Inhibition of Cl- Channel Activation in Chara corallina Membrane by Lanthanum Ion

We examined a role of Ca²⁺ in the activation of the two majorion channels, i.e., Cl^– and K⁺ channels at the excitationof the characean plasmalemma. The current-voltage relation (I-Vcurve) of the Chara membrane was compared under the ramp voltageclamp condition before and after external application of 20µMof La³⁺ (a Ca²⁺ channel blocker). The transient inward currentcomponent, which is carried mainly by the efflux of Cl^–,disappeared almost completely in about 30 min with La³⁺ treatment.On the other hand, no effect was observed on the late largeoutward current, which is mainly carried by the efflux of K⁺in a large depolarization region (less negative than –50mV). These results suggest that the Cl^– channel in theChara plasmalemma is activated by Ca²⁺ influx, while the K⁺channel is simply activated by depolarization. (Received April 7, 1986; Accepted June 6, 1986)     

Excitable Channels in Nitellopsis obtusa as Part of an Active Ion-Transport System

The present paper is a study on the rapid and the slow excitablechannels of Nitellopsis obtusa. The working hypothesis is thatduring the excitation of these cells a Ca²⁺-dependent activeion-transport system in the plasmalemma is activated (Gyenesand Bulychev, 1979; Gyenes, Bulychev, and Kurella, 1980) whichmay interact with a light-dependent active transport systemalso present in the plasmalemma. It is found that under conditionsof maximal light-induced current changes, registered in voltageclamp experiments, the amplitudes of both action current componentsare relatively small (10–15 µA cm²) and they increaseup to 100–150 µA cm^–2 during 15–30 minin the dark. Cells may also be excited chemically under conditionsof unchanged voltage across the plasmalemma. It is suggestedthat in the excitation process of Nitellopsis obtusa two typesof ion channels take part/emdash electrically excitable passiveand chemically excitable active channels-both incorporated inone proteolipid complex of a Ca²⁺-dependent ATPase of the plasmalemma.     

Mechanism of Postinhibitory Rebound in Molluscan Neurons

Postinhibitory rebound (PIR) is an intrinsic property of manyneurons but the underlying mechanism is not well understood.We studied PIR and its relationship to spike adaptation in B-cellsisolated from the buccal ganglia of Aplysia. These neurons exhibitPIR following inhibitory synaptic input and following directmembrane hyperpolarization. Hyperpolarizing and depolarizingvoltage clamp pulses from the resting potential evoke slow changesin membrane current that persist in the form of tail currentsfollowing the pulses. A subtraction method was used to isolateslow tail currents for study. Current-voltage measurements indicatethat slow outward tail currents following depolarizing pulsesresult from increases in membrane conductance, while inwardtail currents following hyperpolarizations to –50 and–60 mV result from conductance decreases. The reversalpotential of both outward and inward tail current is between–60 and –70 mV. Tail currents activated by pulsesmore positive than –60 mV are sensitive to the externalK⁺ concentration and blocked by injection of Cs⁺ and TEA. WhenCa²⁺ influx is prevented by bathing cells in Ca²⁺ free salineor by adding Co²⁺ or Ni²⁺, the tail currents are reduced buta significant fraction of the current is insensitive to thesetreatments. More negative conditioning pulses activate a secondcomponent of inward tail current that is weakly sensitive toK⁺ but more strongly effected by substitution of N-methyl glucamineor Li⁺ for external Na⁺. We conclude that both PIR and adaptationresult from slow changes in a voltage dependent, non-inactivatingK⁺ conductance that is active at voltages near the resting potentialand is not tightly coupled to Ca²⁺ influx. In addition, a secondinward current is activated by large hyperpolarizing pulsesthat results from an increase in Na⁺ and K⁺ conductance. Thissecond process is likely to contribute to PIR under particularcircumstances.     

