Amplification of small signals by voltage-gated sodium channels in drone photoreceptors

Summary Photoreceptor cells of the drone,Apismellifera , have a voltage-gated Na⁺ membrane conductance that can be blocked by tetrodotoxin (TTX) and generates an action potential on abrupt depolarization: an action potential is triggered by the rising phase of a receptor potential evoked by an intense light flash (Autrum and von Zwehl 1964; Baumann 1968). We measured the intracellular voltage response to a small (9%), brief (30 ms) decrease in light intensity from a background, and found that its amplitude was decreased by 1M TTX. The response amplitude was maximal when the background intensity depolarized the cell to –38 mV. With intensities depolarizing the cell membrane to –45 to –33 mV the average response amplitude was decreased by TTX from 1.2mV to 0.5mV. TTX is also known to decrease the voltage noise during steady illumination (Ferraro et al. 1983) but, despite this, the ratio of peak-to-peak signal to noise was, on average, decreased by TTX. The results suggest that drone photoreceptors use voltage-gated Na⁺ channels for graded amplification of responses to small, rapid changes in light intensity.Abbreviations TTX tetrodotoxin - V _i intracellular potential with respect to the bath - V _o extracellular potential - V _m,V _i-V _o approximate transmembrane potential - S amplitude of the voltage response to an 8.9% decrease in light intensity - N voltage noise, usually measured as root mean square voltage deviation as described in Methods     

Whole-cell and single-channel currents across the plasmalemma of corn shoot suspension cells

Summary Whole-cell sealed-on pipettes have been used to measure electrical properties of the plasmalemma surrounding protoplasts isolated from Black Mexican sweet corn shoot cells from suspension culture. In these protoplasts the membrane resting potential (V _m ) was found to be –59±23 mV (n=23) in 1mm K _o ^– . The meanV _m became more negative as [K^–] _o decreased, but was more positive than the K⁺ equilibrium potential. There was no evidence of electrogenic pump activity. We describe four features of the current-voltage characteristic of the plasmalemma of these protoplasts which show voltagegated channel activity. Depolarization of the whole-cell membrane from the resting potential activates time- and voltage-dependent outward current through K⁺-selective channels. A local minimum in the outward current-voltage curve nearV _m =150 mV suggests that these currents are mediated by two populations of K⁺-selective channels. The absence of this minimum in the presence of verapamil suggests that the activation of one channel population depends on the influx of Ca²⁺ into the cytoplasm. We identify unitary currents from two K⁺-selective channel populations (40 and 125 pS) which open when the membrane is depolarized; it is possible that these mediate the outward whole-cell current. Hyperpolarization of the membrane from the resting potential produces time- and voltage-dependent inward whole-cell current. Current activation is fast and follows an exponential time course. The current saturates and in some cases decreases at membrane potentials more negative than –175 mV. This current is conducted by poorly selective K⁺ channels, whereP _Cl/P _K=0.43±0.15. We describe a low conductance (20 pS) channel population of unknown selectivity which opens when the membrane is hyperpolarized. It is possible that these channels mediate inward whole-cell current. When the membrane is hyperpolarized to potentials more negative than –250 mV large, irregular inward current is activated. A third type of inward whole-cell current is briefly described. This activates slowly and with a U-shaped current-voltage curve over the range of membrane potentials –90<V _m <0 mV.     

Effect of Metabolic Inhibitors on Membrane Potential and Ion Conductance of Rat Astrocytes

