptake. Since the uptake incubations were run with saturating concentrations of labelled GABA, the data indicates that GABA_B receptor activation in brain synaptosomes up-regulates their GABA uptake capacity by an increase in V_max. This mechanism appears of physiological relevance under conditions of sustained GABA release and substantial increase of its extracellular concentration.     

Different photoreceptors within the same retina express unique combinations of potassium channels

Single electrode current and voltage clamp recordings in Calliphora, and whole-cell voltage clamp recordings in Drosophila were used to characterise the voltage-gated K channels in both major classes of photoreceptors, R7/8 (long visual fibres, LVFs) and R1-6 (short visual fibres, SVFs). R7/8 were identified by their unique spectral properties, ca. 3–4 fold higher input resistances and 3–4 fold lower cell capacitance. In Calliphora SVFs possess both fast and slow activating delayed rectifier potassium conductances. Drosophila SVFs possess a slowly inactivating delayed rectifier (I_Ks), a very rapidly inactivating A channel encoded by the Shaker gene (I_A), and, in a minority of cells, a third K conductance with intermediate kinetics (I_Kf). In both specs the LVFs lack the slowest component, but exhibit the faster K conductance(s) with properties indistinguishable from those in the SVFs. These findings add to established evidence demonstrating the significant role played by potassium channels in tuning the photoreceptor membrane. The results also suggest that R1-6 photoreceptors and R7/8 form inputs to visual subsystems tuned to different temporal frequencies.Abbreviations LVF long visual fibre - SVF short visual fibre - R1-6 retinular cells 1 to 6 inclusive - R7/8 retinular cell 7 and 8 - I _A rapidly inactivating A type potassium conductance; channel coded by Shaker gene - I _Kf rapidly activating, slowly inactivating delayed rectifier-like potassium conductance - I _Ks slowly activating, slowly inactivating delayed rectifier-like potassium conductance - I _KDs slowly activating delayed rectifier potassium conductance - I _KDf rapidly activating delayed rectifier potassium conductance     

GABAA receptors on rat cerebellar granule cells are potently activated by muscimol but only slightly modulated by the benzodiazepine agonist flunitrazepam

Summary GABA_A receptors present on rat cerebellar granule cells in culture were studied by the whole cell patch clamp technique. Muscimol appeared to be more potent than GABA itself in activating Cl^– currents. A benzodiazepine, flunitrazepam, only slightly (10%) potentiated the GABA action.These results support the previous suggestion that GABA_A receptors containing the subunit, such as those in the cerebellum granule cells, are potently activated by muscimol. The present results also bear out the concept that GABA action on receptors containing the subunit is not potentiated by benzodiazepines.     

Possible Involvement of GABAA and GABAB Receptors in the Inhibitory Action of Lindane on Transmitter Release from Cerebellar Granule Neurons

Cerebellar granule cells in culture express receptors for GABA belonging to the GABA_A and GABA_B classes. In order to characterize the ability of the insecticide lindane to interact with these receptors cells were grown in either plain culture media or media containing 150 M THIP as this is known to influence the properties of both GABA_A and GABA_B receptors. It was found that lindane regardless of the culture condition inhibited evoked (40 mM K⁺) release of neurotransmitter ([³H]D-aspartate as label for glutamate). In naive cells both GABA_A and GABA_B receptor active drugs prevented the inhibitory action of lindane but in THIP treated cultures none of the GABA_A and GABA_B receptor active drugs had any effect on the inhibitory action of lindane. This lack of effect was not due to inability of baclofen itself to inhibit transmitter release. It is concluded that lindane dependent on the state of the GABA_A and GABA_B receptors is able to indirectly interfere with both GABA_A and GABA_B receptors. In case of the latter receptors it was shown using [³H]baclofen to label the receptors that lindane could not displace the ligand confirming that lindane is likely to exert its action at a site different from the agonist binding site.     

Potassium conductance models related to an interactive subunit membrane

Summary The properties of various potassium conductance models have been investigated using an analogue computer. It is shown that the experimental data of Hodgkin and Huxley can be fitted as satisfactorily by a cube (n ³) model of potassium conductance as it is by the Hodgkin-Huxley (n ⁴) and (n ⁶) models.A planar subunit array structure for the membrane has been suggested, where the appearance of a potassium conducting channel depends upon a conformational change to an activated state in each of neighboring subunits. This system is described by the same mathematics as the Hodgkin-Huxley activating particle mechanism and so provides a physical basis for the power (n ) formulae. Introduction of interaction between subunits, such that a conformational change is prohibited unless an adjacent subunit is in the activated state, modifies the mathematics and enables simulation of the delayed potassium currents observed by Cole and Moore (Biophys. J. 1:1, 1960). This innovation avoids the difficulties associated with the higher power (>6) models, by not requiring physical justification for large numbers of simultaneous events, while still providing a good fit to the experimental data. The interactive subunit models satisfactorily describe the potassium conductance changes which occur under voltage clamp or during an action potential.A preliminary account of this work was presented at the Seventh Annual Meeting ofPhysics in Medicine and Biology, Biophysics Group, Australian Institote of Physics, Adelaide, South Australia, May 1967.     