Chloride Transport in Chara: I. KINETICS AND CURRENT-VOLTAGE CURVES FOR A PROBABLE PROTON SYMPORT

Chara cells show an inward positive electric current acrossthe plasmalemma when exposed to Cl^– under voltage-clampconditions. The rapid rise of this current suggests that itis directly associated with the inward transport of Cl^–.The dependence of the current on Cl^– concentration showssaturation, the data fitting the Michaelis-Menten equation withV_m up to 100 nmol m^–2 s^–1 (for Cl^–starvedcells) with K_M 10–20 µM, and with some allowancefor an unstirred layer of water adjacent to the membrane. Theeffects on the current of clamp potential, illumination, withdrawalof alkali metal cations, and addition of amine were also investigated.These results suggest that the mechanism is the symport of 2H⁺ with each Cl^–, and that the actions of light, externalK⁺, and amine in stimulating Cl^–, influx are indirect.     

Divalent cation and anion currents are activated during the dark-induced transient hyperpolarization of the plasma membrane of the green alga Eremosphaera viridis

Darkening after illumination induces a transient hyperpolarizationof the plasma membrane of the unicellular green alga Eremosphaeraviridis de Bary. With electro-physiological methods, in particularthe two electrode voltage-clamp technique, we investigated theion fluxes involved in this transient potential change (TP).The question was whether other ion currents besides those carriedby the known Ca²⁺-dependent K⁺ channel take part in this actionpotential-like, but hyperpolarizing, response. At maximum hyperpolarizationvoltage-clamp measurements resulted in ‘N-shaped’I/V curves, known from other botanical systems. The differentinstantaneous current components of the N-shaped I/V curvesoccurred at different times during a single transient potentialchange (TP). Substitution of alkali metal cations in the bathingsolution by NMG/NO₃ showed that the inward currents in the I/Vcurves were not carried by an influx of K⁺ into the cytoplasm.The voltage amplitude of the TP not only depended on the externalK⁺ concentration, but also on the Mg²⁺ concentration in thebathing solution. Increasing Mg²⁺ concentrations shifted themembrane potential in the top of the TP in the direction ofthe Nernst potential of Mg²⁺ and resulted in an increased inwardcurrent component of the N-shaped I/V curves. Another currentcomponent was found to be carried not by cations but by an effluxof anions. It was a voltage-dependent component with a maximumcurrent amplitude at voltages of about –220 to –240mV, and was blocked by the anion channel inhibitors anthracen-9-carboxylicacid (A9C), (5-nitro-2-3-phenylpropylamino) benzoic acid (NPPB)and ZnCI2. Based on these data a model is proposed which explainsthe N-shape of the I/V curves observed during the transientpotential change of the alga E. viridis by the combination ofan inward cation current with an inward anion current and theoutward cation current carried by the Ca²⁺-dependent K⁺ channels. Key words: Anion current, cation current, Eremosphaera viridis, potassium channel, voltage-clamp     

Excitable Active Electrogenic Ion-Transport Units in the Plasmalemma of Nitellopsis obtusa

With the use of voltage clamp and current clamp techniques thesupposition was proved that during the hyperpolarizing response(HR) N. obtusa cells generate active electromotive force (emf)at the expense of metabolic energy. Threshold inward currentsent through the plasmalemma of the cell which was depolarizedwith 100 mol m^–3 KG resulted in the HR with the transferof the membrane's excitable units from the high-conductive stateto the low-conductive state. During the HR the membrane potentialV_m increased from –135±10 mV to –290±15mV, the membrane resistance increased from 3.3±1.5 kOhmcm² to 5.8±1.2 kOhm cm² and the membrane emf E_m increasedfrom –20±4 mV to –93± 15 mV. Changesin the external concentration of K^–, Na⁺, Cl^– andH^– did not affect the patterns of HR. Cells which weredepolarized by light also generated HR (in normal medium) whichwas accompanied with the increase of V_m, R_m and E_m. The highvalue of Em generated during the HR can be explained only withthe involvement of active electrogenic charge transfer acrossthe membrane. 0.05 mol m^–3 DCCD added to the externalmedium inhibited the HR in both cases. Key words: Active ion transport, Hyperpolarizing response, Nitellopsis obtusa     

