Membrane transport in stomatal guard cells: The importance of voltage control

Potassium uptake and export in the resting conditions and in response to the phytohormone abscisic acid (ABA) were examined under voltage clamp in guard cells of Vicia faba L. In 0.1 mM external K+ (with 5 mM Ca2(+)-HEPES, pH 7.4) two distinct transport states could be identified based on the distribution of the free-running membrane voltage (VM) data in conjunction with the respective I-V and G-V relations. One state was dominated by passive diffusion (mean VM = -143 +/- 4 mV), the other (mean VM = -237 +/- 10 mV) exhibited an appreciable background of primary H+ transport activity. In the presence of pump activity the free-running membrane voltage was negative of the respective K+ equilibrium potential (EK+), in 3 and 10 mM external K+. In these cases VM was also negative of the activation voltage for the inward rectifying K+ current, thus creating a strong bias for passive K+ uptake through inward-rectifying K+ channels. In contrast, when pump activity was absent VM was situated positive of EK+ and cells revealed a bias for K+ efflux. Occasionally spontaneous voltage transitions were observed during which cells switched between the two states. Rapid depolarizations were induced in cells with significant pump activity upon adding 10 microM ABA to the medium. These depolarizations activated current through outward-rectifying K+ channels which was further amplified in ABA by a rise in the ensemble channel conductance. Current-voltage characteristics recorded before and during ABA treatments revealed concerted modulations in current passage through at least four distinct transport processes, results directly comparable to one previous study (Blatt, M.R., 1990, Planta 180:445) carried out with guard cells lacking detectable primary pump activity. Comparative analyses of guard cells in each case are consistent with depolarizations resulting from the activation of an inward-going, as yet unidentified current, rather than an ABA-induced fall in H(+)-ATPase output. Also observed in a number of cells was an inward-directed current which activated in ABA over a narrow range of voltages positive of -150 mV; this and additional features of the current suggest that it may reflect the ABA-dependent activation of an anion channel previously characterized in Vicia guard cell protoplasts, but rule out its function as the primary mechanism for initial depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sodium-pump potentials and currents in guinea-pig ventricular muscles and myocytes.

When guinea-pig papillary muscles were depolarized to ca. -30 mV by superfusion with K+-free Tyrode's solution supplemented with Ba2+, Ni2+, and D600, addition of Cs+ transiently hyperpolarized the membrane in a reproducible manner. The size of the hyperpolarization (pump potential) depended on the duration of the preceding K+-free exposure; peak amplitudes (Epmax) elicited by 10 mM Cs+ after 5-, 10-, and 15-min K+-free exposures were 12.9, 17.7, and 23.2 mV, respectively. Pump potentials were unaffected by external Cl- but suppressed by cardiac glycosides, hyperosmotic conditions, and low-Na+ solution. Using Epmax as an indicator of Na+ pump activation, the half-maximal concentration for activation by Cs+ was 12-16.3 mM. At 6 mM, Cs+ was three times less potent than Rb+ or K+ and five times more potent than Li+. From these findings, and correlative voltage-clamp data from myocytes, we calculate that (i) a pump current of 7.8 nA/cm2 generates an Epmax of 1 mV and (ii) resting pump current in normally polarized muscle (approximately 0.16 microA/cm2) is five times smaller than previously estimated.     

Electrical activity in white adipose tissue of rat

Intracellular recording of white adipocytes was performed in an in vitro preparation. Resting potential, input resistance and membrane time constant averaged: -34 +/- 9 mV, 295 +/- 161 M omega, and 58 +/- 19 ms respectively (mean +/- SD, n = 32). Intracellular injection of positive and negative square current pulses elicited membrane voltage responses, characterized by a rectification of the voltage change evoked by positive pulses, and a slow return to baseline at the offset of hyperpolarizing pulses. The amplitude and duration of the slow return to resting potential was dependent on membrane potential, pulse duration, and extracellular K+ concentration. This response was depressed when external Ca2+ was replaced by Co2+, and by external application of 4-aminopyridine. These results indicate that white adipocytes can generate membrane voltage responses which may mostly be a consequence of the activity of ionic channels. The properties of the slow return to baseline suggest that it may be due to a transient K+ current.     

