Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

Block of a Ca<Superscript>2+</Superscript>-activated Potassium Channel by Cocaine

The primary target for cocaine is believed to be monoamine transporters because of cocaine’s high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K⁺ channel in hippocampal neurons grown in culture (IC₅₀ = ∼30 μM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca²⁺-activated K⁺ channels are 261 ± 37 μM⁻¹s⁻¹ and 11451 ± 1467 s⁻¹. The equilibrium dissociation constant (K_B) for cocaine, determined from single-channel parameters, is 43 μM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca²⁺-dependent K⁺ channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.     

Effect of Extracellular pH on Presteady-State and Steady-State Current Mediated bythe Na<Superscript>+</Superscript>/K<Superscript>+</Superscript> Pump

A ouabain sensitive inward current occurs in Xenopus oocytes in Na⁺ and K⁺ -free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pH_o) produces a conducting pathway through the Na⁺/K⁺ pump that is permeable to H⁺ and blocked by [Na⁺]_o. An alternative suggestion is that the current is mediated by an electrogenic H⁺-ATPase. Here we investigate the effect of pH_o and [Na⁺]_o on both transient and steady-state ouabain-sensitive current. At alkaline or neutral pH_o the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pH_o, as predicted by a model in which protonation of the Na⁺/K⁺ pump reduces the energy barrier between the internal solution and the Na⁺ occluded state. The model also predicts that acidic pH_o increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is 6, suggesting that histidine is involved in induction of the conductance pathway. ²²Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pH_o suggest that both Na⁺ and H⁺ are permeant. The acid-induced inward current is reduced by high [Na⁺]_o, consistent with block by Na⁺. A least squares analysis predicts that H⁺ is four orders of magnitude more permeant than Na⁺, and that block occurs when 3 Na⁺ ions occupy a low affinity binding site (K _0.5=130±30 mM) with a dielectric coefficient of 0.23±0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na⁺ and H⁺ through the Na⁺/K⁺ pump.     

Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

Purinergic signalling in rat GFSHR-17 granulosa cells: an <Emphasis Type="Italic">in vitro</Emphasis> model of granulosa cells in maturing follicles

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca²⁺-imaging and perforated patch-clamp. We observed a resting intracellular Ca²⁺-concentration ([Ca²⁺]_i) of 100 nM and a membrane potential of −40 mV. This was consistent with high K⁺− and Cl⁻ permeability and a high intracellular Cl⁻ concentration of 40 mM. Application of ATP for 5–15 s every 3 min induced repeated [Ca²⁺]_i increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP₃-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y₂ and P2Y₄ receptors and that the [Ca²⁺]_i increase was a prerequisite for hyperpolarization. Inhibitors of Ca²⁺-activated channels or K⁺ channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl⁻ channels hyperpolarized cells to −70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y₂ and P2Y₄ receptors in granulosa cells modulate Cl⁻ permeability by regulating Ca²⁺-release.     

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing <Emphasis Type="Italic">Xenopus</Emphasis> oocytes to Solutions Containing NH<Subscript>3</Subscript>/NH<Subscript>4</Subscript><Superscript>+</Superscript>

Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pH_i), whereas low levels (e.g., 0.5 mM) cause little pH_i change. Here we monitored pH_i and extracellular surface pH (pH_S) while exposing oocytes to 5 or 0.5 mM NH₃/NH₄ ⁺. We confirm that 5 mM causes a paradoxical pH_i fall (−ΔpH_i ≅ 0.2), but also observe an abrupt pH_S fall (−ΔpH_S ≅ 0.2)—indicative of NH₃ influx—followed by a slow decay. Reducing [NH₃/NH₄ ⁺] to 0.5 mM minimizes pH_i changes but maintains pH_S changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpH_S at both levels. During removal of 0.5 or 5 mM NH₃/NH₄ ⁺, failure of pH_S to markedly overshoot bulk extracellular pH implies little NH₃ efflux and, thus, little free cytosolic NH₃/NH₄ ⁺. A new analysis of the effects of NH₃ vs. NH₄ ⁺ fluxes on pH_S and pH_i indicates that (a) NH₃ rather than NH₄ ⁺ fluxes dominate pH_i and pH_S changes and (b) oocytes dispose of most incoming NH₃. NMR studies of oocytes exposed to ¹⁵N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH_i and pH_S demonstrate that NH₃ flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

Differential Contribution of L-, N-, and P/Q-type Calcium Channels to [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> Changes Evoked by Kainate in Hippocampal Neurons

We investigated the contribution of L-, N- and P/Q-type Ca²⁺ channels to the [Ca²⁺]_i changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca²⁺ imaging. Selective Ca²⁺ channel blockers, namely nitrendipine, ω-Conotoxin GVIA (ω-GVIA) and ω-Agatoxin IVA (ω-AgaIVA) were used. The [Ca²⁺]_i changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but the N-methyl-d-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca²⁺ channel blocker caused differential inhibitory effects on [Ca²⁺]_i responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above 90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by ω-GVIA or ω-AgaIVA. Thus, in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case of ω-GVIA or ω-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what concerns their L-, N- and P/Q- type Ca²⁺ channels activated by stimulation of the AMPA/kainate receptors. Special issue article in honor of Dr. Ricardo Tapia.     

