Biomedical subjects

F W Bentrup

Publications and source records attributed to F W Bentrup.

15 recordsLinked to original sources

PubMed1996

Potassium and voltage dependence of the inorganic pyrophosphatase of intact vacuoles from Chenopodium rubrum.

The activity and the voltage dependence of the inorganic pyrophosphatase (PPase) was measured on intact vacuoles of Chenopodium rubrum cells using the patch-clamp technique. With K+ at the cytoplasmic side a negative current representing the forward mode of the pump was measured after addition of pyrophosphate (PPi). The pump was reversed and a positive current was detected after addition of orthophosphate (Pi) in the presence of K+ at the vacuolar side when a pH gradient across the tonoplast was applied. The PPase operates as a constant current source, because no voltage dependence was observed (-60 to 60 mV). The K+ dependence of the PPi-induced current was investigated by substitution of cytoplasmic K+ by other cations. The selectivity sequence was: K+ > or = Rb+ > NH4+ = Cs+ > Na+ > Li+ = choline+, and was independent of the membrane voltage and pHcyt. With Cs+ or Li+ in the bath and K+ inside the vacuole the PPi-induced current became voltage-dependent, and positive currents were observed even if the pump was geared to operate in the forward mode. We suggest a "tunneling' effect through a channel-like domain in the PPase molecule which, under defined electrochemical gradient conditions and in the presence of PPi, allows K+ ions to cross the energy barrier usually separating the cytoplasmic from the vacuolar face of the pump.

Cations, Monovalent↗

PubMed1994

Voltage- and Ca(2+)-dependence of the K+ channel in the vacuolar membrane of Chenopodium rubrum L. suspension cells.

Voltage- and Ca(2+)-dependence of the slow-activating SV-K+ channel in the vacuolar membrane of Chenopodium rubrum suspension cells has been analyzed using the patch clamp technique in the vacuole-attached, outside-out and whole-vacuolar configuration. Patch-pipette perfusion was applied to measure Ca2+ dependence of single channels in the attached-configuration. Using the PCLAMP-software (Axon Instruments), an algorithm was developed to extract reliable individual channel data from multi-channel activity records, including open probability, mean open and closed times, as well as time constants for open and closed distributions. The channel conductance of the major open state was about 83 pS (seal resistance > 8 G omega) at 30 mV (transmembrane voltage Vm, vacuole negative), and symmetrical 100 mM KCl. the channel exhibited a strong voltage- and a weak Ca(2+)-activation: increasing Vm from 40 to 100 mV is equivalent to a Ca2+ concentration change from 10(-7) to 10(-4) M. Mean open probabilities at Vm = 30 mV were 0.03 with 1 microM and 0.09 with 100 microM Ca2+. Mean open times were approx. 7 ms, and almost independent of both, voltage and Ca2+. Mean closed times, however, varied in a strongly voltage- and Ca(2+)-dependent manner, e.g., at Vm = 30 mV dropped from 205 to 67 ms, if Ca2+ was raised from 10(-6) to 10(-4) M. Open and closed distributions of events within bursts could be fitted by the sum of two exponentials with time constants between 0.3 and 11 ms.

Calcium↗

PubMed1993

Pharmacology of the SV channel in the vacuolar membrane of Chenopodium rubrum suspension cells.

Single channel performance and deactivation currents have been analyzed in the presence of cation channel blockers to reveal pharmacological properties of the slow-activating (SV) cation-selective ion channel in the vacuolar membrane (tonoplast) isolated from suspension cells of Chenopodium rubrum L. At a holding potential of -100 mV, the SV channel showed half-maximal inhibition with 20 mM tetraethylammonium (TEA), 7 microM 9-amino-acridine, 6 microM (+)-tubocurarine, 300 nM quinacrine, and 35 microM quinine, respectively. The SV channel is also blocked by charybdotoxin (20 nM at -80 mV) but not by apamine. 9-Amino-acridine, (+)-tubocurarine and quinacrine act in a voltage-dependent fashion, binding to the open channel and to different sites along the transmembrane voltage profile according to Woodhull (J. Gen. Physiol. 61:687-708, 1973). No binding site could be specified for charybdotoxin, which binds to the closed channel, and for quinine. Except for quinine, all tested blockers were effective only if added to the cytoplasmic side of the tonoplast. A structural relationship between the SV channel and Maxi-K channels in animal systems is inferred.