1. The aim of this study was to elucidate the effect of metabolic inhibition on the membrane potential and ion conductance of rat astrocytes. The metabolic inhibitors investigated were dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), cyanide, and oligomycin.2. Primary cultures of astroglial cells from newborn rat cerebral cortex were cultivated for 13–20 days on chamber slides. The effect of metabolic inhibitors on the cellular ATP concentration was estimated from the decrease in peak chemiluminescence from the luciferin/luciferase reaction. The membrane potential and ion conductances were measured from whole-cell recordings with the patch-clamp technique.3. After 2.0 min of incubation ATP decreased from the control level to 43%with cyanide (2 mM), 58% with DNP (1 mM), 47% with FCCP (1 M), and 69% with oligomycin (10 M).4. Under normal conditions V was –74.4±1.0 mV. DNP and FCCP both caused a rapid and reversible depolarization equivalent to a shift in the I/V curve of 8.2±1.3 and 19.7±3.8 mV, respectively. DNP decreased the slope conductance (g) by 22.1% but FCCP had no significant effect on g. In contrast, neither oligomycin nor cyanide had any significant effect on the I/V curve.5. Tetraethylammonium (TEA; 10 mM) depolarized the cells by 7.1±2.0 mV but had no significant effect on g. In the presence of TEA, DNP caused a depolarization of 52.8±3.5 mV and increased g by 45.5±9.6%. The action of FCCP was not affected by the presence of TEA.6. Perfusion of the astrocytes with a Cl^– free solution inhibited the action of DNP and FCCP. Thus the depolarization was only 4.2±1.5mV in DNP and 3.7±0.3 mV in FCCP, which were significantly smaller effects than in the presence of a high intracellular [Cl^–].7. Block of tentative K_ATP channels with tolbutamide (1 mM) or Cl^– channels with Zn²⁺ (1 mM) did not inhibit the depolarization caused by DNP or FCCP.8. In conclusion, DNP and FCCP have specific effects on the plasmalemma in rat astrocytes which may be due to opening of Cl^– channels. This effect was not seen with cyanide or oligomycin and should be considered as a possible complication when DNP and FCCP are used for metabolic inhibition.     

Ionic mechanisms for the antiarrhythmic action of cinnamophilin in rat heart

We investigated the electrophysiological effect and antiarrhythmic potential of cinnamophilin (Cinn), a thromboxane A₂ antagonist isolated fromCinnamomum philippinense, on rat cardiac tissues. Action potential and ionic currents in single rat ventricular cells were examined by current clamp or voltage clamp in a whole-cell configuration. In 9 episodes of ischemia-reperfusion arrhythmia, 10 µM Cinn converted 6 of them to normal sinus rhythm. Cinn suppressed the maximal rate of rise of the action potential upstroke (V_max) and prolonged the action potential duration at 50% repolarization (APD₅₀). Voltage clamp study showed that the suppression of V_max by Cinn was associated with an inhibition of sodium inward current (I_Na, IC₅₀=10.0 ± 0.4 µM). At 30 µM, V_1/2 for the steady-state inactivation curve of I_Na was shifted from –84.1 ± 0.2 to –93.0 ± 0.5 mV. Cinn also reduced calcium inward current (I_Ca) dose-dependently with an IC₅₀ value of 9.5 ± 0.3 µM. Cinn (10 µM) reduced the I_Ca with a negative shift of V_1/2 for the steady-state inactivation curve of I_Ca from –32.2 ± 0.3 to –50.7 ± 0.4 mV. The prolongation of APD₅₀ was associated with an inhibition of the integral of potassium outward current with IC₅₀ values between 4.8 and 7.1 µM. At 10 µM, Cinn reduced I_Na without a negative shift of its voltage-dependent steady-state inactivation curves. The inhibition of transient outward current (I_to) by Cinn (3–30 µM) was associated with an acceleration of its time constant of inactivation and negative shift of its potential-dependent steady-state inactivation curves. The equilibrium dissociation constant (K_d) of Cinn to inhibit open state I_to channels, as calculated from the time constant of developing block, was 18.3 µM. The time constant of recovery of I_to from inactivation state was unaffected by Cinn. The rate constant for the relief from the depolarization-dependent block of I_to was calculated to be 23.9 ms. As compared with its effect on I_to, Cinn exerted about half the potency to block I_Na and I_Ca. These results indicate that the inhibition of I_Na, I_Ca and I_to may contribute to the antiarrhythmic activity of Cinn against ischemia-reperfusion arrhythmia.     

High-conductance K+ channel in pancreatic islet cells can be activated and inactivated by internal calcium

Summary The Ca²⁺-activated K⁺ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than –40 mV. In symmetrical K⁺-rich solutions the single-channelI/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca²⁺-free solution containing 2mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca²⁺ concentration in contact with the membrane inside ([Ca²⁺]_i) to 1.5×10^–7 m had little effect on the relationship between membrane potential andP. When [Ca²⁺]_i was increased to 3×10^–7 m and 6×10^–7 m smaller potential changes were required to open the channels. Increasing [Ca²⁺]_i further to 8×10^–7 m again activated the channels, but the relationship between membrane potential andP was complex. Changing the membrane potential from –50 mV to +20 mV increasedP from near 0 to 0.6 but further polarization to +50 mV decreasedP to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca²⁺]_i=1 and 2 m. In this situation a membrane potential change from –70 to +20 mV increasedP from near 0 to about 0.7 but further polarization to +80 mV reducedP to less than 0.1. The high-conductance K⁺ channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca²⁺]_i within the range 0.1 to 1 m which suggests a physiological role for this channel in regulating the membrane potential and Ca²⁺ influx through voltage-activated Ca²⁺ channels.     

