Ion Permeabilities in Mouse Sperm Reveal an External Trigger for SLO3-Dependent Hyperpolarization

Unlike most cells of the body which function in an ionic environment controlled within narrow limits, spermatozoa must function in a less controlled external environment. In order to better understand how sperm control their membrane potential in different ionic conditions, we measured mouse sperm membrane potentials under a variety of conditions and at different external K⁺ concentrations, both before and after capacitation. Experiments were undertaken using both wild-type, and mutant mouse sperm from the knock-out strain of the sperm-specific, pH-sensitive, SLO3 K⁺ channel. Membrane voltage data were fit to the Goldman-Hodgkin-Katz equation. Our study revealed a significant membrane permeability to both K⁺ and Cl⁻ before capacitation, as well as Na⁺. The permeability to both K⁺ and Cl⁻ has the effect of preventing large changes in membrane potential when the extracellular concentration of either ion is changed. Such a mechanism may protect against undesired shifts in membrane potential in changing ionic environments. We found that a significant portion of resting membrane potassium permeability in wild-type sperm was contributed by SLO3 K⁺ channels. We also found that further activation of SLO3 channels was the essential mechanism producing membrane hyperpolarization under two separate conditions, 1) elevation of external pH prior to capacitation and 2) capacitating conditions. Both conditions produced a significant membrane hyperpolarization in wild-type which was absent in SLO3 mutant sperm. Hyperpolarization in both conditions may result from activation of SLO3 channels by raising intracellular pH; however, demonstrating that SLO3-dependent hyperpolarization is achieved by an alkaline environment alone shows that SLO3 channel activation might occur independently of other events associated with capacitation. For example sperm may undergo stages of membrane hyperpolarization when reaching alkaline regions of the female genital tract. Significantly, other events associated with sperm capacitation, occur in SLO3 mutant sperm and thus proceed independently of hyperpolarization.     

Interaction of the B subunit of cholera toxin with endogenous ganglioside GM1 causes changes in membrane potential of rat thymocytes

Summary The fluorescent anionic dye, bisoxonol, and flow cytometry have been used to monitor changes in the membrane potential of rat thymocytes exposed to the B subunit of cholera toxin. The B subunit induced a rapid hyperpolarization, which was due to activation of a Ca²⁺-sensitive K⁺ channel. Reduction of extracellular Ca²⁺ to <1 m by the addition of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid immediately abolished the hyperpolarization caused by the B subunit. Cells treated with quinine and tetraethylammonium lost their ability to respond to the B subunit, whereas 4-aminopyridine did not have any effect. Thus, calcium-sensitive and not voltage-gated K⁺ channels appeared to be responsible for the hyperpolarization. The results of ion substitution experiments indicated that extracellular Na⁺ was not essential for changes in membrane potential. Further studies with ouabain, amiloride and furosemide demonstrated that electrogenic Na⁺/K⁺ ATPase, Na⁺/H⁺ antiporter and Na⁺/K⁺/Cl^– cotransporter, respectively, were not involved in the hyperpolarization process induced by the B subunit. Thus, crosslinking of several molecules of ganglioside GM1 on the cell surface of rat thymocytes by the pentavalent B subunit of cholera toxin modulated plasma membrane permeability to K⁺ by triggering the opening of Ca²⁺-sensitive K⁺ channels. A role for gangliosides in regulating ion permeability would have important implications for the function of gangliosides in various cellular phenomena.     

SLO3 K+ Channels Control Calcium Entry through CATSPER Channels in Sperm

Here we show how a sperm-specific potassium channel (SLO3) controls Ca²⁺ entry into sperm through a sperm-specific Ca²⁺ channel, CATSPER, in a totally unanticipated manner. The genetic deletion of either of those channels confers male infertility in mice. During sperm capacitation SLO3 hyperpolarizes the sperm, whereas CATSPER allows Ca²⁺ entry. These two channels may be functionally connected, but it had not been demonstrated that SLO3-dependent hyperpolarization is required for Ca²⁺ entry through CATSPER channels, nor has a functional mechanism linking the two channels been shown. In this study we show that Ca²⁺ entry through CATSPER channels is deficient in Slo3 mutant sperm lacking hyperpolarization; we also present evidence supporting the hypothesis that SLO3 channels activate CATSPER channels indirectly by promoting a rise in intracellular pH through a voltage-dependent mechanism. This mechanism may work through a Na⁺/H⁺ exchanger (sNHE) and/or a bicarbonate transporter, which utilizes the inward driving force of the Na⁺ gradient, rendering it intrinsically voltage-dependent. In addition, the sperm-specific Na⁺/H⁺ exchanger (sNHE) possess a putative voltage sensor that might be activated by membrane hyperpolarization, thus increasing the voltage sensitivity of internal alkalization.     