Ion permeation and block of M-type and delayed rectifier potassium channels. Whole-cell recordings from bullfrog sympathetic neurons

Ion permeation and conduction were studied using whole-cell recordings of the M-current (I(M)) and delayed rectifier (IDR), two K+ currents that differ greatly in kinetics and modulation. Currents were recorded from isolated bullfrog sympathetic neurons with 88 mM [K+]i and various external cations. Selectivity for extracellular monovalent cations was assessed from permeability ratios calculated from reversal potentials and from chord conductances for inward current. PRb/PK was near 1.0 for both channels, and GRb/GK was 0.87 +/- 0.01 for IDR but only 0.35 +/- 0.01 for I(M) (15 mM [Rb+]o or [K+]o). The permeability sequences were generally similar for I(M) and IDR: K+ approximately Rb+ > NH4+ > Cs+, with no measurable permeability to Li+ or CH3NH3+. However, Na+ carried detectable inward current for IDR but not I(M). Nao+ also blocked inward K+ current for IDR (but not IM), at an apparent electrical distance (delta) approximately 0.4, with extrapolated dissociation constant (KD) approximately 1 M at 0 mV. Much of the instantaneous rectification of IDR in physiologic ionic conditions resulted from block by Nao+. Extracellular Cs+ carried detectable inward current for both channel types, and blocked I(M) with higher affinity (KD = 97 mM at 0 mV for I(M), KD) approximately 0.2 M at 0 mV for IDR), with delta approximately 0.9 for both. IDR showed several characteristics reflecting a multi-ion pore, including a small anomalous mole fraction effect for PRb/PK, concentration-dependent GRb/GK, and concentration- dependent apparent KD's and delta's for block by Nao+ and Cso+. I(M) showed no clear evidence of multi-ion pore behavior. For I(M), a two- barrier one-site model could describe permeation of K+ and Rb+ and block by Cso+, whereas for IDR even a three-barrier, two-site model was not fully adequate.     

Na-K pump current in the Amphiuma collecting tubule

There is strong evidence supporting the hypothesis of an electrogenic Na-K pump in the basolateral membrane of several epithelia. Thermodynamic considerations and results in nonepithelial cells indicate that the current carried by the pump could be voltage dependent. In order to measure the pump current and to determine its voltage dependence in a tight epithelium, we have used the isolated perfused collecting tubule of Amphiuma and developed a technique for clamping the basolateral membrane potential (Vbl) through transepithelial current injection. The transcellular current was calculated by subtracting the paracellular current (calculated from the transepithelial conductance measured in the presence of luminal amiloride) from the total transepithelial current. Basolateral membrane current-voltage (I-V) curves were obtained in conditions where the ratio of the pump current to the total basolateral membrane current had been maximized by loading the cells with Na+ (exposure to low-K+ bath), and by blocking the basolateral K+ conductance with barium. The pump current was defined as the difference of the current across the basolateral membrane measured before and 10-15 s after the addition of strophanthidin (20 microM) to the bath solution. With a bath solution containing 3 mM K+, the pump current was nearly constant in the Vbl range of -20 to -80 mV (52 +/- 5 microA.cm-2 at -60 mV) but showed a marked voltage dependence at higher negative Vbl (pump current decreased to 5 +/- 9 microA.cm-2 at -180 mV). In a 1.0 mM K bath, the shape of the pump I-V curve was similar but the amplitude of the current was decreased (24 +/- 4 microA.cm-2 at -60 mV). In a 0.1 mM K bath, the pump current was not significantly different from 0. Our results indicate that the basolateral Na-K pump generates a current which depends on the extracellular potassium concentration. With physiological peritubular concentration of K+ and in the physiological range of potential, the pump activity, measured as the pump-generated current, was independent of the membrane potential.     

Electrical and biochemical properties of an enzyme model of the sodium pump

The electrochemical properties of a widely accepted six-step reaction scheme for the Na+, K+-ATPase have been studied by computer simulation. Rate coefficients were chosen to fit the nonvectorial biochemical data for the isolated enzyme and a current-voltage (I-V) relation consistent with physiological observations was obtained with voltage dependence restricted to one (but not both) of the two translocational steps. The vectorial properties resulting from these choices were consistent with physiological activation of the electrogenic sodium pump by intracellular and extracellular sodium (Na+) and potassium (K+) ions. The model exhibited K+/K+ exchange but little Na+/Na+ exchange unless the energy available from the splitting of adenosine triphosphate (ATP) was reduced, mimicking the behavior seen in squid giant axon. The vectorial ionic activation curves were voltage dependent, resulting in large shifts in apparent Km's with depolarization. At potentials more negative than the equilibrium or reversal potential transport was greatly diminished unless the free energy of ATP splitting was reduced. While the pump reversal potential is at least 100 mV hyperpolarized relative to the resting potential of most cells, the voltage-dependent distribution of intermediate forms of the enzyme allows the possibility of considerable slope conductance of the pump I-V relation in the physiological range of membrane potentials. Some of the vectorial properties of an electrogenic sodium pump appear to be inescapable consequences of the nonvectorial properties of the isolated enzyme. Future application of this approach should allow rigorous quantitative testing of interpretative ideas concerning the mechanism and stoichiometry of the sodium pump.     