Conditioning prepulses and kinetics of potassium conductance in the frog node

Summary The kinetics of potassium conductance were analyzed in response to voltage-clamp steps with holding potential (–75 mV) as initial condition and after a positive prepulse to-wards +45 mV of 10-msec duration. As the potassium reversal potentialE _K altered during potassium current flow, a method to obtain the conductance independent ofE _K was used. Conductance kinetics at 15°C were analyzed according to the Hodgkin-Huxley (HH) model. The time constant of potassium activation, with holding potential as initial condition, is a monotonous decreasing function of membrane potential. Its value ofca. 9 msec at –50 mV decreases to 1 msec at +30 mV. Changes inE _K did not affect the voltage dependency of this time constant. The time constant of potassium deactivation, i.e. the off-response following a 10-msec prepulse towards +45 mV, shows a completely different voltage dependency. At a membrane potential of –90 mV it is approximately 2 msec and gradually increases for more positive voltages towards a maximum value of about 6 msec, that is reached between –5 and 0 mV. At still larger values of membrane voltage this time constant starts to fall again. It is concluded that a HH-model, as applied for a single population of potassium channels, has to be rejected. Computer simulations indicate that an extension to two populations of independent potassium channels, each with HH-kinetics, is also inconsistent with the observed results.     

Polypeptide neurotoxins modify gating and apparent single-channel conductance of veratridine-activated sodium channels in planar lipid bilayers

Summary The effects of scorpion and sea anemone polypeptide toxins on partially purified veratridine (VER)-activated Na channels from rat brain were studied at the single-channel level in planar lipid bilayers. The probability of the VER-activated channel being open (P _o ) increased with depolarization;P _o was 0.5 at –40 to –50 mV. Saxitoxin (STX) blocked VER-activated channels with an apparent dissociation constant of about 1nm at –45 mV. The apparent single-channel conductance was approximately 9 pS, similar to that seen in VER-activated Na channels from skeletal muscle transverse tubules. Addition of sea anemone or scorpion polypeptide toxins to VER-activated Na channels resulted in a 19% increase in apparent single-channel conductance and a hyperpolarizing shift in theP _o vs. V _m relation such that the channels were more likely to be open at potentials <40 mV. These effects of the polypeptide toxins on the single-channel properties of VER-activated Na channels may account for the previously described potentiation of VER action by polypeptide toxins.     

Expression of low voltage-activated calcium channels inXenopus oocytes

Calcium channels were expressed inXenopus oocytes by means of messenger RNA extracted from the rat thalamo-hypothalamic complex, mRNA(h). Inward barium currents,I _Ba, were recorded in Cl^–-free extracellular solution with 40 mM Ba²⁺ as a charge carrier, using two-microelectrode technique. Depolarizations from a very negative holding potential (V _h=–120 mV) began to activateI _Ba at about –80 mV; this current peaked at –30 to –20 mV and reversed at +50 mV, indicating that I _Ba may be transferred through the low voltage-activated (LVA) calcium channels. The time-dependent inactivation of the current during a prolonged depolarization to –20 mV was quite slow, followed a single exponential decay with a time constant of 1550 msec, and contained a residual component constituting 30% of the maximum amplitude. The current could not be completely inactivated at any holding potential. As expected for LVA current, a steady-state inactivation curve was shifted towards negative potentials. It could be described by the Boltzmann's equation with the half-inactivation potential of –78 mV, slope factor of 15 mV, and residual level of 0.3. ExpressedI _Ba could be blocked by flunarizine (K _d=0.42 µM), nifedipine (K _d=10 µM), and amiloride at a 500 µM concentration. Among the inorganic Ca²⁺ channel blockers, the most potent was La³⁺ (K _d=0.48 µM), while Cd²⁺ and Ni²⁺ were not very selective and almost thousand-fold less effective (K _d=0.52 mM andK _d=0.62 mM, respectively) than La³⁺. Our data show that mRNA(h) induces expression in the oocytes of almost exclusively LVA Ca²⁺ channels with voltage-dependent and pharmacological properties very similar to those observed for T-type Ca²⁺ current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 183–189, May–June, 1995.     

Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat

Summary Chloride channels from rat colonic enterocytes were studied using the patch-clamp technique. After removal of mucus, inside-out patches were excised from the apical membrane of intact epithelium located at the luminal surface. They contained spontaneously switching Cl^– channels with a conductance of 35–40 pS. The channels were blocked reversibly by anthracene-9-carboxylic acid (1mm).In excised patches from single enterocytes, isolated by calcium removal, the Cl^– channels were studied in more detail. TheI–V relation was linear between ±80 mV. The selectivity was I^–>Br^–>Cl^–=NO ₃ ^– >F^–=HCO ₃ ^– .Thirty pS Cl^– channels were also found on the basolateral membrane of crypts isolated by brief calcium removal. TheI–V curve of these Cl^– channels was also linear.The results provide direct evidence for the existence of Cl^– channels in the apical membrane of surface cells in colonic mucosa. The properties of these channels are similar to those previously observed when incorporating membrane vesicles into planar lipid bilayers. Both results support the validity of the theoretical models describing intestinal secretion.     

Microelectrode studies of the effect of lanthanum on the electrical potential and resistance of outer and inner cell membranes of isolated frog skin

Summary Microelectrodes were used to investigate the effect of 0.5mm mucosal lanthanum (La³⁺) on the intracellular potential and the resistance of outer and inner isolated frog skin (Rana esculenta) cell membranes. Under short-circuit conditions, the transapical membrane potentialV _o ^sc (mean value=–65.4±3.2 mV, inside negative) hyperpolarized to –108.7±2.3 mV in control skins, after addition of the sodium blocker amiloride. Current-voltage curves for the outer and inner membranes were constructed from the amiloride-inhibitable current versus the outer membrane potentialV _o or the inner membrane potentialV _t . The outer, and to a lesser degree the inner, membrane showed a characteristic nonlinearity with two slope resistances. Addition of La³⁺ to the outer medium increased the short-circuit current to 190% of the control value.V _o ^sc concomitantly changed to –28±3.5 mV and outer and inner membrane resistances fell, considerably attenuating the nonlinearity seen in control skins. La³⁺ is suggested to raise the conductance by its effect on the surface potential. A secondary long-term inhibitory effect of La³⁺ on short-circuit current has been observed. It is ascribed to the penetration of La³⁺ into the sodium channels.     

The ionic basis of the resting potential of molluscan unstriated muscle

Summary The membrane potential (Vm) of unstriated, non-spiking fibres from the buccal retractor muscle of the opisthobranch molluscPhiline aperta is primarily determined by the distribution of the potassium ion across the membrane. In salines where potassium is varied and chloride remains constant or nearly so, the membrane potential varied with log external K⁺ with a slope of 50.6 (±2.3) mV per decade. In chloride-free salines the slope was 48.5 mV per decade. The experiments were conducted at temperatures of 18–20° C.A ten-fold reduction in external chloride concentration depolarised the fibres by around 10 mV, indicating that chloride permeability makes some contribution to Vm. In salines where [K]₀·[Cl]₀ is constant the Nernst slope was 55.8 mV per decade compared with the theoretical value of 58 mV.The experimental data suggest that the internal potassium concentration of the fibres is 247±31 mM and pNa/pK is 0.01, giving a predicted value of Vm in sea water of –72 mV. The membrane potential of 90 fibres measured in sea water was –74.2±1.3 mV. The membrane contains an electrogenic sodium pump which contributes 4–5 mV to the membrane potential.     

Excitatory action of γ-aminobutyric acid (GABA) on crustacean neurosecretory cells

Summary 1. Intracellular and voltage-clamp recordings were obtained from a selected population of neuroscretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of -aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 µM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity.2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 µm from the cell body. Responses were larger when the application was made at the neuropil level.3. Topical application of Cd²⁺ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA.4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a –50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells.5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10^–6 M [Ca²⁺]_i, GABA (50 µM) increased the membrane conductance more than threefold and shifted the zero-current potential from–25 to–10 mV. At 10^–9 M [Ca²⁺]_i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential.6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.     

