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C L Slayman

Publications and source records attributed to C L Slayman.

At least 37 records · Page 2Linked to original sources
PubMed1992

Ion channels in Arabidopsis plasma membrane : transport characteristics and involvement in light-induced voltage changes.

White light (25 watts per square meter) induced an increase in plasma membrane K(+)-channel activity and a 30- to 70-millivolt transient membrane depolarization (completed in 2-3 minutes) in Arabidopsis thaliana leaf mesophyll cells. Transport characteristics of three types of ion channels in the plasma membrane were determined using inside-out patches. With 220 millimolar K(+) on the cytoplasmic side of the patch and 50 millimolar K(+) in the pipette, (220/50 K), the open-channel current-voltage curves of these channels were sigmoidal and consistent with an enzyme kinetic model. Two channel types were selective for K(+) over Na(+) and Cl(-). One (named PKC1) had a maximum conductance (G(max)) of 44 picosiemens at a membrane voltage (V(m)) of -65 mV in (220/50 K) and is stimulated by light. The other (PKC2) had G(max) = 66 picosiemens at V(m) = 60 millivolts in (220/50 K). The third channel type (PCC1) transported K(+) and Na(+) about equally well but not Cl(-). It had G(max) = 109 picosiemens at V(m) = 55 millivolts in (250/50 K) with 10 millimolar Ca(2+) on the cytoplasmic side. Reducing Ca(2+) to 0.1 millimolar increased PCC1 open-channel currents by approximately 50% in a voltage-independent manner. Averaged over time, PKC2 and PCC1 currents strongly outward rectified and PKC1 currents did so weakly. Reductants (1 millimolar dithiothreitol or 10 millimolar beta-mercaptoethanol) added to the cytoplasmic side of an excised patch increased the open probability of all three channel types.

Journal Article↗
PubMed1992

Complex modulation of cation channels in the tonoplast and plasma membrane of Saccharomyces cerevisiae: single-channel studies.

Detailed patch-clamp studies have been made of ion channels in the plasma membrane and tonoplast of the yeast Saccharomyces cerevisiae. The predominant tonoplast channel is a high-conductance cation-selective inward rectifier (passing ions easily into the cytoplasm from the vacuole), with its open probability (Po) peaking at about -80 mV (cytoplasm negative) and falling to near zero at +80 mV. It has a maximal slope conductance of approximately 150 pS in 100 mmol l-1 KCl, and conducts Na+, K+ and Ca2+. Elevated cytoplasmic Ca2+ concentration, alkaline pH and reducing agents can activate the channel, its likely physiological function being to adjust cytoplasmic Ca2+ concentration from the vacuolar reservoir. The predominant plasma-membrane channel is a strongly outward rectifying K+ channel (passing K+ easily out of the cytoplasm to the extracellular medium), which is activated by positive-going membrane voltages as well as by elevated cytoplasmic Ca2+ concentration and alkaline pH. Interaction between membrane voltage and [Ca2+]cyt is complex and defines three parallel closed states for the channel: a Ca(2+)-independent brief closure (I), a calcium-inhibited long closure (G) and, at large positive voltages, a calcium-induced brief blockade (B). This channel is likely to function in steady-state turgor regulation and in charge balancing during proton-coupled substrate uptake.

Cations↗
PubMed1990

Cation-selective channels in the vacuolar membrane of Saccharomyces: dependence on calcium, redox state, and voltage.

The vacuolar membrane of the yeast Saccharomyces cerevisiae, which is proposed as a system for functional expression of membrane proteins, was examined by patch-clamp techniques. Its most conspicuous feature, in the absence of energizing substrates, is a cation channel with a characteristic conductance of approximately 120 pS for symmetric 100 mM KCl solutions and with little selectivity between K+ and Na+ (PNa+/PK+ approximately 1) but strong selectivity for cations over anions (PCl-/PK+ less than 0.1). Channel gating is voltage-dependent; open probability, Po, reaches maximum (approximately 0.7) at a transmembrane voltage of -80 mV (cytoplasmic surface negative) and declines at both more negative and more positive voltages (i.e., to 0 around +80 mV). The time-averaged current-voltage curve shows strong rectification, with negative currents (positive charges flowing from vacuolar side to cytoplasmic side) much larger than positive currents. The open probability also depends strongly on cytoplasmic Ca2+ concentration but, for ordinary recording conditions, is high only at unphysiologically high (greater than or equal to 1 mM) Ca2+. However, reducing agents such as dithiothreitol and 2-mercaptoethanol poise the channels so that they can be activated by micromolar cytoplasmic Ca2+. The channels are blocked irreversibly by chloramine T, which is known to oxidize exposed methionine and cysteine residues specifically.