Influences of Lovastatin on membrane ion flow and intracellular signaling in breast cancer cells

Lovastatin, an inhibitor of cellular cholesterol synthesis, has an apparent anti-cancer property, but the detailed mechanisms of its anti-cancer effects remain poorly understood. We investigated the molecular mechanism of Lovastatin anti-tumor function through the study of its effect on membrane ion flow, gap junctional intercellular communication (GJIC), and the pathways of related signals in MCF-7 mammary cancer cells. After treatment for 24–72 h with 4, 8 or 16 μmol/L Lovastatin, cellular proliferation was examined via the MTT assay, and changes in membrane potential and cellular [Ca²⁺]_i were monitored using confocal laser microscopy. In addition, the expression of plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA was analyzed via RT-PCR, the GJIC function was examined using the scrape-loading dye transfer (SLDT) technique, and MAPK phosphorylation levels were tested with the kinase activity assay. The results showed that Lovastatin treatment significantly inhibited the growth of MCF-7 breast cancer cells. It also increased the negative value of the membrane potential, leading to the hyperpolarization of cells. Moreover, Lovastatin treatment continuously enhanced [Ca²⁺]_i, although the levels of PMCA1 mRNA were unchanged. GJIC was also upregulated in MCF-7 cells, with transfer of LY Fluorescence reaching 4 to 5 rows of cells from the scraped line after treatment with 16 μmol/L Lovastatin for 72 h. Finally, downregulation of ERK1 and p38^MAPK phosphorylation were found in Lovastatin-treated MCF-7 cells. It could be deduced that Lovastatin can induce changes in cellular hyperpolarization and intracellular Ca²⁺ distributions, and increase GJIC function. These effects may result in changes in the downstream signal cascade, inhibiting the growth of MCF-7 cells.     

Chemoreception inParamecium tetraurelia: acetate and folate-induced membrane hyperpolarization

Summary Acetic and folic acids hyperpolarize the membrane potential ofParamecium tetraurelia in a concentration-dependent manner. The membrane responses are accompanied by small changes in cell resistance, and are significantly reduced by increasing extracellular cation concentrations, suggesting that the attractants bring about the membrane potential change by increasing cell permeability to cations. The inability to show a reversal potential for the hyperpolarization to attractants suggests that the effects of cations on the response are non-specific, however. The possible roles of Ca⁺⁺, K⁺, and Na⁺ in the attractant-induced responses were further investigated by applying acetate and folate to cells with genetic defects in specific ion conductances, by collapsing the driving forces for these ions, and by testing the effects of ion channel blockers on the responses. These studies suggest that the membrane responses to attractants are not due to the direct effects of increased or decreased membrane permeability to cations.Attempts to block the acetate and folate-induced hyperpolarization by collapsing surface potential or using a mutant with reduced surface charge were inconclusive, as were studies on the possible role of attractant transport in the membrane responses.We hypothesize that the membrane hyperpolarization may be due to either the indirect effects of increased calcium permeability, to extrusion of calcium through activation of a calcium pump, or to a proton efflux.     

Photogeneration of membrane potential hyperpolarization and depolarization in non-excitable cells

We monitored femtosecond laser induced membrane potential changes in non-excitable cells using patchclamp analysis. Membrane potential hyperpolarization of HeLa cells was evoked by 780 nm, 80 fs laser pulses focused in the cellular cytoplasm at average powers of 30–60 mW. Simultaneous detection of intracellular Ca²⁺ concentration and membrane potential revealed coincident photogeneration of Ca²⁺ waves and membrane potential hyperpolarization. By using non-excitable cells, the cell dynamics are slow enough that we can calculate the membrane potential using the steady-state approximation for ion gradients and permeabilities, as formulated in the GHK equations. The calculations predict hyperpolarization that matches the experimental measurements and indicates that the cellular response to laser irradiation is biological, and occurs via laser triggered Ca²⁺ which acts on Ca²⁺ activated K⁺ channels, causing hyperpolarization. Furthermore, by irradiating the cellular plasma membrane, we observed membrane potential depolarization in combination with a drop in membrane resistance that was consistent with a transient laser-induced membrane perforation. These results entail the first quantitative analysis of location-dependent laser-induced membrane potential modification and will help to clarify cellular biological responses under exposure to high intensity ultrashort laser pulses.     