Membrane potentials and intracellular Cl− activity of toad skin epithelium in relation to activation and deactivation of the transepithelial Cl− conductance

Summary The potential dependence of unidirectional³⁶Cl fluxes through toad skin revealed activation of a conductive pathway in the physiological region of transepithelial potentials. Activation of the conductance was dependent on the presence of Cl^– or Br^– in the external bathing solution, but was independent of whether the external bath was NaCl-Ringer's, NaCl-Ringer's with amiloride, KCl-Ringer's or choline Cl-Ringer's To partition the routes of the conductive Cl^– ion flow, we measured in the isolated epithelium with double-barrelled microelectrodes apical membrane potentialV _a , and intracellular Cl^– activity,a _Cl ^c , of the principal cells indentified by differential interference contrast microscopy. Under short-circuit conditionsI _sc=27.0±2.0 A/cm², with NaCl-Ringer's bathing both surfaces,V _a was –67.9±3.8mV (mean ±se,n=24, six preparations) anda _Cl ^c was 18.0±0.9mM in skins from animals adapted to distilled water. BothV _a anda _Cl ^a were found to be positively correlated withI _sc (r=0.66 andr=0.70, respectively). In eight epithelia from animals adapted to dry milieu/tap waterV _a anda _Cl ^c were measured with KCl Ringer's on the outside during activation and deactivation of the transepithelial Cl^– conductance (G _Cl) by voltage clamping the transepithelial potential (V) at 40 mV (mucosa positive) and –100 mV. AtV=40 mV; i.e. whenG _Cl was deactivated,V _a was –70.1±5.0 mV (n=15, eight preparations) anda _Cl ^c was 40.0±3.8mm. The fractional apical membrane resistance (fR _a) was 0.69±0.03. Clamping toV=–100 mV led to an instantaneous change ofV _a to 31.3±5.6 mV (cell interior positive with respect to the mucosal bath), whereas neithera _Cl ^c norfR _a changed significantly within a 2 to 5-min period during whichG _Cl increased by 1.19±0.10 mS/cm². WhenV was stepped back to 40 mV,V _a instantaneously shifted to –67.8±3.9 mV whilea _Cl ^c andfR _a remained constant during deactivation ofG _Cl. Similar results were obtained in epithelia impaled from the serosal side. In 12 skins from animals adapted to either tap water or distilled water the density of mitochondria-rich (D _MRC) cells was estimated and correlated with the Cl current (I _Cl though the fully activated (V=–100mV) Cl^– conductance). A highly significant correlation was revealed (r=–0.96) with a slope of –2.6 nA/m.r. (mitochondria-rich cell and an I-axis intercept not significantly different from zero. In summary, the voltage-dependent Cl currents were not reflected infR _a anda _Cl ^a of the principal cells but showed a correlation with the m.r. cell density. We conclude that the pricipal cells do not contribute significantly to the voltage-dependent Cl conductance.     

Effects of low concentrations of tetradotoxin on rat trigeminal ganglion neurons

Voltage clamping and intracellular perfusion methods were used to investigate ionic currents produced by depolarizing shifts of –120 mV from holding potential during experiments on neurons isolated from the trigeminial ganglion of one-month-old rats. It was found that tetradotoxin at low (external) concentrations of 10^–12–10¹⁰ M produced an increase in the amplitude and alternations in the kinetics of inward ionic currents. Similar effects were observed in 8 test cells out of 29. The extent to which the increase in the amplitude of inward ionic currents depended on concentration level could be described by Langmuir's isotherm, with a dissociation constant of the order of 5·10^–12 M. No such tetrodotoxin effects were observed when chloride ions were replaced by a non-penetrating anion in the intracellular solution. It is suggested that tetrodotoxin-sensitive channels exist in the neuronal membrane of the rat trigeminal ganglion, letting through chloride ions during depolarization of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 723–729, November–December, 1986.     