Calcium↗
PubMed1987

The plasma membrane ATPase of Neurospora: a proton-pumping electroenzyme.

Probably the best marker enzyme for plasma membranes of eukaryotic cells is a magnesium-dependent, vanadate-inhibited ATPase whose primary function is the transmembrane transport of cations. In animal cells, different species of the enzyme transport different cations: sodium ions released in unequal exchange for potassium ions, calcium ions extruded alone (perhaps), or protons secreted in equal exchange for potassium ions. But in plants and fungi only proton secretion has been clearly demonstrated. A useful model cell for studying the proton-secreting ATPase has been the ascomycete fungus Neurospora, in which the enzyme drives an outward current of protons that can exceed 50 microA/cm2 and can support membrane potentials greater than 300 mV. Both thermodynamic and kinetic studies have shown that the proton-pumping ATPase of Neurospora normally transports only a single proton for each ATP molecule split; and kinetic modelling studies have suggested (contrary to conventional assumptions) that the fast steps in the overall reaction are transmembrane transit of the proton and its dissociation following transport, while the slow steps are the binding of protons and/or ATP. The primary structure of the Neurospora enzyme, recently deduced by gene sequencing, is very close to that of the yeast (Saccharomyces) enzyme, and the hydropathic patterns for both closely resemble those for the animal-cell plasma-membrane ATPases. All of these enzymes appear to have 6-10 membrane-spanning alpha-helices, plus a large cytoplasmic headgroup which bears the catalytic nucleotide-binding site. Structural data, taken together with the electrical-kinetic behavior, suggest that the catalytic headgroup functions as an energized gate for protons. From a geometric point of view, action of such a gate would transfer the membrane field across the "transported" ion, rather than vice versa.

Adenosine Triphosphatases↗
PubMed1987

Potassium-proton symport in Neurospora: kinetic control by pH and membrane potential.

Active transport of potassium in K+-starved Neurospora was previously shown to resemble closely potassium uptake in yeast, Chlorella, and higher plants, for which K+ pumps or K+/H+-ATPases had been proposed. For Neurospora, however, potassium-proton cotransport was demonstrated to operate, with a coupling ratio of 1 H+ to 1 K+ taken inward so that K+, but not H+, moves against its electrochemical gradient (Rodriguez-Navarro et al., J. Gen. Physiol. 87:649-674). In the present experiments, the current-voltage (I-V) characteristic of K+-H+ cotransport in spherical cells of Neurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in the Neurospora plasma membrane. Addition of 5-200 microM K+ to the bathing medium causes 10-150 mV depolarization of the unclamped membrane, and yields a sigmoid I-V curve with a steep slope (maximal conductance of 10-30 microS/cm2) for voltages of -300 to -100 mV, i.e., in the normal physiologic range. Outside that range the apparent I-V curve of the K+-H+ symport saturates for both hyperpolarization and depolarization. It fails to cross the voltage axis at its predicted reversal potential, however, an effect which can be attributed to failure of the I-V difference method under reversing conditions. In the absence of voltage clamping, inhibitors-such as cyanide or vanadate-which block the primary proton pump in Neurospora also promptly inhibit K+ transport and K+-H+ currents. But when voltage clamping is used to offset the depolarizing effects of pump blockade, the inhibitors have no immediate effect on K+-H+ currents. Thus, the inhibition of K+ transport usually observed with these agents reflects the kinetic effect of membrane depolarization rather than any direct chemical action or the cotransport system itself. Detailed study of the effects of [K+]o and pHo on the I-V curve for K+-H+ symport has revealed that increasing membrane potential systematically decreases the apparent affinity of the transporter for K+, but increases affinity for protons (Km range: for [K+]o, 15-45 microM; for [H+]o, 10-35 nM). This behavior is consistent with two distinct reaction-kinetic models, in which (i) a neutral carrier binds K+ first and H+ last in the forward direction of transport, or (ii) a negatively charged carrier (-2) binds H+ first and K+ last.

Carrier Proteins↗
PubMed1987

Role of "active" potassium transport in the regulation of cytoplasmic pH by nonanimal cells.