Animals↗

PubMed1991

Charybdotoxin blocks cation-channels in the vacuolar membrane of suspension cells of Chenopodium rubrum L.

Using the patch-clamp technique, we studied the action of charybdotoxin which blocks Ca(2+)-activated large-conductance K+ channels in animal tissue on the slow-activating (SV), Ca(2+)-activated cation channel in the vacuolar membrane of suspension-cells of Chenopodium rubrum L. The toxin reversibly reduced the vacuolar current with EC50 approximately 20 nM suggesting structural similarities between ion channels in animal and plant membranes.

Cations↗

PubMed1990

(+)-Tubocurarine is a potent inhibitor of cation channels in the vacuolar membrane of Chenopodium rubrum L.

The effect of the acetylcholine antagonist and channel blocker (+)-tubocurarine on the calcium-dependent slow vacuolar (SV) cation channels in the tonoplast of suspension-cultured cells of Chenopodium rubrum L. was examined using the patch-clamp technique. In whole-vacuolar recordings the drug strongly suppressed the potassium conductance (EC50: 6 microM) and altered the kinetics of channel inactivation. In excised membrane patches (+)-tubocurarine evokes channel-'flickering' without affecting the single-channel conductance (approx. 80 pS).

Cations↗

PubMed1986

ATP-dependent acidification and tonoplast hyperpolarization in isolated vacuoles from green suspension cells of Chenopodium rubrum L.

The tonoplast of isolated vacuoles from photoautotrophic suspension cells of Chenopodium rubrum L. was studied by means of the patch-clamp technique. In a symmetrical K(+) concentration of 46 mM, similar to in vivo conditions, the tonoplast displayed a membrane potential near zero and a linear current-voltage relationship with a mean slope of 1.0 S/m(2). ATP at 2 mM hyperpolarized the tonoplast (vacuole positive) by 15-20 mV and, in a parallel experiment, acidified the vacuole (outside pH 7.0) to pH 5.0, as monitored by accumulation of acridine orange. Analysis of the voltage-clamp current indicates a 2-fold, ATP-dependent increase of the membrane capacitance, from 4 to 8 mF/m(2), and an ATP-independent, unidentified ion channel having a mean opening time of about 5 msec and a conductivity of 0.5-1.0 pS.

Journal Article↗

PubMed1984

Amine Transport in Riccia fluitans: Cytoplasmic and Vacuolar pH Recorded by a pH-Sensitive Microelectrode.

The cytoplasmic and vacuolar pH and changes thereof in the presence of ammonia (NH(4)Cl) and methylamine (CH(3)NH(3)Cl) have been measured in rhizoid cells of Riccia fluitans by means of a pH-sensitive microelectrode.On addition of 1 micromolar NH(4)Cl, the cytoplasmic pH of 7.2 to 7.4 drops by 0.1 to 0.2 pH units, but shifts to pH 7.8 in the presence of 50 micromolar NH(4)Cl or 500 micromolar CH(3)NH(3)Cl. The pH of the vacuole increases drastically from 4.5 to 5.7 with these latter concentrations. Since a NH(4) (+)/CH(3)NH(3) (+) uniporter has been demonstrated in the plasmalemma of R. fluitans previously (Felle 1983 Biochim Biophys Acta 602:181-195), the concentration-dependent shifts of cytoplasmic pH are interpreted as results of two processes: first, acidification through deprotonation of the actively transported NH(4) (+); and second, alkalinization through protonation of NH(3) which is taken up to a significant extent from high external concentrations. Furthermore, it is concluded that the determination of intracellular pH by means of methylamine distribution is not a reliable method for eucaryotic systems.