Exocytotic release of ATP and activation of P2X receptors in dissociated guinea pig stellate neurons

Activation of P2X receptors by a Ca²⁺- and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent release of ATP was measured using patch-clamp recordings from dissociated guinea pig stellate neurons. Asynchronous transient inward currents (ASTICs) were activated by depolarization or treatment with the Ca²⁺ ionophore ionomycin (1.5 and 3 µM). During superfusion with a HEPES-buffered salt solution containing 2.5 mM Ca²⁺, depolarizing voltage steps (–60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger, transient inward current. Equimolar substitution of Ba²⁺ for Ca²⁺ augmented the postdepolarization frequency of ASTICs, while eliminating the larger transient current. Perfusion with an ionomycin-containing solution elicited a sustained activation of ASTICs, allowing quantitative analysis over a range of holding potentials. Under these conditions, increasing extracellular [Ca²⁺] to 5 mM increased ASTIC frequency, whereas no events were observed following replacement of Ca²⁺ with Mg²⁺, demonstrating a Ca²⁺ requirement. ASTICs were Na⁺ dependent, inwardly rectifying, and reversed near 0 mV. Treatment with the nonselective purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 µM) blocked all events under both conditions, whereas the ganglionic nicotinic antagonist hexamethonium (100 µM and 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 µM). The presence of botulinum neurotoxin E (BoNT/E) in the whole-cell recording electrode significantly attenuated the ionomycin-induced ASTIC activity, whereas phorbol ester treatment potentiated this activity. These results suggest that ASTICs are mediated by vesicular release of ATP and activation of P2X receptors. sympathetic; purinergic; neurotransmission; phorbol ester; botulinum toxin     

Effects of Calmodulin on Ca2+-Dependent Cl--Sensitive Anion Channels in the Chara Plasmalemma: a Patch-Clamp Study

The effects of calmodulin (from spinach) on the Ca²⁺-dependentCl^--sensitive anion channel in the Chara plasmalemma (Okiharaet al. 1991) were studied by the inside-out patch-clamp technique.The current of Cl^- ions, which flowed through the channel at1.0 µM Ca²⁺, tended to decrease irregularly with time.This tendency toward a decrease in the current was no longerapparent after application of calmodulin for some time. Theactivity of the channel was restored to a small extent or tendedto increase during the early stages of application of calmodulin.Such a transient action of calmodulin on the channel activitywas evident, at voltages more negative than –100 mV. (Received August 20, 1992; Accepted October 19, 1992)     

Evidence for electrogenic active ion transport across the frog olfactory mucosa in vitro

We present the first electrophysiological evidence for electrogenicion transport across the frog olfactory mucosa in vitro. Whenthe isolated dorsal mucosa was placed in an Ussing chamber andbathed symmetrically in amphibian Ringer's, the ciliated sidebecame electronegative (V = –5.2 mV ± 0.7 mV).The resistance of the mucosal preparation was 148 ± 4 cm². The true short-circuit current was obtained as the intersectionof the I–V curve with the current axis after correctingfor the series solution resistance. The average value of theshort-circuit current was 35.9 µA/cm². The I–V relationwas linear over the applied potential range of ± 16mV.The magnitude of the specific resistance of the olfactory mucosais comparable to values reported for various actively transportingrespiratory and oral cavity epithelia. Because the geometricalarea of the aperture used to normalize both the short-circuitcurrent and the resistance undoubtedly underestimates the actualarea of the dorsal olfactory epithelium, the specific resistanceand the short-circuit current are probably underestimated andoverestimated, respectively. Therefore, the nominally low resistanceneed not imply a leaky epithelium. Substitution of NO₃^–for Cl^– caused the current to increase and the resistanceto decrease. These results suggest that cation absorption playsa role in the sign of the short-circuit current. The in vitropreparation responded to the odorant ethyl n-butyrate by givingan electro-olfactogram (EOG)-like voltage transient which wassuperimposed on the steady-state potential created by activeion transport. The significance of these results is discussedfrom the perspective of the peripheral events surrounding olfactorytransduction.     