KCa3.1-Dependent Hyperpolarization Enhances Intracellular Ca2+ Signaling Induced by fMLF in Differentiated U937 Cells

Formylated peptides are chemotactic agents generated by pathogens. The most relevant peptide is fMLF (formyl-Met-Leu-Phe) which participates in several immune functions, such as chemotaxis, phagocytosis, cytokine release and generation of reactive oxygen species. In macrophages fMLF-dependent responses are dependent on both, an increase in intracellular calcium concentration and on a hyperpolarization of the membrane potential. However, the molecular entity underlying this hyperpolarization remains unknown and it is not clear whether changes in membrane potential are linked to the increase in intracellular Ca²⁺. In this study, differentiated U937 cells, as a macrophage-like cell model, was used to characterize the fMLF response using electrophysiological and Ca²⁺ imaging techniques. We demonstrate by means of pharmacological and molecular biology tools that fMLF induces a Ca²⁺-dependent hyperpolarization via activation of the K⁺ channel K_Ca3.1 and thus, enhancing fMLF-induced intracellular Ca²⁺ increase through an amplification of the driving force for Ca²⁺ entry. Consequently, enhanced Ca²⁺ influx would in turn lengthen the hyperpolarization, operating as a positive feedback mechanism for fMLF-induced Ca²⁺ signaling.     

Src Kinase Is the Connecting Player between Protein Kinase A (PKA) Activation and Hyperpolarization through SLO3 Potassium Channel Regulation in Mouse Sperm

Plasma membrane hyperpolarization is crucial for mammalian sperm to acquire acrosomal responsiveness during capacitation. Among the signaling events leading to mammalian sperm capacitation, the immediate activation of protein kinase A plays a pivotal role, promoting the subsequent stimulation of protein tyrosine phosphorylation that associates with fertilizing capacity. We have shown previously that mice deficient in the tyrosine kinase cSrc are infertile and exhibit improper cauda epididymis development. It is therefore not clear whether lack of sperm functionality is due to problems in epididymal maturation or to the absence of cSrc in sperm. To further address this problem, we investigated the kinetics of cSrc activation using anti-Tyr(P)-416-cSrc antibodies that only recognize active cSrc. Our results provide evidence that cSrc is activated downstream of PKA and that inhibition of its activity blocks the capacitation-induced hyperpolarization of the sperm plasma membrane without blocking the increase in tyrosine phosphorylation that accompanies capacitation. In addition, we show that cSrc inhibition also blocks the agonist-induced acrosome reaction and that this inhibition is overcome by pharmacological hyperpolarization. Considering that capacitation-induced hyperpolarization is mediated by SLO3, we evaluated the action of cSrc inhibitors on the heterologously expressed SLO3 channel. Our results indicate that, similar to SLO1 K⁺ channels, cSrc blockers significantly decreased SLO3-mediated currents. Together, these results are consistent with findings showing that hyperpolarization of the sperm plasma membrane is necessary and sufficient to prepare the sperm for the acrosome reaction and suggest that changes in sperm membrane potential are mediated by cSrc activation.     

Membrane potential changes associated with pinocytosis of serum lipoproteins in L cells

L cells exhibit spontaneous oscillations of membrane potential in accord with fluctuations of the cytoplasmic Ca²⁺ concentration. Upon addition of low-density lipoprotein (LDL), L cells show a prolonged hyperpolarization which is followed by an increase in the frequency of membrane potential oscillations. These membrane potential changes induced by LDL were inhibited by Ca²⁺-channel blockers. LDL-induced membrane potential changes were accompanied by a vigorous pinocytosis which was coupled with the formation of ring-like ridge structures on the cell surface. These electrical and morphological changes were also induced by high-density lipoprotein (HDL) but not by very-low-density lipoprotein (VLDL). These results suggest that the application of LDL or HDL to the membrane surface elicits a rapid influx of Ca²⁺ into the cytosol, resulting in membrane hyperpolarization. A rise in cytoplasmic Ca²⁺ may be implicated in the primary factor for the pinocytic process.     