Purification,properties, and metabolic roles of NAD+-glutamate dehydrogenase in Clostridium botulinum 113B

Cell-free extracts of proteolytic strains of Clostridium botulinum types A, B and F (group I) were found to have unusually high specific activities of NAD⁺-dependent L-glutamate dehydrogenase (NAD-GDH). In comparison, nonproteolytic strains of types B, E and F (group II) had low specific activities. The enzyme was purified 131-fold from C. botulinum 113B to a final specific activity of >1,092 molxmin^-1xmg protein^-1. The enzyme is a hexamer of a polypeptide of Mr=42,500, and the native molecular weight is 250,800. The apparent K _m values for substrates were 5.3 mM for glutamate and 0.028 mM for NAD⁺ in the deamination reaction, and 7.2 mM for -ketoglutarate, 243 mM for NH ₄ ⁺ and 0.028 mM for NADH in the reverse reaction. NADP⁺ did not serve as a hydrogen acceptor for the enzyme. Activity in the animation direction was inhibited by fumarate, oxalacetate, aspartate, glutamate and glutamine. The results suggest that GDH is important in group I (proteolytic) C. botulinum to generate -ketoglutarate as a substrate for transamination reactions. We have also found that the high activity decreases significantly when cells are exposed to sodium chloride. Therefore GDH probably has several important physiological roles in group I proteolytic C. botulinum.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Summary In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the simultaneous mechanism the cotransporterC forms a ternary complexNCS with Na⁺ and the substrateS; coupled transport of Na⁺ andS involves a conformational transition between statesNCS andNCS with inward- and outward-facing binding sites. In the consecutive (or ping-pong) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the simultaneous model, but incompatible with the consecutive mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the simultaneous model can be estimated from the experimental results. The equilibrium dissociation constants of Na⁺ and alanine at the extracellular side are determined to beK _N <-64mm andK _S <-18mm. Furthermore, the ratioK _N /K _N ^S of the dissociation constants of Na⁺ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na⁺ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na⁺ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.     

A kinetic analysis of the electrogenic pump ofChara corallina: III. Pump activity during action potential

Summary The current-voltage curve (I–V curve) of theChara membrane was obtained by applying a slow ramp hyper- and depolarization by use of voltage clamp. By inhibiting the electrogenic pump with 50m DCCD (dicyclohexylcarbodiimide), theI–V curve approached a steadyI–V curve within two hours, which gave thei _d -V curve of the passive diffusion channel. Thei _p -V curve of the electrogenic pump channel was obtained by subtracting the latter from the former. The sigmoidali _p -V curve could be simulated satisfactorily with a simple reaction kinetic model which assumes a stoichiometric ratio of 2. The emf of the pump (E _p ) is given as the voltage at which the pump current changes its sign. The conductance of the pump (g _p ) can be calculated as the chord conductance from thei _p -V curve, which is highly voltage dependent having a peak at a definite voltage. The changes of emf and conductance during excitation were determined by use of the current clamp (I=0). Since theE _p andg _p (V) are known, the changes, during excitation, of emf (E _d ) and conductance (g _d ) of the passive diffusion channel can be calculated. The marked increase of the membrane conductance and the large depolarization during the action potential are caused by the marked increase of the conductance of the passive diffusion channel and the large depolarization of its emf. The conductance of the electrogenic pump decreases to about half at the peak of action potential, while the pump current increases almost to a saturated level.     

A new mouse T-cell receptorα chain variable region family

Sequence analysis of the rearranged T-cell receptor a chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribedV gene family. We have designated this familyV 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously describedV haplotypes:V ^a ,V ^b , andV ^c .     