High-affinity potassium uptake in Neurospora occurs by symport with protons [Km (apparent) = 15 microM at pH 5.8], for which a large inward gradient (approximately 400 mV) is generated by the H+-extruding ATPase of the plasma membrane. Operating in parallel, the two transport systems yield a net 1:1 exchange of K+ for cytoplasmic H+. Since this exchange could play a role in cytoplasmic pH (pHi) regulation, the coordinated functioning of the K+-H+ symport and H+ pump has been examined during acid stress. Cytoplasmic acid loads were imposed by injection and by exposure to extracellular permeant weak acid. Multibarrelled microelectrodes were used to monitor membrane potential (Vm), pHi, and the current-voltage (I-V) characteristics of the cells. The behaviors of the H+ pump and K+-H+ symport were resolved, respectively, by fitting whole membrane I-V curves to an explicit kinetic model of the Neurospora membrane and by subtracting I-V curves obtained in the absence from those obtained in the presence of 5-200 microM K+ outside. Proton pumping accelerates nearly in proportion with the cytoplasmic H+ concentration, but pHi recovery from imposed acid loads is dependent on micromolar K+ outside. Potassium import via the symport leads to a measurable alkalinization of the cytoplasm in accordance with stoichiometric (1:1) K+/H+ exchange. Potassium transport is accelerated at low pHi, but in a manner consistent with its inherent voltage sensitivity and changes in Vm resulting from an increased rate of H+ extrusion by the pump. The primary response to acid stress thus rests with the H+ pump, but K+ transport introduces an essential kinetic "valve" that can regulate net H+ export.

Biological Transport, Active↗
PubMed1986

A potassium-proton symport in Neurospora crassa.

Combined ion flux and electrophysiological measurements have been used to characterized active transport of potassium by cells of Neurospora crassa that have been moderately starved of K+ and then maintained in the presence of millimolar free calcium ions. These conditions elicit a high-affinity (K1/2 = 1-10 microM) potassium uptake system that is strongly depolarizing. Current-voltage measurements have demonstrated a K+-associated inward current exceeding (at saturation) half the total current normally driven outward through the plasma membrane proton pump. Potassium activity ratios and fluxes have been compared quantitatively with electrophysiological parameters, by using small (approximately 15 micron diam) spherical cells of Neurospora grown in ethylene glycol. All data are consistent with a transport mechanism that carries K ions inward by cotransport with H ions, which move down the electrochemical gradient created by the primary proton pump. The stoichiometry of entry is 1 K ion with 1 H ion; overall charge balance is maintained by pumped extrusion of two protons, to yield a net flux stoichiometry of 1 K+ exchanging for 1 H+. The mechanism is competent to sustain the largest stable K+ gradients that have been measured in Neurospora, with no direct contribution from phosphate hydrolysis or redox processes. Such a potassium-proton symport mechanism could account for many observations reported on K+ movement in other fungi, in algae, and in higher plants.

Biological Transport↗
PubMed1985

Membrane voltage, resistance, and channel switching in isolated mouse fibroblasts (L cells): a patch-electrode analysis.

The whole-cell patch-electrode technique of Fenwick, Marty & Neher (1982) has been applied to single suspension-cultured mouse fibroblasts. Seals in the range of 10-50 G omega were obtained without special cleaning of the cell membranes. Rupture of the membrane patch inside the electrode was accompanied by a shift of measured potential into the range -10 to -25 mV, but in most cases with little change in the recorded resistance. The latter fact implied that the absolute resistance of the cell membrane must be in the same range as the seal resistance and the recorded potential is a poor measure of actual cell membrane potential. Steady-state current-voltage curves (range -160 mV to +80 mV) were generated before and after rupture of the membrane patch, and the difference between these gave (zero-current) membrane potentials of -50 to -75 mV, which represents a leak-corrected estimate of the true cell-membrane potential. The associated slope conductivity of the cell membrane was 5-15 microS/cm2 (assumed smooth-sphere geometry, cells 13-15 microns in diameter) and was K+-dominated. With 0.1 mM (or more) free Ca2+ filling the patch electrode, membrane potentials in the range -60 to -85 mV were observed following patch rupture, with associated slope conductivities of 200-400 microS/cm2, also K+-dominated. Similar voltages and conductivities were observed at the peak of pulse-induced 'hyperpolarizing activation' (Nelson, Peacock, & Minna, 1972), and the two phenomena probably reflect the behaviour of Ca2+-activated K+ channels. Both the pulse-induced conductance and the Ca2+-activated conductance spontaneously decayed, the latter over periods of 5-15 min following patch rupture. Sr2+, Ba2+, and Co2+ could also activate the putative K+ channels, but only Sr2+ really mimicked Ca2+. Co2+ and Ba2+ activated with a delay of several minutes following patch rupture, and deactivated quickly with a small decrease of conductance and a large decrease of membrane potential. Evidently, Co2+ and Ba2+ affect channel specificity as well as channel opening and closing kinetics.