Effect of extracellular Ca2+, K+ and OH− on erythrocyte membrane potential as monitored by the fluorescent probe 3,3′-dipropylthiodicarbocyanine

Changes in fluorescence intensity of thiodicarbocyanine, DiS-C₃(5), were correlated with direct microelectrode potential measurements in red blood cells from Amphiuma means and applied qualitatively to evaluate the effects of extracellular Ca²⁺, K⁺ and pH on the membrane potential of human red cells. Increasing extracellular [Ca²⁺] from 1.8 to 15 mM causes a K⁺-dependent hyperpolarization and decrease in fluorescence intensity in Amphiuma red cells. Both the hyperpolarization and fluorescence change disappear when the temperature is raised from 17 to 37°C. No change in fluorescence intensity is observed in human red cells with comparable increase in extracellular Ca²⁺ in the temperature range 5–37°C. Increasing the extracellular pH, however, causes human red cells to respond to an increase in extracellular Ca²⁺ with a significant but temporary loss in fluorescence intensity. This effect is blocked by EGTA, quinine or by increasing extracellular [K⁺], indicating that at elevated extracellular pH, human erythrocytes respond to an increase in extracellular Ca²⁺ with an opening of K⁺ channels and associated hyperpolarization of the plasma membrane.     

Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

Analyzing and Modeling the Kinetics of Amyloid Beta Pores Associated with Alzheimer’s Disease Pathology

Amyloid beta (Aβ) oligomers associated with Alzheimer’s disease (AD) form Ca²⁺-permeable plasma membrane pores, leading to a disruption of the otherwise well-controlled intracellular calcium (Ca²⁺) homeostasis. The resultant up-regulation of intracellular Ca²⁺ concentration has detrimental implications for memory formation and cell survival. The gating kinetics and Ca²⁺ permeability of Aβ pores are not well understood. We have used computational modeling in conjunction with the ability of optical patch-clamping for massively parallel imaging of Ca²⁺ flux through thousands of pores in the cell membrane of Xenopus oocytes to elucidate the kinetic properties of Aβ pores. The fluorescence time-series data from individual pores were idealized and used to develop data-driven Markov chain models for the kinetics of the Aβ pore at different stages of its evolution. Our study provides the first demonstration of developing Markov chain models for ion channel gating that are driven by optical-patch clamp data with the advantage of experiments being performed under close to physiological conditions. Towards the end, we demonstrate the up-regulation of gating of various Ca²⁺ release channels due to Aβ pores and show that the extent and spatial range of such up-regulation increases as Aβ pores with low open probability and Ca²⁺ permeability transition into those with high open probability and Ca²⁺ permeability.     

Iberiotoxin and clofilium regulate hyperactivation,acrosome reaction,and ion homeostasis synergistically during human sperm capacitation

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K⁺ currents in human sperm (I_KSper) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca²⁺, K⁺, Cl⁻, and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K⁺]_i, [Cl⁻]_i, and pH_i, but a decrease in [Ca²⁺]_i. Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K⁺]_i, [Cl⁻]_i, and pH_i, and the decrease in [Ca²⁺]_i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.     

Possible involvement of the membrane potential in the gravitactic orientation of Euglena gracilis

Euglena gracilis, a unicellular photosynthetic flagellate, uses light and gravity as environmental hints to reach and stay in regions optimal for growth and reproduction. The current model of gravitaxis (the orientation with respect to the earth's gravitational field) is based on the specific density difference between cell body and medium. The resulting sedimentation of the cell body applies a force to the lower membrane. This force activates mechano-sensitive ion channels. The resulting ion flux changes the membrane potential, which in turn triggers reorientational movements of the trailing flagellum. One possibility for recording the predicted membrane potential changes during reorientation is the use of potential-sensitive dyes, such as Oxonol VI. The absorption changes of the dye indicating potential changes were recorded with a custom-made photometer, which allows a high precision measurement with a high temporal resolution. After a gravitactic stimulation, a short period of hyperpolarization was detected, followed by a massive depolarization of the cell. The membrane potential returned to initial values after a period of approximately 200 s. Parallel measurements of the precision of orientation and the membrane potential showed a close relationship between both phenomena. The obtained results support the current model of gravitaxis of Euglena gracilis     

Ion transport through dimethyl sulfoxide (DMSO) induced transient water pores in cell membranes