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

Inward current in single smooth muscle cells of the guinea pig taenia coli

Using the tight-seal voltage-clamp method, the ionic currents in the enzymatically dispersed single smooth muscle cells of the guinea pig taenia coli have been studied. In a physiological medium containing 3 mM Ca2+, the cells are gently tapering spindles, averaging 201 (length) x 8 microns (largest diameter in center of cell), with a volume of 5 pl. The average cell capacitance is 50 pF, and the specific membrane capacitance 1.15 microF/cm2. The input impedance of the resting cell is 1-2 G omega. Spatially uniform voltage-control prevails after the first 400 microseconds. There is much overlap of the inward and outward currents, but the inward current can be isolated by applying Cs+ internally to block all potassium currents. The inward current is carried by Ca2+. Activation begins at approximately -30 mV, maximum ICa occurs at +10-+20 mV, and the reversal potential is approximately +75 mV. The Ca2+ channel is permeable to Sr2+ and Ba2+, and to Cs+ moving outwards, but not to Na+ moving inwards. Activation and deactivation are very rapid at approximately 33 degrees C, with time-constants of less than 1 ms. Inactivation has a complex time course, resolvable into three exponential components, with average time constants (at 0 mV) of 7, 45, and 400 ms, which are affected differently by voltage. Steady-state inactivation is half-maximal at -30 mV for all components combined, but -36 mV for the fast component and -26 and -23 mV for the other two components. The presence of multiple forms of Ca2+ channel is inferred from the inactivation characteristics, not from activation properties. Recovery of the fast channel occurs with a time-constant of 72 ms (at +10 mV). Ca2+ influx during an action potential can transfer approximately 9 pC of charge, which could elevate intracellular Ca2+ concentration adequately for various physiological functions.     

Identification of a spontaneously active, Na+-permeable channel in guinea pig gallbladder smooth muscle

The action potential in gallbladder smooth muscle (GBSM) is caused by Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), which contributes to the GBSM contractions. Action potential generation in GBSM is critically dependent on the resting membrane potential (about -50 mV), which is approximately 35 mV more positive of the K+ equilibrium potential. We hypothesized that a tonic, depolarizing conductance is present in GBSM and contributes to the regulation of the resting membrane potential and action potential frequency. GBSM cells were isolated from guinea pig gallbladders, and the whole cell patch-camp technique was used to record membrane currents. After eliminating the contribution of VDCC and K+ channels, we identified a novel spontaneously active cation conductance (I(cat)) in GBSM. This I(cat) was mediated predominantly by influx of Na+. Na+ substitution with N-methyl-D-glucamine (NMDG), a large relatively impermeant cation, caused a negative shift in the reversal potential of the ramp current and reduced the amplitude of the inward current at -50 mV by 65%. Membrane potential recordings with intracellular microelectrodes or in current-clamp mode of the patch-clamp technique indicated that the inhibition of I(cat) conductance by NMDG is associated with membrane hyperpolarization and inhibition of action potentials. Extracellular Ca2+, Mg2+, and Gd3+ attenuated the I(cat) in GBSM. Muscarinic stimulation did not activate the I(cat). Our results indicate that, in GBSM, an Na+-permeable channel contributes to the maintenance of the resting membrane potential and action potential generation and therefore plays a critical role in the regulation of GBSM excitability and contractility.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Voltage-dependent stimulation of Na+/K(+)-pump current by external cations: selectivity of different K+ congeners.