Pacemaker potentials generated by interstitial cells of Cajal in the murine intestine

Pacemaker potentials were recorded in situ from myenteric interstitial cells of Cajal (ICC-MY) in the murine small intestine. The nature of the two components of pacemaker potentials (upstroke and plateau) were investigated and compared with slow waves recorded from circular muscle cells. Pacemaker potentials and slow waves were not blocked by nifedipine (3 µM). In the presence of nifedipine, mibefradil, a voltage-dependent Ca²⁺ channel blocker, reduced the amplitude, frequency, and rate of rise of upstroke depolarization (dV/dt_max) of pacemaker potentials and slow waves in a dose-dependent manner (1–30 µM). Mibefradil (30 µM) changed the pattern of pacemaker potentials from rapidly rising, high-frequency events to slowly depolarizing, low-frequency events with considerable membrane noise (unitary potentials) between pacemaker potentials. Caffeine (3 mM) abolished pacemaker potentials in the presence of mibefradil. Pinacidil (10 µM), an ATP-sensitive K⁺ channel opener, hyperpolarized ICC-MY and increased the amplitude and dV/dt_max without affecting frequency. Pinacidil hyperpolarized smooth muscle cells and attenuated the amplitude and dV/dt_max of slow waves without affecting frequency. The effects of pinacidil were blocked by glibenclamide (10 µM). These data suggest that slow waves are electrotonic potentials driven by pacemaker potentials. The upstroke component of pacemaker potentials is due to activation of dihydropyridine-resistant Ca²⁺ channels, and this depolarization entrains pacemaker activity to create the plateau potential. The plateau potential may be due to summation of unitary potentials generated by individual or small groups of pacemaker units in ICC-MY. Entrainment of unitary potentials appears to depend on Ca²⁺ entry during upstroke depolarization. pacemaker activity; slow waves; gastrointestinal motility; calcium channel     

Light-Induced H+ Accumulation in the Vacuole of Nitellopsis obtusa

The effects of light on the pH in the vacuole and the electricpotential difference across the plasmalemma and the tonoplastof Nitellopsis obtusa were investigated by means of conventionaland H⁺-specific glass or antimony microelectrodes. Illuminationis found to bring about a decrease in the pH of the vacuolarsap by 0.1–0.5 units concomitant with a depolarizationof the cell. The light-induced changes of the potential differenceand the vacuolar pH depend in different ways on the pH of theexternal medium (pH_o). At pH_o 9.0 cells exhibit great light-inducedpotential changes (up to 100 mV), but only small pH changesof the vacuolar sap. At neutral or slightly acidic pH_o valuesthe amplitude of the light-induced pH changes in the vacuoleincreases up to 0.3–0.5 pH units, but the amplitudes ofthe potential changes at the plasmalemma are relatively small.At pH_o 9.0 a transient acidification of the medium is observedupon illumination whereas at lower pH values light-induced alkalinizationwas only seen. Transfer of the cells from pH_o 9.0 to pH_o 7.5results in a cell hyperpolarization by 60–80 mV and adecrease of the vacuolar pH by 0.4–0.5 units under lightconditions but has no significant effect on the potential andthe vacuolar pH in the darkness. It is proposed that mechanismsof active H⁺ extrusion from the cytoplasm are located both inthe plasmalemma and the tonoplast. The observed acidificationin the vacuole appears to be determined by a light-induced increaseof the concentration of H⁺ in the cytoplasm. The H⁺ conductionof the plasmalemma seems to increase on illumination. The patternof the light-induced H⁺ fluxes across the tonoplast and theplasmalemma depends crucially on the extent of the light-inducedchanges in the H⁺ conductance and on the electrochemical gradientfor H⁺ at the plasmalemma.     