Abstract

It is well known that dimethyl sulphoxide (DMSO) increases membrane permeability, which makes it widely used as a vehicle to facilitate drug delivery across biological membranes. However, the mechanism of how DMSO increases membrane permeability has not been well understood. Recently, molecular dynamics simulations have demonstrated that DMSO can induce water pores in biological membranes, but no direct experimental evidence is so far available to prove the simulation result. Using FluxOR Tl⁺ influx assay and intracellular Ca²⁺ imaging technique, we studied the effect of DMSO on Tl⁺ and Ca²⁺ permeation across cell membranes. Upon application of DMSO on CHO-K1 cell line, Tl⁺ influx was transiently increased in a dose-dependent manner. The increase in Tl⁺ permeability induced by DMSO was not changed in the presence of blockers for K⁺ channel and Na⁺-K⁺ ATPase, suggesting that Tl⁺ permeates through transient water pores induced by DMSO to enter into the cell. In addition, Ca²⁺ permeability was significantly increased upon application of DMSO, indicating that the transient water pores induced by DMSO were non-selective pores. Furthermore, similar results could be obtained from RAW264.7 macrophage cell line. Therefore, this study provided experimental evidence to support the prediction that DMSO can induce transient water pores in cell membranes, which in turn facilitates the transport of active substances across membranes.     

Effect of entomocidal proteins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> on ion permeability of apical membranes of <Emphasis Type="Italic">Tenebrio molitor</Emphasis> larvae gut epithelium

Effects of entomocidal Cry-type proteins, δ-endotoxins Cry3A and Cry11A produced by Bacillus thuringiensis, on ion permeability of the apical membranes of intestinal epithelium from Tenebrio molitor larvae midgut were studied. Using potential-sensitive dyes safranine O and oxonol VI and δpH indicator acridine orange, it was shown that placing brush border membrane vesicles (BBMV) (loaded with Mg²⁺ during their preparation) into a salt-free buffer medium resulted in spontaneous generation of transmembrane electric potential on the vesicular membrane (negative inside the vesicles) accompanied by acidification of the aqueous phase inside the vesicles. The generation of transmembrane ion gradients on the vesicular membrane was a result of an electrogenic efflux of Mg²⁺ from the vesicles as shown by abolishing of the membrane potential by such agents as MgSO₄ or CaCl₂ in centimolar concentrations, a highly lipophilic cation tetraphenylphosphonium, and some blockers of cell membrane Ca²⁺-channels in submillimolar concentrations. A passive generation of membrane potential on the vesicular membrane (but positive inside the vesicles) was also observed upon addition of centimolar concentrations of K₂SO₄. Addition of δ-endotoxins Cry3A and Cry11A to the vesicle suspension in a salt-free buffer medium or in the same medium supplemented with centimolar concentrations of K₂SO₄ exerted a pronounced hyperpolarization of the vesicular membrane. This hyperpolarization was sensitive to the same agents, which abolished the membrane potential generation in the absence of δ-endotoxin. It is concluded that Cry proteins induced in BBMV from T. molitor opening pores or ion channels, which were considerably more permeable for alkaline- and alkaline-earth metal cations than for the accompanying anions.     

Ca2+ influx and clearance at hyperpolarized membrane potentials modulate spontaneous and stimulated exocytosis in neuroendocrine cells

Neuroendocrine adrenal chromaffin cells release neurohormones catecholamines in response to Ca²⁺ entry via voltage-gated Ca²⁺ channels (VGCCs). Adrenal chromaffin cells also express non-voltage-gated channels, which may conduct Ca²⁺ at negative membrane potentials, whose role in regulation of exocytosis is poorly understood. We explored how modulation of Ca²⁺ influx at negative membrane potentials affects basal cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and exocytosis in metabolically intact voltage-clamped bovine adrenal chromaffin cells. We found that in these cells, Ca²⁺ entry at negative membrane potentials is balanced by Ca²⁺ extrusion by the Na⁺/Ca²⁺ exchanger and that this balance can be altered by membrane hyperpolarization or stimulation with an inflammatory hormone bradykinin. Membrane hyperpolarization or application of bradykinin augmented Ca²⁺-carrying current at negative membrane potentials, elevated basal [Ca²⁺]_i, and facilitated synchronous exocytosis evoked by the small amounts of Ca²⁺ injected into the cell via VGCCs (up to 20 pC). Exocytotic responses evoked by the injections of the larger amounts of Ca²⁺ via VGCCs (> 20 pC) were suppressed by preceding hyperpolarization. In the absence of Ca²⁺ entry via VGCCs and Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger, membrane hyperpolarization induced a significant elevation in [Ca²⁺]_i and asynchronous exocytosis. Our results indicate that physiological interferences, such as membrane hyperpolarization and/or activation of non-voltage-gated Ca²⁺ channels, modulate basal [Ca²⁺]_i and, consequently, segregation of exocytotic vesicles and their readiness to be released spontaneously and in response to Ca²⁺ entry via VGCCs. These mechanisms may play role in homeostatic plasticity of neuronal and endocrine cells.     