Currents generated by the endogenous Na+/K+ pump in the oocytes of Xenopus laevis were determined under voltage-clamp as currents activated by different K+ congeners. The voltage dependence of the pump current reflects voltage-dependent steps in the reaction cycle. The decrease of K(+)-activated pump current at positive potentials has been attributed to voltage-dependent stimulation by the external K+ (Rakowski, Vasilets, LaTona and Schwarz (1991) J. Membr. Biol. 121, 177-187). In Na(+)-free solution, activation of the pump by external cations seems to be the dominating voltage-dependent and rate-determining step in the reaction cycle. Under these conditions, the voltage dependence of apparent Km values for pump activation can be analyzed. The dependence suggests voltage-dependent binding of extracellular cations assuming that an effective charge of about 0.4 of an elementary charge is moved in the electrical field during a step associated with the cation binding. The apparent Km values at 0 mV differ for various cations that stimulate pump activity. The values are in mM: 0.10 for Tl+, 0.63 for K+, 0.71 for Rb+, 9.3 for NH4+, and 12.9 for Cs+. The corresponding apparent affinities follow the same sequence as the cation permeability of the K(+)-selective delayed rectifier channel of nerve cells. The results are compatible with the interpretation that the cations have to pass an ion-selective access channel to reach their binding sites in the pump molecule.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

Unidirectional K+ fluxes through recombinant Shaker potassium channels expressed in single Xenopus oocytes

We describe a method to evaluate the ratio of ionic fluxes through recombinant channels expressed in a single Xenopus oocyte. A potassium channel encoded by the Drosophila Shaker gene tested by this method exhibited flux ratios far from those expected for independent ion movement. At a fixed extracellular concentration of 25 mM K+, this channel showed single-file diffusion with an Ussing flux-ratio exponent, n', of 3.4 at a membrane potential of -30 mV. There was an apparent, small voltage dependence of this parameter with n' values of 2.4 at -15 and -5 mV. These results indicate that the pore in these channels can simultaneously accommodate at least four K+ ions. If each of these K+ ions is in contact with two water molecules, the minimum length of the pore is 24 A.     

Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes

Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and time-dependent properties of these currents are interpreted here in terms of a model in which Ito underlies the initial, rapid repolarization phase of the action potential (AP), and IK is responsible for the slower phase of AP repolarization back to the resting membrane potential, in adult rat ventricular myocytes.     

Elevation of a potassium current in differentiating human leukemic (HL-60) cells

Human promyelocytic leukemia (HL-60) cells display a novel voltage-dependent outward current under voltage clamp. This current is present at low levels in the proliferative state and in granulocytes derived from HL-60 cells which were induced to differentiate with retinoic acid. It is elevated in macrophages derived from HL-60 cells after exposure to phorbol-12-myristate-13-acetate (PMA). The current is carried primarily by K+, is blocked by Cs+ and by increased intracellular concentrations of Cl-. From a holding potential of -80 mV, significant activation required depolarization to +20 mV membrane potential. Activation was not influenced by intracellular Ca2+ (1-2 X 10(-6) M). These properties appear to differ significantly from the Ca2+-activated K+ channel and the delayed rectifier. The increase of this voltage-activated current in differentiation toward the macrophage, but not the granulocyte, suggests that this current is correlated specifically with macrophage differentiation.     

Effect of acetylcholine on postjunctional membrane permeability in eel electroplaque

Acetylcholine (ACh) was applied iontophoretically to the innervated face of isolated eel electroplaques while the membrane potential was being recorded intracellularly. At the resting potential (about -85 mV) application of the drug produced depolarizations (ACh potentials) of 20 mV or more which became smaller when the membrane was depolarized and reversed in polarity at about zero membrane potential. The reversal potential shifted in the negative direction when external Na+ was partially replaced by glucosamine. Increasing external K+ caused a shift of reversal potential in the positive direction. It was concluded that ACh increased the permeability of the postjunctional membrane to both ions. Replacement of Cl- by propionate had no effect on the reversal potential. In Na+-free solution containing glucosamine the reversal potential was positive to the resting potential, suggesting that ACh increased the permeability to glucosamine. Addition of Ca++ resulted in a still more positive reversal potential, indicating an increased permeability to Ca++ as well. Analysis of the results indicated that the increases in permeability of the postjunctional membrane to K+, Na+, Ca++, and glucosamine were in the ratios of approximately 1.0:0.9:0.7:0.2, respectively. With these permeability ratios, all of the observed shifts in reversal potential with changes in external ionic composition were predicted accurately by the constant field equation.     