Ionic Currents across the Plasmalemma of Chara inflata Cells: I. OSMOTIC EFFECTS OF SORBITOL ON K+, CI- AND LEAK CURRENTS

This paper describes experiments designed to investigate theeffects of increases in external osmotic pressure on the electrophysiologicalbehaviour of the plasmalemma in cells of the brackish-wateralga, Chara inflata. The electrical conductance of the plasmalemmaof these cells of, due to the diffusion of ions, consists mainlyof K⁺, Cl^– and leak components. The addition of sorbitolat concentrations in the range 40–280 mol m^–3 tothe external solution bathing the cells, progressively and reversiblydepolarized the cell membrane and increased the total membraneconductance, chiefly due to increases in each of the separateionic conductances. At concentrations higher than about 280mol m^–3 when the cells became plasmolysed, the effectsof sorbitol on the electrical properties of the cell ceasedto be fully reversible. When the membrane electrical potentialdifference is stepped in a negative direction with a voltage-clamp,the resulting inward current has voltage-dependent componentsconsisting of an inactivating K⁺ current, an activating Cl^–current and a constant leak current. The addition of sorbitolto the external solution modified these currents by increasingtheir magnitude, by increasing the half-time of the inactivationof the K⁺ current, and by decreasing the half-time of activationof the Cl^– current. Key words: Chara inflata, osmotic effects, K⁺ and Cl^– currents     

Electrophysiological Evidence for an Electrogenic Proton Pump and the Proton Symport of Glucose in the Marine Fungus Dendryphiella salina

The marine hyphomycete Dendryphiella salina (Suth.) Nicot &Pugh has a resting membrane potential of –250 mV (insidenegative). The respiratory inhibitors sodium azide and FCCPinduced a rapid but reversible depolarization of the membraneof at least 180 mV; sodium azide also caused alkalinizationof the medium. Vanadate brought about significant depolarizationbut this was not always reversible. EDTA induced depolarizationthough to a lesser extent. DIDS and SITS caused a depolarizationof around 30–70 mV which was readily reversible, N-ethylmaleimideirreversibly depolarized the membrane by 180–200 mV. Ouabainhad no effect. When external concentrations of H⁺ , K⁺ , Na⁺or Cl^– were changed singly, only changes in H⁺ affectedmembrane potential, with shifts decreasing with increasing pH.Glucose and 3-O-methyl glucose depolarized the membrane in aconcentration-dependent manner which was enhanced by starvationof the hyphae. Recovery occurred in the presence of the hexose.Glucose caused an alkalinization of the medium, with time characteristicssimilar to the membrane potential changes. It is concluded thatthere is an electrogenic proton pump and a proton—glucosesymporter in D. salina. The retention of proton-based transportsystems suggests a terrestrial origin for the fungus. Key words: Marine fungi, Dendryphiella salina, membrane potential, electrogenic proton pump, proton symport, hexose     

Cytoplasmic pH of Root Hair Cells of Sinapis alba Recorded by a pH-sensitive Micro-electrode. Does Fusicoccin Stimulate the Proton Pump by Cytoplasmic Acidification?

Bertl, A. and Felle, H. 1985. Cytoplasmic pH of root hair cellsof Sinapsis alba recorded by a pH-sensitive micro-electrode.Does fusicoccin stimulate the proton pump by cytoplasmic acidification?—J. exp. Bot. 36: 1142–1149. pH-sensitive micro-electrodes, filled with ion-exchanger resinhave been fabricated with a turgorinsensitive tip and have beenapplied to test the intracellular pH and changes thereof inroot hair cells of Sinapis alba. (1) The cytoplasmic pH of Sinapisroot hairs was determined to be 7.3 ±0.2 (at neutralexternal pH). (2) 10 mol m^–3 sodium azide depolarizesthe membrane potential by about 100 mV and acidifies the cytoplasmby 0.8 pH-units. (3) The change from 1.0 mol m^–3 to 10mol m^–3 external potassium causes a depolarization ofabout 45 mV, but no change in internal pH. (4) At an externalpH of 5.0, sodium acetate hyperpolarizes the plasmalemma byabout 60 mV and acidifies the cytoplasmic pH by 0.2 to 0.3 units.(5) 2.0 mmol m^–3 fusicoccin (FC) hyperpolarizes the plasmalemmaby 20–25 mV, acidifies the cytoplasm by 0.1 to 0.2 pH-units,and acidifies the external medium by about 0–3 pH-units.It is concluded that cytoplasmic acidification stimulates theelectrogenic proton pump in Sinapis root hairs, and it is suggestedthat the FC-induced effects, viz. hyperpolarization and externalacidification, can also be interpreted in this way. Key words: —Cytoplasmic pH, pH-sensitive micro-electrode, fusicoccin     

