Electrical Measurements on Endomembranes

Bertl, Adam; Blumwald, Eduardo; Coronado, R.; Eisenberg, Robert S.; Findlay, Goeff; Gradmann, Dietrich; Hille, Bertil; Köhler, Kurt; Kolb, Hans-Peter; MacRobbie, Enid; Meissner, Gerhard; Miller, Christopher J.; Neher, Erwin; Palade, Philip; Pantoja, Omar; Sanders, Dale; Schroeder, Julian I.; Slayman, Clifford L.; Spanswick, Roger M.; Walker, Alan; Williams, Alan L.

doi:10.1126/science.1439795

Cited by 193 publications

(124 citation statements)

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“…In accord with a recent proposal (Bertl et al, 1992), transvacuolar membrane potentials in this study are referenced to the extra-cytosolic medium (i.e., the vacuolar lumen). As the two primary pumps at the vacuolar membrane pass current (positive charge) out of the cytosol (Rea and Sanders, 1987), negative potentials correspond to the physiological situation (cytosol negative, vacuolar lumen positive).…”

Section: Reference and Sign Of Voltages And Currentsmentioning

confidence: 49%

Two Voltage-Gated, Calcium Release Channels Coreside in the Vacuolar Membrane of Broad Bean Guard Cells

Allen¹,

Sanders²

1994

The Plant Cell

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Voltage-gated, Ca2+ release channels have been characterized at the vacuolar membrane of broad bean guard cells using patch clamps of excised, inside-out membrane patches. The most prevalent Ca2+ release channel had a conductance of 27 pS over voltages negative of the reversal potential(€,ev) (cytosol referenced to vacuole), with 5, 10, or 20 mM Ca2+ as the charge carrier on the vacuolar side and 50 mM K+ on the cytosolic side. The single-channel current saturated at -2.6 pA. The relative permeability of the channel was in the range of a Pcaa+:Ptp ratio of 6:l. Divalent cations could act as charge carriers on the vacuolar.side with a conductance series of Ba2+ > Mg2+ > Sr2+ > Ca2+ and a selectivity sequence of Ca2+ = Ba2+ = Sr2+ > Mg2+. The channel was gated open by cytosol-negative (physiological) transmembrane voltages, increases in vacuolar Ca2+ concentration, and increases in the vacuolar pH. The channel was potently inhibited by the Ca2+ channel blockers Gd3+ (half-maximal inhibition at 10.3 pM) and nifedipine (half-maximal inhibition at 77 pM). The stilbene derivative 4,4'-diisothiocyano9,2'-stilbene disulfonate was also inhibitory (half-maximal inhibition for a 4-min incubation period at 6.3 pM). The 27-pS channel coresides in individual guard cell vacuoles with a less frequently observed 14-pS Ca2+ release channel that had similar, although not identical, voltage dependence and gating characteristics and a lower selectivity for Ca2+ over K+. The requirement for two channels with a similar function at the vacuolar membrane of guard cells is discussed.

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Section: Reference and Sign Of Voltages And Currentsmentioning

confidence: 49%

Two Voltage-Gated, Calcium Release Channels Coreside in the Vacuolar Membrane of Broad Bean Guard Cells

Allen¹,

Sanders²

1994

The Plant Cell

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“…Data were digitized by an ITC-16 interface (Instrutech), stored on a computer, and analyzed using the software programs Pulse and PulseFit (HEKA Elektronik) and IGORPro (Wave Metrics). Recordings were performed in agreement with the convention for electrical measurements on endomembranes (39). The whole-vacuolar currents were measured at the clamped voltage of 0 mV.…”

Section: Methodsmentioning

confidence: 99%

Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation

Krebs

Beyhl

Görlich

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

286

335

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The productivity of higher plants as a major source of food and energy is linked to their ability to buffer changes in the concentrations of essential and toxic ions. Transport across the tonoplast is energized by two proton pumps, the vacuolar H + -ATPase (VATPase) and the vacuolar H + -pyrophosphatase (V-PPase); however, their functional relation and relative contributions to ion storage and detoxification are unclear. We have identified an Arabidopsis mutant in which energization of vacuolar transport solely relies on the activity of the V-PPase. The vha-a2 vha-a3 double mutant, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but shows day-length-dependent growth retardation. Nitrate content is reduced whereas nitrate assimilation is increased in the vha-a2 vha-a3 mutant, indicating that vacuolar nitrate storage represents a major growth-limiting factor. Zinc is an essential micronutrient that is toxic at excess concentrations and is detoxified via a vacuolar Zn 2+ /H + -antiport system. Accordingly, the double mutant shows reduced zinc tolerance. In the same way the vacuolar Na + /H + -antiport system is assumed to be an important component of the system that removes sodium from the cytosol. Unexpectedly, salt tolerance and accumulation are not affected in the vhaa2 vha-a3 double mutant. In contrast, reduction of V-ATPase activity in the trans-Golgi network/early endosome (TGN/EE) leads to increased salt sensitivity. Taken together, our results show that during gametophyte and embryo development V-PPase activity at the tonoplast is sufficient whereas tonoplast V-ATPase activity is limiting for nutrient storage but not for sodium tolerance during vegetative and reproductive growth.proton-pump | pH-homeostasis | vacuole | nitrate | salt tolerance T he productivity of higher plants as a major source of food and renewable energy is linked to their ability to cope with fluctuations in essential as well as toxic ion and metabolite concentrations. In plants the large central vacuoles function as reservoirs for ions and metabolites that allow buffering of changes in nutrients as well as challenges by toxic components that plants, as sessile, photoautotrophic organisms, frequently encounter. Furthermore, vacuoles are essential for plant growth and development. Expansion of plant cells is achieved by osmotically driven water influx into the vacuole that, in combination with the cell wall, generates turgor, the driving force for hydraulic stiffness and plant growth.All vacuolar functions require massive fluxes of ions and metabolites that are channeled by a battery of vacuolar transport proteins, many of which have been well characterized physiologically, but the nature of several transporters remains to be identified (1). Among those characterized on the molecular level are, e.g., those transporting NO 3 − , Na + , and protons. Nitrate, a major plant nutrient, is accumulated and stored in the vacuole from where it can be retrieved according to metabolic deman...

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“…Sign conventions throughout this report define the vacuolar interior as Ref. 26, so that positive membrane voltages mean that the cytoplasmic electric potential is positive to the vacuolar potential, and a positive current represents positive charges moving from the cytoplasm to the inside of a vacuole. Experiments were performed at room temperatures (20 -23°C).…”

Section: Methodsmentioning

confidence: 99%

Patch Clamp Studies on V-type ATPase of Vacuolar Membrane of Haploid Saccharomyces cerevisiae

Yabe¹,

Horiuchi²,

Nakahara³

et al. 1999

Journal of Biological Chemistry

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Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(⌬vma1::TRP)). The K m value for the ATP dose response of the current was 159 M and the H ؉ /ATP ratio estimated from the reversible potential of the V-I curve was 3.5 ؎ 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter in S. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems.

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Electrical Measurements on Endomembranes

Cited by 193 publications

References 0 publications

Two Voltage-Gated, Calcium Release Channels Coreside in the Vacuolar Membrane of Broad Bean Guard Cells

Two Voltage-Gated, Calcium Release Channels Coreside in the Vacuolar Membrane of Broad Bean Guard Cells

Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation

Patch Clamp Studies on V-type ATPase of Vacuolar Membrane of Haploid Saccharomyces cerevisiae

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