Action Potentials↗
PubMed1985

Steady-state kinetic analysis of an electroenzyme.

Primary active transport of ions through the plasma membranes of plants and fungi is driven by a proton-dependent ATPase, which consists of a membrane-embedded (Mr 104,000) polypeptide, forms a beta-aspartylphosphate intermediate and is blocked by orthovanadate. It can be extracted from cell membranes and reactivated in native lipid micelles or in exogenous phospholipid vesicles. For the fungus Neurospora, vesicle preparations directly display proton-pumping, and can develop membrane potentials (delta psi) of 120 mV or pH differences (delta pH) of 2 units, with a stoichiometry of 1 H+ transported per ATP molecule split. In vivo, the proton pump sustains delta psi values of 150-350 mV (cytoplasm negative) and delta pH values up to 3.5 units (pHi congruent to 7, with pHo = 3.5). Since the total proton-motive force thus can exceed 400 mV, compared with a delta GATP of 500 mV, the stoichiometry must be 1 H+/ATP, with little leeway for neutralizing ions. Kinetic analysis of pump-currents measured during forcing of [ATP]i, pHo, pHi, and delta psi yields three main conclusions: again, the stoichiometry is 1 H+/ATP; energy conversion occurs during transmembrane charge transfer, which therefore is probably the E1 approximately P--E2 X P transition (see Na+,K+-ATPase); protons are strongly dissociated at both membrane surfaces, with pKi congruent to 5.4 versus pHi = 7.2, and pKo congruent to 2.9 versus pHo = 5.8. Considerations of structure and partial-reaction chemistry (by analogy with the Na+,K+-ATPase) suggest a kinetically testable model for the transport mechanism: a sequential, double-gated channel, through which the membrane field is transported across the ion, rather than vice versa.

Adenosine Triphosphatases↗
PubMed1984

Kinetics and pH-dependence of glycine-proton symport in Saccharomyces cerevisiae.

Interactions between intracellular pH (pHi) and H+-coupled transmembrane transport of glycine have been studied by means of 31P-NMR, using both aerobic and 'energy starved' cells of the yeast Saccharomyces cerevisiae. The general features of glycine transport in the yeast strain used (NCYC 239) are similar to those already reported for Saccharomyces carlsbergensis and S. cerevisiae, there being two kinetically distinct glycine uptake systems, with pH-independent K1/2 values near 14 and 0.4mM, respectively, but pH-dependent maximal velocities. Glycine transport itself has no measurable effect on pHi in aerobic cells, and only a marginal effect in energy-starved cells, but changes of pHi, imposed by extracellular addition of butyric acid, strongly influence glycine transport. Indeed, the dependence of glycine influx (in energy-starved cells) upon cytoplasmic H+ concentration appears to be third order, showing Hill slopes of 2.7-3.0. A crucial kinetic role for cytoplasmic pH in glycine transport is further indicated by a proportionality between the decline of flux and the decline of pHi produced by various metabolic inhibitors and uncouplers. Extracellular pH (pHo), by contrast, has only a weak effect on glycine influx, showing a Hill slope of 0.5. The major observations can be accommodated by a simple cyclic carrier scheme, in which 2 or more protons are transported along with glycine, but only one extracellular proton binding site dissociates in the testing range, with a pK near 5.5. The model requires a finite membrane potential, which must be somewhat sensitive to both pHi and pHo, and accommodates the discrepancy between measured net proton flux (one per glycine) and the kinetically required proton flux (two or more per glycine) by shunting through other proton-conducting pathways in the yeast membrane.

Biological Transport↗
PubMed1984

Generalized kinetic analysis of ion-driven cotransport systems: a unified interpretation of selective ionic effects on Michaelis parameters.