Klotho attenuates isoproterenol-induced hypertrophic response in H9C2 cells by activating Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase and inhibiting the reverse mode of Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript>-exchanger

Cardiac hypertrophy plays a major role in heart failure and is related to patient morbidity and mortality. Calcium overloading is a main risk for cardiac hypertrophy, and Na⁺/K⁺-ATPase (NKA) has been found that it could not only regulate intracellular Na⁺ levels but also control the intracellular Ca²⁺ ([Ca²⁺]_i) level through Na⁺/Ca²⁺-exchanger (NCX). Recent studies have reported that klotho could affect [Ca²⁺]_i level. In this study, we aimed at exploring the role of klotho in improving isoproterenol-induced hypertrophic response of H9C2 cells. The H9C2 cells were randomly divided into control and isoproterenol (ISO) (10 μM) groups. Klotho protein (10 μg/ml) or NKAα2 siRNA was used to determine the changes in isoproterenol-induced hypertrophic response. The alterations of [Ca²⁺]_i level were measured by spectrofluorometry. Our results showed that H9C2 cells which were treated with isoproterenol presented a higher level of [Ca²⁺]_i and hypertrophic gene expression at 24 and 48 h compared with the control group. Moreover, the expressions of NKAα1 and NKAα2 were both increased in control and ISO groups after treating with klotho protein; meanwhile, the NKA activity was increased and NCX activity was decreased after treatment. Consistently, the [Ca²⁺]_i level and hypertrophic gene expression were decreased in ISO group after klotho protein treatment. However, these effects were both prevented by transfecting with NKAα2 siRNA. In conclusion, these findings demonstrated that klotho inhibits isoproterenol-induced hypertrophic response in H9C2 cells by activating NKA and inhibiting the reverse mode of NCX and this effect may be associated with the upregulation of NKAα2 expression.     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Electrogenic H<Superscript>+</Superscript> Transport and pH Gradients Generated by a V-H<Superscript>+</Superscript>-ATPase in the Isolated Perfused Larval <Emphasis Type="Italic">Drosophila</Emphasis> Midgut

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (V_t) of 38 to 45 mV and high resistance (R_t) of 800 to 1400 Ω • cm² measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10⁻⁴ M, ouabain 10⁻⁴ M, bumetanide 10⁻⁴ M), K⁺-free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect V_t or R_t. Bafilomycin-A₁ (3 to 5 μM) decreased V_t by ≈ 40% and short-circuit current (I_sc) by ≈ 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H⁺-ATPase located in the basal membrane. Gradients of H⁺ were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO₃⁻), driven by the basal V-H⁺-ATPase.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

Ca<Superscript>2+</Superscript> binding protein-1 inhibits Ca<Superscript>2+</Superscript> currents and exocytosis in bovine chromaffin cells

Summary Calcium binding protein-1 (CaBP1) is a calmodulin like protein shown to modulate Ca²⁺ channel activities. Here, we explored the functions of long and short spliced CaBP1 variants (L- and S-CaBP1) in modulating stimulus-secretion coupling in primary cultured bovine chromaffin cells. L- and S-CaBP1 were cloned from rat brain and fused with yellow fluorescent protein at the C-terminal. When expressed in chromaffin cells, wild-type L- and S-CaBP1s could be found in the cytosol, plasma membrane and a perinuclear region; in contrast, the myristoylation-deficient mutants were not found in the membrane. More than 20 and 70% of Na⁺ and Ca²⁺ currents, respectively, were inhibited by wild-type isoforms but not myristoylation-deficient mutants. The [Ca²⁺] _i response evoked by high K⁺ buffer and the exocytosis elicited by membrane depolarizations were inhibited only by wild-type isoforms. Neuronal Ca²⁺ sensor-1 and CaBP5, both are calmodulin-like proteins, did not affect Na⁺, Ca²⁺ currents, and exocytosis. When expressed in cultured cortical neurons, the [Ca²⁺] _i responses elicited by high-K⁺ depolarization were inhibited by CaBP1 isoforms. In HEK293T cells cotransfected with N-type Ca²⁺ channel and L-CaBP1, the current was reduced and activation curve was shifted positively. These results demonstrate the importance of CaBP1s in modulating the stimulus-secretion coupling in excitable cells. M.-L. Chen and Y.-C. Chen contributed equally to this study     