Electrical characteristics of stomatal guard cells: The contribution of ATP-dependent, “Electrogenic” transport revealed by current-voltage and difference-current-voltage analysis

Summary The steady-state, current-voltage (I–V) characteristics of stomatal guard cells fromVicia faba L. were explored by voltage clamp using conventional electrophysiological techniques, but with double-barrelled microelectrodes containing 50mm K⁺-acetate. Attention was focused, primarily, on guard cell response to metabolic blockade. Exposures to 0.3–1.0mm NaCN and 0.4mm salicylhydroxamic acid (SHAM) lead consistently to depolarizing (positive-going) shifts in guard cell potentials (V _m ), as large as +103 mV, which were generally complete within 60–90 sec (mean response half-time, 10.3±1.7 sec); values forV _m in NaCN plus SHAM were close or positive to –100 mV and well removed from the K⁺ equilibrium potential. Guard cell ATP content, which was followed in parallel experiments, showed a mean half-time for decay of 10.8±1.9 ([ATP] _t=0, 1.32±0.28mm; [ATP] _{t=60–180sec}, 0.29±0.40mm). In respiring cells, theI–V relations were commonly sigmoid aboutV _m or gently concave to the voltage axis positive toV _m . Inward- and outward-rectifying currents were also observed, especially near the voltage extremes (nominally –350 and +50 mV). Short-circuit currents (atV=0 mV) were typically about 200–500 mA m^–2. The principal effect of cyanide early on was to linearize theI–V characteristic while shifting it to the right along the voltage axis, to decrease the membrane conductance, and to reduce the short-circuit current by approx. 50–75%. The resulting difference-current-voltage (dI–V) curves (±cyanide) showed a marked sensitivity to voltages negative from –100 mV and, when clamp scans had been extended sufficiently, they revealed a distinct minimum near –300 mV before rising at still more negative potentials. The difference currents, along with changes in guard cell potential, conductance and ATP content are interpreted in context of a primary, ATP-consuming ion pump. FittingdI–V curves to reaction kinetic model for the pump [Hansen, U.-P., et al. (1981)J. Membrane Biol. 63:165; Blatt, M.R. (1986)J. Membrane Biol. 92:91] implicates a stoichiometry of one (+) charge transported outward for each ATP hydrolyzed, with pump currents as high as 200 mA m^–2 at the free-running potential. The analysis indicates that the pump can comprise more than half of the total membrane conductance and argues against modulations of pump activity alone, as an effective means to controlling K⁺ transport for stomatal movements.     

Sialic acid and the surface charge associated with hyperpolarization-activated,inward rectifying channels

Summary The whole-cell configuration of the patch-clamp technique was used with cultured pacemaker cells from the rabbit sinoatrial node to test the hypothesis that sialic acid residues (NANA) constitute much of the negative surface charge associated with hyperpolarization-activated, inward rectifying channels. Activation-voltage relationships (between –70 and –140 mV) were determined for hyperpolarization-activated (inward rectifying) current (i _f). Addition of 10mm Ca²⁺ shifted the half-activation potential (V _1/2) from –89.5±0.9 mV to –77.9±2.6 mV (P<0.01), confirming the presence of negative fixed charges on the myocytes after 3 to 5 days in culture. Addition of 20mm dimethonium, an organic divalent cation that screens but does not bind to negative surface charge, shiftedV _1/2 from –86.8±1.4 mV to –75.0±1.7 mV (P<0.001) without affecting the amplitude of the current. In contrast, 10mm Ca²⁺ reduced the amplitude ofi _f significantly. Incubation of cells with a highly purified preparation of neuraminidase (0.1–2.0 U/ml, 1 hr, 37°C), an enzyme that selectively removes NANA from glycoproteins and glycolipids, failed to alterV _1/2 or the amplitude ofi _f significantly. Pretreatment of cells with neuraminidase (1.0 U/ml, 1 hr, 37°C) failed to alter the positive shift ofV _1/2 produced by dimethonium. The results suggest that NANA does not constitute the negative surface charge associated with hyperpolarization-activated, inward rectifying channels.     

Diversity of K+ channels in the basolateral membrane of restingnecturus oxyntic cells

Summary Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa ofNecturus maculosa. In cell-attached patches with pipette solution containing 100mm KCl, four major classes of K⁺ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec fora, and 8 msec forb). Some channels showed subconductance levels. The maximal inward conductanceg _max was 31±5 pS (n=13) and the reversal potentialE _rev was atV _p=–34±6 mV (n=9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels withg _max=40 ±18 pS (n=3) andE _rev was atV _p=–31±5 mV (n=3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1mm) to the bath solution in cell-attached patches increased the open probabilityP _o of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels withg _max=21±3 pS (n=4) andE _rev was atV _p=–24±9 mV (n=4). These channels exhibited very high open probability (P _o=0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26±2 pS (n=3) andE _rev atV _p=–33±3 mV (n=3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K⁺ in the pipette solution replaced by Na⁺, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K⁺ over Na⁺ and Cl^–. A variety of K⁺ channels contributes to the basolateral K⁺ conductance of resting oxyntic cells.     