Electrogenic Pump Current and ATP-Dependent H+ Efflux Across the Plasma Membrane of Nitellopsis obtusa

The correlation between the pump current and the ATP-dependentH⁺ efflux was examined in internodal cells of Nitellopsis obtusa.To control the cytoplasmic pH and ATP concentration, the tonoplastwas removed by intracellular perfusion with an EGTA-containingmedium. Two groups of perfused cells were prepared, one with1 mM ATP (+ATP cells) and the other without ATP but with hexokinaseand glucose (–ATP cells). The ATP-dependent H⁺ effluxwas calculated as the difference in H⁺ efflux between the +ATPand –ATP cells. Based on an electrically equivalent circuitmodel of the plasma membrane, the pump current was calculatedfrom the membrane potentials and the membrane resistances ofboth +ATP and –ATP cells. When the membrane potentialwas not too high (–220 mV), the ATP-dependent H⁺ current(19 mA m^–2) was almost equal to the pump current (20 mAm^–2) calculated from the electrical data. This indicatesthat the electrogenic pump current across the plasma membraneof Nitellopsis obtuse was mostly carried by H⁺. But when themembrane potential was high (–280 mV), the H⁺ currentwas lower than the pump current. The possible cause of thisdiscrepancy is discussed. (Received November 5, 1984; Accepted February 28, 1985)     

Pacemaker activity in urethral interstitial cells is not dependent on capacitative calcium entry

The aim of the present study was to investigate the properties and role of capacitative Ca²⁺ entry (CCE) in interstitial cells (IC) isolated from the rabbit urethra. Ca²⁺ entry in IC was larger in cells with depleted intracellular Ca²⁺ stores compared with controls, consistent with influx via a CCE pathway. The nonselective Ca²⁺ entry blockers Gd³⁺ (10 µM), La³⁺ (10 µM), and Ni²⁺ (100 µM) reduced CCE by 67% (n = 14), 65% (n = 11), and 55% (n = 9), respectively. These agents did not inhibit Ca²⁺ entry when stores were not depleted. Conversely, CCE in IC was resistant to SKF-96365 (10 µM), wortmannin (10 µM), and nifedipine (1 µM). Spontaneous transient inward currents were recorded from IC voltage-clamped at –60 mV. These events were not significantly affected by Gd³⁺ (10 µM) or La³⁺ (10 µM) and were only slightly decreased in amplitude by 100 µM Ni²⁺. The results from this study demonstrate that freshly dispersed IC from the rabbit urethra possess a CCE pathway. However, influx via this pathway does not appear to contribute to spontaneous activity in these cells. smooth muscle; patch clamp; spontaneous transient inward currents     

Involvement of anion channel(s) in the modulation of the transient outward K(+) channel in rat ventricular myocytes