Phosphorylation Regulates an Inwardly Rectifying ATP-sensitive K<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript>- Conductance in Proximal Tubule Cells of Frog Kidney

K⁺ channels in the renal proximal tubule play an important role in salt reabsorption. Cells of the frog proximal tubule demonstrate an inwardly rectifying, ATP-sensitive K⁺ conductance that is inhibited by Ba²⁺, G_Ba. In this paper we have investigated the importance of phosphorylation state on the activity of G_Ba in whole-cell patches. In the absence of ATP, G_Ba decreased over time; this fall in G_Ba involved phosphorylation, as rundown was inhibited by alkaline phosphatase and was accelerated by the phosphatase inhibitor F⁻(10 mM). Activation of PKC using the phorbol ester PMA accelerated rundown via a mechanism that was dependent on phosphorylation. In contrast, the inactive phorbol ester PDC slowed rundown. Inclusion of the PKC inhibitor PKC-ps in the pipette inhibited rundown. These data indicate that PKC-mediated phosphorylation promotes channel rundown. Rundown was prevented by the inclusion of PIP-2 in the pipette. PIP-2 also abrogated the PMA-mediated increase in rundown, suggesting that regulation of G_Ba by PIP-2 occurred downstream of PKC-mediated phosphorylation. G-protein activation inhibited G_Ba, with initial currents markedly reduced in the presence of GTPγs. These properties are consistent with G_Ba being a member of the ATP-sensitive K⁺ channel family.     

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.     

Tachyphylaxis to the inhibitory effect of L-type channel blockers on ACh-induced [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> oscillations in porcine tracheal myocytes

Summary Discrepancies about the role of L-type voltage-gated calcium channels (VGCC) in acetylcholine (ACh)-induced [Ca²⁺]_i oscillations in tracheal smooth muscle cells (TSMCs) have been seen in recent reports. We demonstrate here that ACh-induced [Ca²⁺]_i oscillations in TMCS were reversibly inhibited by three VGCC blockers, nicardipine, nifedipine and verapamil. Prolonged (several minutes) application of VGCC blockers, led to tachyphylaxis; that is, [Ca²⁺]_i oscillations resumed, but at a lower frequency. Brief (15–30 s) removal of VGCC blockers re-sensitized [Ca²⁺]_i oscillations to inhibition by the agents. Calcium oscillations tolerant to VGCC blockers were abolished by KB-R7943, an inhibitor of the reverse mode of Na⁺/Ca²⁺ exchanger (NCX). KB-R7943 alone also abolished ACh-induced [Ca²⁺]_i oscillations. Enhancement of the reverse mode of NCX via removing extracellular Na⁺ reversed inhibition of ACh-induced [Ca²⁺]_i oscillations by VGCC blockers. Inhibition of non-selective cation channels using Gd³⁺ slightly reduced the frequency of ACh-induced [Ca²⁺]_i oscillations, but did not prevent the occurrence of tachyphylaxis. Altogether, these results suggest that VGCC and the reverse mode of NCX are two primary Ca²⁺ entry pathways for maintaining ACh-induced [Ca²⁺]_i oscillations in TSMCs. The two pathways complement each other, and may account for tachyphylaxis of ACh-induced [Ca²⁺]_i oscillations to VGCC blockers.     

Vasotocin has the potential to inhibit basolateral Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-pump current across isolated skin of tree frog in vitro<Emphasis Type="Italic">,</Emphasis> via its V<Subscript>2</Subscript>-type receptor/cAMP pathway

Adult frog skin transports Na⁺ from the apical to the basolateral side across the skin. Antidiuretic hormone (ADH) is involved in the regulation of Na⁺ transport in both mammals and amphibians. We investigated the effect of arginine vasotocin (AVT), the ADH of amphibians, on the short-circuit current (SCC) across intact skin and on the basolateral Na⁺/K⁺-pump current across apically nystatin-permeabilized skin of the tree frog, Hyla japonica, in which the V₂-type ADH receptor is expressed in vitro. In intact skin, 1 pM AVT had no effect on the SCC, but 10 nM AVT was sufficient to stimulate the SCC since 10 nM and 1 μM of AVT increased the SCC 3.2- and 3.4-fold, respectively (P > 0.9). However, in permeabilized skin, AVT (1 μM) decreased the Na⁺/K⁺-pump current to 0.79 times vehicle control. Similarly, 500 μM of 8Br-cAMP increased the SCC 3.2-fold, yet 1 mM of 8Br-cAMP decreased the Na⁺/K⁺-pump current to 0.76 times vehicle control. Arachidonic acid (10⁻⁵ M) tended to decrease the Na⁺/K⁺-pump current. To judge from these in vitro experiments, AVT has the potential to inhibit the basolateral Na⁺/K⁺-pump current via the V₂-type receptor/cAMP pathway in the skin of the tree frog.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.