Pre- and post-synaptic actions of the cerebral ventral giant cell on buccal muscles ofPhiline

Summary The cerebral ventral giant cell ofPhiline exterts a selective presynaptic inhibitory modulatory action on the terminals of a buccal excitatory motoneuron in two buccal muscles. Other excitatory inputs to the muscles are not affected. The ventral giant cell also makes direct synaptic contacts on the fibres of the same muscles. In the retractor muscle M4, 5 the junction potentials are usually depolarising when measured in sea water, but in the fibres of M6 they may have either polarity. The mean membrane potential of the fibres of M4, 5 and M6 was –74.7±0.65 mV and –64±0.95 mV respectively. Depolarization of the muscles fibres by around 15 mV by immersion in 20 mM K saline abolished the junction potential in M4, 5 and converted the depolarizing potential in M6 to a hyperpolarizing response.It is concluded that the VGC junction potential results from an increase in membrane conductance to an ion with a reversal potential between –60 and –70 mV.     

Novel actions of ryanodine and analogues—Perturbers of potassium channels

The effects of ryanodine, 9,21-didehydroryanodine and 9,21-didehydroryanodol on two types of K⁺ channel (a maxi, Ca²⁺-activated, 170 pS channel (BK channel) and an inward rectifier, stretch-sensitive channel of 35 pS conductance (IK channel) found in the plasma membrane of locust skeletal muscle have been investigated. 10^–9M-10^–5M ryanodine irreversibly induced a dose-dependent reduction of the reversal potential (V_rev) of the currents of both channels, i.e. from 60 mV in the absence of the alkaloid to 15 mV for 10^–5M ryanodine, measured under physiologically normal K⁺ and Na⁺ gradients. In both cases the change in the ionic selectivity was Ca²⁺-independent. 9,21-didehydroryanodine and 9,21-didehyroryanodol also reduced V_rev, but only to 35 mV during application of 10^–5M of these compounds. Additionally, 9,21-didehydroryanodine reversibly diminished the conductances of the two K⁺ channels. To test the hypothesis that ryanoids increase Na⁺ permeability by enlarging the K⁺ channels, the channels were probed with quaternary ammonium ions during ryanoid application. When applied to the cytoplasmic face of inside-out patches exised from locust muscle membrane, TEA blocked the K⁺ channels in a voltage-dependent fashion. The dissociation constant (K_d(0)) for TEA block of the IK channel was reduced from 44 mM to 1 mM by 10^–7 M ryanodine, but the voltage-dependence of the block was unaffected. Qualitatively similar data were obtained for the BK channel. Ryanodine had no effect on the K_d for cytoplasmically-applied TMA. However, the voltage-dependence for TMA block was increased for both K⁺ channels, from 0.47 to 0.8 with 10^–6M ryanodine. The effects of ryanodine on TEA and TMA block support the hypothesis that ryanodine enlarges the K⁺ channels so as to facilitate permeation of partially hydrated Na⁺ ions.     

Chloride currents inChara—A patch-clamp study

Summary Ionic current steps were recorded with the patch-clamp technique from algal cells that had been prepared without enzyme treatment. Inward current steps with different conductance levels occurred, the lowest level being 7 pS. There were complex transitions between levels indicating either a lack of independence between single channels, or sublevels of a much larger conductance unit. The reversal potential was consistent with the permeant ion being Cl^–. Furthermore, when a different concentration of Cl^– was used in the patch electrode the reversal potential of the inward current shifted in a manner consistent with a Nernstian change in the Cl^– reversal potential. The frequency of the current steps was voltage dependent and suggestive of the hyperpolarization-activated Cl^– currents reported in voltage-clamp studies. Outward current steps, with conductances of 38 pS, were recorded when the membrane patch was depolarized by more than +120 mV. Their amplitude and frequency increased at more positive potentials. The current was probably carried by an efflux of cations through a different set of channels. The resting membrane potential, measured unambiguously without contamination from the tonoplast, was –190±5 mV.