The cardiac Ca²⁺-independent transient outward K⁺ current (I_to), a major repolarizing ionic current, is markedly affected by Cl^– substitution and anion channel blockers. We reexplored the mechanism of the action of anions on I_to by using whole cell patch-clamp in single isolated rat cardiac ventricular myocytes. The transient outward current was sensitive to blockade by 4-aminopyridine (4-AP) and was abolished by Cs⁺ substitution for intracellular K⁺. Replacement of most of the extracellular Cl^– with less permeant anions, aspartate (Asp^–) and glutamate (Glu^–), markedly suppressed the current. Removal of external Na⁺ or stabilization of F-actin with phalloidin did not significantly affect the inhibitory action of less permeant anions on I_to. In contrast, the permeant Cl^– substitute Br^– did not markedly affect the current, whereas F^– substitution for Cl^– induced a slight inhibition. The I_to elicited during Br^– substitution for Cl^– was also sensitive to blockade by 4-AP. The ability of Cl^– substitutes to induce rightward shifts of the steady-state inactivation curve of I_to was in the following sequence: NO₃^– > Cl^– Br^– > gluconate^– > Glu^– > Asp^–. Depolymerization of actin filaments with cytochalasin D (CytD) induced an effect on the steady-state inactivation of I_to similar to that of less permeant anions. Fluorescent phalloidin staining experiments revealed that CytD-pretreatment significantly decreased the intensity of FITC-phalloidin staining of F-actin, whereas Asp^– substitution for Cl^– was without significant effect on the intensity. These results suggest that the I_to channel is modulated by anion channel(s), in which the actin cytoskeleton may be implicated. transient outward potassium current; anion channel; actin cytoskeleton; myocyte; potassium ion     

cAMP-activated maxi-Cl(-) channels in native bovine pigmented ciliary epithelial cells

The eye’s aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl^– enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl^– channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl^– channels with a unitary conductance of 272 ± 2 pS (n = 80). Voltage steps from 0 to ±80 mV, but not to ±40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl^– channels. cAMP also activated the maxi-Cl^– channels in outside-out patches. In both cases, maxi-Cl^– channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl^– concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl^– concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at ±80 but not at ±40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl^– channels of native PE cells that contribute to Cl^– release particularly from Cl^–-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl^– reabsorption into the ciliary stroma. cell volume; chloride secretion; aqueous humor formation     

Transient Cytoplasmic pH Changes in Correlation with Opening of Potassium Channels in Eremosphaera

Steigner, W. Khler, K., Simonis, W. and Urbach, W. 1988. Transientcytoplasmic pH changes in correlation with opening of potassiumchannels in Eremosphaera.—J. exp. Bot. 39: 23–36. The role of the cytoplasmic pH (pH_c) of Eremosphaera viridisin the signal transduction chain after light-off from the chloroplaststo the K⁺ channels in the plasmalemma of this unicellular algawas investigated. The temporary opening of K⁺ channels is indicatedby a transient hypcrpolarization (TP). To record rapid changesof pH_c, continuous measurements with pH sensitive micro-electrodeswere carried out. (i) Under normal conditions pH_c in the light(7·56 ±0·2) did not differ from pHc inthe dark (7·62 ±0·2). (ii) The vacuolepH ranged between 4·8 and 5·2. (iii) After light-offa rapid transient acidification of pHc O19±0·07occurred and a TP was released, (iv) In every case, the startof the transient acidification after light-off preceded thehyperpolarization by about 3s. (v) Light-on caused a rapid transientalkalinization but never a TP. (vi) Change to acid externalmedium (3.2) transiently acidified the cytoplasm and was ableto release a TP. (vii) After addition of NH₄Cl, pH_c again showeda rapid transient acidification and the release of a TP. The origin of the protons appearing in the cytoplasm after light-offis discussed critically with respect to the buffer capacity.Either direct or indirect translocation is a possible mechanismfor the movement of H⁺ from the chloroplasts into the cytoplasm.The intracellular acidification and its relation to the openingof potassium channels in the plasmalemma leads us to suggestthat a sudden change of pH_c is a potent internal signal factorin Eremosphaera viridis. Key words: Cytoplasmic pH, transient potential, K⁺–channels, Eremosphaera viridis     

Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods

The Na⁺-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K_0.5) was in the low micromolar range (12–34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I_max) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I_max. Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I_max. Fludarabine and formycin B were also transported with higher I_max than uridine and moderate affinity (102 ± 10 and 66 ± 6 µM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about –39 mV and a valence of about –0.8. K_0.5 for Na⁺ was 2.3 mM at –50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na⁺ per nucleoside. This discrepancy in the number of Na⁺ molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na⁺ binding sites. two-electrode voltage clamp; concentrative nucleoside transport; presteady-state currents