Journal Article↗

PubMed1979

A study of dielectric membrane breakdown in the Fucus egg.

Unfertilized eggs and zygotes of the marine brown alga, Fucus serratus, have been subjected to single external electric field pulses of 1 to 1760 musec duration (tau p) and 50 to 400 V field strength (Upcm-1). During exposure, the difference in electric potential across the plasmalemma (Vm) was recorded intracellularly from single eggs, and the efflux of 86Rb+(K+) from the cytoplasm was measured on egg populations. A given single pulse instantaneously depolarizes the plasmalemma by a few (i.e., 6) millivolts and releases a certain fraction (i.e., 5%) of the cytoplasmic 86Rb+(K+). The dependence of these responses upon Up and tau p is fully consistent with the assumption that the membrane undergoes a localized reversible dielectric breakdown and reseals within less than 3 sec. The data are treated in terms of the electro-mechanical model for a compressible membrane by H.G.L. Coster and U. Zimmermann (1975, J. Membrane Biol. 22:73) and verify this model on a nonvacuolated plant cell. A threshold Vm for membrane breakdown (Vc) of 0.58 and 0.51 V is estimated for the turgorless unfertilized eggs and the turgescent (4.8 bar) zygotes, respectively. Using these values for Vc, and a reasonable value of the membrane's elastic modulus (i.e., Ym approximately 10(6) Nm-2), possible sites of membrane breakdown are discussed in terms of membrane thickness and relative permittivity.

Electric Stimulation↗

PubMed1979

Serine transport and membrane depolarization in the liverwort Riccia fluitans.

The plasmalemma of thallus cells of the aquatic liverwort, Riccia fluitans, is reversibly depolarized by L- and D-serine. At 0.1 mM K+ in the medium, the depolarization saturates at 50 mV; half-maximal depolarization occurs at 13 microM L-serine and 30 microM D-serine, respectively. Uptake of 14C-labelled L-serine depends upon the K+ concentration and is sensitive to the membrane potential as indicated by its reduction through 1 mM sodium cyanide. We propose that serine binds to and is transported by an electrogenic carrier. However, an interaction of serine with K+ channels of the membrane seems also possible.

Biological Transport↗

PubMed1977

A study of the primary effect of the uncoupler carbonyl cyanide m-chlorophenylhydrazone on membrane potential and conductance in Riccia fluitans.

1. In the presence of 10(-5) to 10 (-8) M carbonyl cyanide m-chlorophenylhydrazone (CCCP) the membrane potential of thallus cells of the aquatic liverwort Riccia fluitans responds to changes of the external pH between 5.5 and 8.3. This occurs in the light and dark, and also if respiration is abolished by addition of 10(-4) M KCN and 10 (-5) M salicyl-hydroxamic acid. 2. The ATP-level of the thallus is reduced, independently of the external pH, by greter than or equal to 10 (-6) M CCCP to 30--40% of the control level of about 1.1 nmol ATP per mg dryweight within 5 min. 3. Upon addition of 10 (-4) M CCCP at 20 degrees C the ATP-level declines exponentially with a half time of about 20--30 s, whereas the membrane potential declines exponentially with a half time of about 2--3 s. 4. At pH 7.2 the electrical membrane conductance yields a sigmoid curve as a function of the logarithm of the CCCP concentration between 10 (-8) and 3 - 10 (-6) M. On the other hand, at 3 - 10(-7) M M CCCP the gm (electrical slope conductance, muS - cm-2 = 10 (-6)-omega-1-cm-2) versus pH-curve displays an optimum between pH 6.5 and 7.5. 5. We conclude that CCCP acts upon membrane potential and conductance in Riccia predominantly by inducing a passive proton permeability of the cell membrane, i.e. CCCP raises the permeability ratio, PH/PK, more than 100-fold above its control level of about 10.

Adenosine Triphosphate↗

PubMed1973