Recording of single K+ channels in the membrane of cytoplasmic drop ofChara australis

Summary The cytoplasmic drop formed of effused cytoplasm fromChara internodes is enclosed by a membrane. Patch clamp experiments have been carried out on this membrane, revealing a K⁺ channel as the most frequently detected ion translocator. The K⁺ channel is saturated at a level of about 20 pA inward and 10 pA outward current. The channel conductance is dependent on the accessability of K⁺ ions, its maximum value amounts to about 165 pS. The discrimination of Na⁺ and Cl^– is significant, permeability ratios P_Na/P_K and P_Cl/P_K were estimated to be 0.01 either. Binding experiments with the fluorescent probe concanavalin A/FITC suggest that the membrane is derived from the tonoplast.Abbreviations E_K K⁺ equilibrium potential - FITC fluorescein isothiocyanat - V_m membrane voltage - V_pip pipette clamp voltage - V_r reversal voltage     

Multiple-channel conductance states and voltage regulation of embryonic chick cardiac gap junctions

Summary We used the double whole-cell voltage-clamp technique on ventricle cell pairs isolated from 7-day chick heart to measure the conductance of their gap junctions (G _j) and junctional channels ( _j) with a steady-state voltage difference (V _j) applied across the junction. Currents were recorded from single gap junction channels (i _j) as symmetrical rectangular signals of equal size and opposite sign in the two cells, and _j was measured from i _j/V _j. We observed channel openings at six reproducible conductance levels with means of 42.6, 80.7, 119.6, 157.7, 200.4 and 240.3 pS. More than half of all openings were to the 80-and 160-pS conductance levels. The probability that a high conductance event (e.g., 160 or 240 pS) results from the random simultaneous opening of several 40-pS channels is small, based on their frequency of occurrence and on the prevalence of shifts between small and large conductance states with no intervening 40-pS steps. Our results are consistent with three models of embryonic cardiac gap junction channel configuration: a homogeneous population of 40-pS channels that can open cooperatively in groups of up to six; a single population of large channels with a maximal conductance near 240 pS and five smaller substates; or several different channel types, each with its own conductance. G _j was determined from the junctional current (I _j) elicited by rectangular pulses of applied transjunctional voltage as I _j/V _j. It was highest near 0 V _j and was progressively reduced by application of V _j between 20 and 80 mV or –20 and –80 mV. In response to increases in V _j, G _j decayed in a voltage-and timedependent fashion. After a 6-sec holding period at 0 V _j, the initial conductance (G _init) measured immediately after the onset of an 80-mV step in V _j was nearly the same as that measured by a 10-mV prepulse. However, during 6-sec pulses of V _j>±20 mV, G _j declined over several seconds from G _init to a steady-state value (G _ss). At potentials greater than ±20 mV the current decay could be fit with biexponential curves with the slow decay time constant ( ₂) 5–20 times longer than ₁. For the response to a step to 80 mV V _j, for example, ₁=127 msec and ₂=2.6 sec. The rate of current decay in response to smaller positive or negative steps in V _j was slower, the magnitude of the decline was smaller, and the ratio ₂/ ₁ decreased. The relationship between G _init and V _j was approximately linear between 0 and 80 mV or –80 mV. whereas the relationship between G _ss and V _j was nonlinear beyond ±20 mV. Upon returning to 0 V _j, G _j recovered with a biexponential time course, reaching its maximal value after several seconds; recovery time constants after a step in V _j from 80 to 0 mV were 225 msec and 1.9 sec. In the resting state, at low junctional voltage, high conductance channel activity (160–240 pS) is favored. Voltage-dependent decline of G _j results in part from a shift from high to lower conductance states.We thank Ms. B.J. Duke for technical assistance and for preparation of the cell cultures and Drs. L.J. DeFelice and D. Eaton for stimulating and helpful discussions of the results.     

A kinetic analysis of the electrogenic pump ofChara corallina: II. Dependence of the pump activity on external pH

Summary The current-voltage curve of theChara membrane was obtained by applying a slow ramp de- and hyperpolarization by use of voltage clamp. By inhibiting the electrogenic pump with 50m DCCD (dicyclohexylcarbodiimide), theI–V curve approached a steady state within 100 min, which gave thei _d -V curve of the passive diffusion channel. Thei _p -V curve of the electrogenic pump channel was obtained by subtracting the latter from the former. With the increase of external pH, thei _d -V curve showed only a slight change, while thei _p -V curve of the pump channel showed almost a parallel shift, in the hyperpolarizing direction, along the voltage axis in the pH range between 6.5 and 7.5. The sigmoidali _p -V curve in this pH range could be simulated satisfactorily with the five-state model reported previously (U. Kishimoto, N. Kami-ike, Y. Takeuchi & T. Ohkawa,J. Membrane Biol. 80:175–183, 1984) as well as with a lumped two-state model presented in this report. The analysis based on these models suggests that the electrogenic pump of theChara membrane is mainly a 2H⁺/1ATP pump. The forward rate constant in the voltage-dependent step increased with the increase of external pH, while the backward one decreased. On the other hand, the forward rate constant in the voltage-independent step remained almost unchanged with the increase of external pH, while the backward one increased markedly. The pump conductance at the resting membrane potential showed either a slight increase or a decrease with the increase of external pH, depending on the sample. Nevertheless, the pump current showed generally a slight increase with the increase of external pH.     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.