A patch-clamp study of histamine-secreting cells

The ionic conductances in rat basophilic leukemia cells (RBL-2H3) and rat peritoneal mast cells were investigated using the patch-clamp technique. These two cell types were found to have different electrophysiological properties in the resting state. The only significant conductance of RBL-2H3 cells was a K+-selective inward rectifier. The single channel conductance at room temperature increased from 2-3 pS at 2.8 mM external K+ to 26 pS at 130 mM K+. This conductance, which appeared to determine the resting potential, could be blocked by Na+ and Ba2+ in a voltage-dependent manner. Rat peritoneal mast cells had a whole-cell conductance of only 10-30 pS, and the resting potential was close to zero. Sometimes discrete openings of channels were observed in the whole-cell configuration. When the Ca2+ concentration on the cytoplasmic side of the membrane was elevated, two types of channels with poor ion specificity appeared. A cation channel, observed at a Ca2+ concentration of approximately 1 microM, had a unit conductance of 30 pS. The other channel, activated at several hundred micromolar Ca2+, was anion selective and had a unit conductance of approximately 380 pS in normal Ringer solution and a bell-shaped voltage dependence. Antigenic stimulation did not cause significant changes in the ionic conductances in either cell type, which suggests that these cells use a mechanism different from ionic currents in stimulus-secretion coupling.     

Voltage-activated currents in guinea pig pancreatic alpha 2 cells. Evidence for Ca2+-dependent action potentials

Glucagon-secreting alpha 2 cells were isolated from guinea pig pancreatic islets and used for electrophysiological studies of voltage- activated ionic conductances using the patch-clamp technique. The alpha 2 cells differed from beta cells in producing action potentials in the absence of glucose. The frequency of these potentials increased after addition of 10 mM arginine but remained unaffected in the presence of 5- 20 mM glucose. When studying the conductances underlying the action potentials, we identified a delayed rectifying K+ current, an Na+ current, and a Ca2+ current. The K+ current activated above -20 mV and then increased with the applied voltage. The Na+ current developed at potentials above -50 mV and reached a maximal peak amplitude of 550 pA during depolarizing pulses to -15 mV. The Na+ current inactivated rapidly (tau h approximately 0.7 ms at 0 mV). Half-maximal steady state inactivation was attained at -58 mV, and currents could no longer be elicited after conditioning pulses to potentials above -40 mV. The Ca2+ current first became detectable at -50 mV and reached a maximal amplitude of 90 pA (in extracellular [Ca2+] = 2.6 mM) at about -10 mV. Unlike the Na+ current, it inactivated little or not at all. Membrane potential measurements demonstrated that both the Ca2+ and Na+ currents contribute to the generation of the action potential. Whereas there was an absolute requirement of extracellular Ca2+ for action potentials to be elicited at all, suppression of the much larger Na+ current only reduced the upstroke velocity of the spikes. It is suggested that this behavior reflects the participation of a low-threshold Ca2+ conductance in the pacemaking of alpha 2 cells.     

Changes in internal ionized Ca2+ and H+ in voltage clamped squid axons

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na+, K+, and some inert cation, at either physiological or zero bath Ca2+ concentration [( Ca2+]o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca2+ level, [( Ca2+]i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K+]o (i.e. approximately -15 mV). In the absence of [Ca2+]o a significant steady depolarization brought about by current flow increased [Ca2+]i (and acidified the axoplasm). Changes in internal hydrogen activity, [H+]i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca2+]i could be made. However, there are effects on [Ca2+]i that can be ascribed to membrane potential. Thus, in the absence of [Ca2+]o, hyperpolarization can reduce [Ca2+]i, implying that a Ca2+ efflux mechanism is enhanced. It is also observed that [Ca2+]i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na+ for Ca2+ in squid giant axon.     

Slow cholinergic and peptidergic transmission in sympathetic ganglia

Experiments of voltage-clamped bullfrog sympathetic neurons suggest that the "slow depolarization" produced by orthodromic stimulation, by muscarinic agonists, or by the peptide luteinizing hormone-releasing hormone (LHRH), results from the suppression of a time- and voltage-dependent outward K+ current, the "M current" (IM). This current is activated between -60 and -10mV, with a half-maximal activation voltage of -35 mV, a minimum time constant (TM) of 150 ms at -35 mV, and a voltage sensitivity corresponding to a single gating particle with a minimum valency of 4.IM does not show time-dependent inactivation within its activation range and provides the sole potential-sensitive component of the steady outward membrane conductances between -60 and -25 mV. Muscarinic agonists and LHRH selectively depress IM via different receptors, without altering their voltage sensitivity. Although not dependent on external Ca2+ ion, IM is also selectively depressed by Ba2+ ions, so accounting for the cholinomimetic action of Ba2+. It is suggested that IM acts as a braking control on spike discharges and that removal of this control during slow cholinergic and peptidergic transmission provides a unique synaptic tuning mechanism.