A major obstacle to the understanding of gradient-driven transport systems has been their apparently wide kinetic diversity, which has seemed to require a variety of ad hoc mechanisms. Ordinary kinetic analysis, however, has been hampered by one mathematically powerful but physically dubious assumption: that rate limitation occurs in transmembrane transit, so that ligand-binding reactions are at equilibrium. Simple models lacking that assumption turn out to be highly flexible and are able to describe most of the observed kinetic diversity in co- and counter-transport systems. Our "minimal" model of cotransport consists of a single transport loop linking six discrete states of a carrier-type molecule. The state transitions include one transmembrane charge-transport step, and one step each for binding of substrate and cosubstrate (driver ion) at each side of the membrane. The properties of this model are developed by sequential use of realistic experimental simplifications and generalized numerical computations, focussed to create known effects of substrate, driver ion, and membrane potential upon the apparent Michaelis parameters (Jmax, Km) of isotopic substrate influx. Specific behavior of the minimal model depends upon the arrangement of magnitudes of individual reaction constants among the whole set (12) in the loop. Well defined arrangements have been found which permit either increasing membrane potential or increasing external driver-ion selectively to reduce the substrate Km, elevate Jmax, jointly raise both Km and Jmax, or lower Km while raising Jmax. Other arrangements allow rising internal driver ion to act like either a competitive or a noncompetitive inhibitor of entry, or allow internal substrate to shut down ("transinhibit") influx despite large inward driving forces. These findings obviate most postulates of special mechanisms in cotransport: e.g., stoichiometry changes, ion wells, carrier-mediated leakage, and gating - at least as explanations for existing transport kinetic data. They also provide a simple interpretation of certain kinds of homeostatic regulation, and lead to speculation that the observed diversity in cotransport kinetics reflects control-related selection of reaction rate constants, rather than fundamental differences of mechanism.

Biological Transport↗
PubMed1983

Stoichiometry of H+/amino acid cotransport in Neurospora crassa revealed by current-voltage analysis.

Coupling of ions to the uptake of neutral and basic amino acids via a general amino acid transport system (System II), was studied in a mutant of Neurospora crassa (bat mtr) which lacks other transport systems for these solutes. All amino acids tested--including ones bearing no net charge--elicited rapid membrane depolarization, as expected for ion-coupled transport. (Since amino acid transport in Neurospora is not dependent on extracellular Na+ or K+, the associated ion is presumed to be H+.) Although the 14C-labeled amino acid fluxes through System II are largely independent of the identity of the amino acid, the depolarization caused by basic amino acids (L-lysine and L-ornithine) is 60-70% greater than that for neutral amino acids (e.g. L-leucine). This difference is consistent with a constant H+/amino acid stoichiometry of 2, the extra charge for lysine and ornithine being that on the amino acid itself, so that the charge ratio basic:neutral amino acids is 3:2. When actual membrane charge flow associated with amino acid uptake was compared with measured 14C-labeled amino acid influx, ratios of 2.07 charges/mol L-leucine and 3.40 charges/mol L-lysine were obtained, again in accord with a constant translocation stoichiometry of 2H+/amino acid. The advantages of this electrical method for estimating H+/solute stoichiometry in cotransport are discussed in relation to more familiar methods.

Amino Acids↗
PubMed1982

Control of intracellular pH. Predominant role of oxidative metabolism, not proton transport, in the eukaryotic microorganism Neurospora.

Recessed-tip microelectrodes were used to measure internal pH (pHi) in the fungus Neurospora, and to examine the response of pHi to several kinds of stress: changes of extracellular pH (pHo), inhibition of the principal proton pump in the plasma membrane, and inhibition of respiration. Under control conditions, at pHo = 5.8, pHi in Neurospora is 7.19 +/- 0.04. Changes of pHo between 3.9 and 9.3 affect pHi linearly but with a slope of only approximately 0.1 unit pHi per unit pHo, stable pHi being reached within 3 min of changed pHo. Despite a postulated high passive permeability of the Neurospora membrane to protons (Slayman, 1970), neither active nor passive H+ transport appears critical to pHi because (alpha) specific inhibition of the proton pump by orthovanadate has little effect on pHi, and (b) cytoplasmic acidification produced by respiratory blockade is unaffected by the size or direction of proton gradient. To convert measured changes in pHi into net proton fluxes, intracellular buffering capacity (beta i) was measured by the weak acid/weak base technique. At pHi = 7.2, beta i was (-) 35 mmol H+ (liter cell water)-1 (pH unit)-1, but beta i increased substantially in both the acid and alkaline directions, which suggests that amino acid side chains are the principal source of buffer.

Butyrates↗