Characterization of the plasma-membrane H+-ATPase from Vicia faba guard cells

Stomatal movement is controlled by external and internal signals such as light, phytohormones or cytoplasmic Ca²⁺. Using Vicia faba L., we have studied the dose-dependent effect of auxins on the modulation of stomatal opening, mediated through the activity of the plasma-membrane H⁺-ATPase. The patch-clamp technique was used to elucidate the electrical properties of the H⁺-ATPase as effected by growth regulators and seasonal changes. The solute composition of cytoplasmic and extracellular media was selected to record pump currents directly with high resolution. Proton currents through the ATPase were characterized by a voltage-dependent increase in amplitude, positive to the resting potential, reaching a plateau at more depolarized values. Upon changes in extracellular pH, the resting potential of the cell shifted with a non-Nernst potential response (±21 mV), indicating the contribution of a depolarizing ionic conductance other than protons to the permeability of the plasma membrane. The use of selective inhibitors enabled us to identify the currents superimposing the H⁺-pump as carried by Ca²⁺. Auxinstimulation of this electroenzyme resulted in a rise in the outwardly directed H⁺ current and membrane hyperpolarization, indicating that modulation of the ATPase by the hormone may precede salt accumulation as well as volume and turgor increase. Annual cycles in pump activity (1.5–3.8 μA · cm^-2) were expressed by a minimum in pump current during January and February. Resting potentials of up to -260 mV and plasmamembrane surface area, on the other hand, did not exhibit seasonal changes. The pump activity per unit surface area was approximately 2- to 3-fold higher in guard cells than in mesophyll cells and thus correlates with their physiological demands.     

BK Channels Reveal Novel Phosphate Sensitivity in SNr Neurons

Whether large conductance Ca²⁺-activated potassium (BK) channels are present in the substantia nigra pars reticulata (SNr) is a matter of debate. Using the patch-clamp technique, we examined the functional expression of BK channels in neurons of the SNr and showed that the channels were activated or inhibited by internal high-energy phosphates (IHEPs) at positive and negative membrane potentials, respectively. SNr neurons showed membrane potential hyperpolarization under glucose-deprivation conditions which was attenuated by paxilline, a specific BK channel blocker. In addition, Fluo-3 fluorescence recording detected an increase in the level of internal free calcium ([Ca²⁺]_i) during ischemic hyperpolarization. These results confirm that BK channels are present in SNr neurons and indicate that their unique IHEP sensitivity is requisite in neuronal ischemic responses. Bearing in mind that the K_ATP channel blocker tolbutamide also attenuated the hyperpolarization, we suggest that BK channels may play a protective role in the basal ganglia by modulating the excitability of SNr neurons along with K_ATP channels under ischemic stresses.     

Resting potential of rat cerebellar granule cells during early maturation in vitro

The survival of rat cerebellar granule cells maintained in vitro is enhanced by a KCl-enriched medium. This effect is classically interpreted as resulting from a higher cytosolic calcium concentration. This implies the presence of voltage-dependent Ca²⁺ channels and a membrane potential that can respond to changes in external K⁺. Since previous studies cast a doubt on these two conditions, we reinvestigated the resting membrane potential and Ca²⁺ influxes in rat cerebellar granule neurones during the first week in vitro using a fluorescence imaging approach. Membrane potential was assessed with the fluorescent dye bis-oxonol, and intracellular free calcium with Fura-2. Resting potential was shown to progressively decrease from −40 mV at the first day in vitro to −60 mV at day 7. At all times in culture, as early as day 0, cells were depolarized when external KCl concentration was increased from 5 to 30 mM. This depolarization resulted in an increased cytosolic calcium concentration due to Ca²⁺ influx through L-type and N-type voltage-activated Ca²⁺ channels, functional at day 0. Gross estimations of the permeabilities of Na⁺ and Cl⁻ were obtained at various times in culture by measuring the changes in resting potential brought about by a reduction of their external concentration. A progressive increase of the relative permeability to K⁺ ions seems to underlie the evolution of the resting potential with time. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 11–21, 1997.