Clifford L. Slayman

The relationship between ATP and an electrogenic pump in the plasma membrane ofNeurospora crassa

SummarySudden respiratory blockade has been used to study rapid changes of the resting membrane potential, of intracellular adenosine 5′-triphosphate (ATP) levels, and of pyridine nucleotide reduction inNeurospora crassa. Membrane depolarization occurs with a first-order rate constant of 0.167 sec−1, following a lag period of about 4 sec, at 24°C (ambient temperature). This depolarization is several-fold too slow to be directly linked to electron transfer, as judged from the rate of pyridine nucleotide reduction, but has essentially the same rate constant as the decay of ATP. The latter process, however, shows no lag period after the respiratory inhibitor is introduced. Plots of membrane potential versus the intracellular ATP concentration yield saturation curves which are readily fitted by a Michaelis equation, to which is added a constant term representing the diffusion component of membrane potential. Parameters obtained from such fits indicate the maximal voltage which the pump can develop at high ATP levels to be 300 to 350 mV, with an apparentK1/2 of 2.0mm. The data strongly suggest that an electrogenic ion pump in the plasma membrane ofNeurospora is fueled by ATP; comparison of the measured membrane potentials with the energy available from hydrolysis of ATP indicates that two ions could be pumped for each molecule of ATP split.

Interpretation of current-voltage relationships for "active" ion transport systems: I. Steady-state reaction-kinetic analysis of class-I mechanisms.

SummaryThis paper develops a simple reaction-kinetic model to describe electrogenic pumping and co- (or counter-) transport of ions. It uses the standard steady-state approach for cyclic enzyme- or carrier-mediated transport, but does not assume rate-limitation by any particular reaction step. Voltage-dependence is introduced, after the suggestion of Läuger and Stark (Biochim. Biophys. Acta211:458–466, 1970), via a symmetric Eyring barrier, in which the charge-transit reaction constants are written ask12=k120exp(zFΔΨ/2RT) andk21=k210exp(−zFΔΨ/2RT). For interpretation of current-voltage relationships, all voltage-independent reaction steps are lumped together, so the model in its simplest form can be described as a pseudo-2-state model. It is characterized by the two voltage-dependent reaction constants, two lumped voltage-independent reaction constants (K12,K21), and two reserve factors (ri,r0) which formally take account of carrier states that are indistinguishable in the current-voltage (I–V) analysis. The model generates a wide range ofI–V relationships, depending on the relative magnitudes of the four reaction constants, sufficient to describe essentially allI–V data now available on “active” ion-transport systems. Algebraic and numerical analysis of the reserve factors, by means of expanded pseudo-3-, 4-, and 5-state models, shows them to be bounded and not large for most combinations of reaction constants in the lumped pathway. The most important exception to this rule occurs when carrier decharging immediately follows charge transit of the membrane and is very fast relative to other constituent voltage-independent reactions. Such a circumstance generates kinetic equivalence of chemical and electrical gradients, thus providing a consistent definition of ion-motive forces (e.g., proton-motive force, PMF). With appropriate restrictions, it also yields both linear and log-linear relationships between net transport velocity and either membrane potential or PMF. The model thus accommodates many known properties of proton-transport systems, particularly as observed in “chemiosmotic” or energy-coupling membranes.

Current-voltage relationships for the plasma membrane and its principal electrogenic pump inNeurospora crassa: I. Steady-state conditions

SummaryThe nonlinear membrane current-voltage relationship (I–V curve) for intact hyphae ofNeurospora crassa has been determined by means of a 3-electrode voltage-clamp technique, plus “quasi-linear” cable theory. Under normal conditions of growth and respiration, the membraneI–V curve is best described as a parabolic segement convex in the direction of depolarizing current. At the average resting potential of −174 mV, the membrane conductance is ≈190 μmhos/cm2; conductance increases to ≈240 μmhos/cm2 at −300 mV, and decreases to ≈130 μmhos/cm2 at 0 mV. Irreversible membrane breakdown occurs at potentials beyond this range.Inhibition of the primary electrogenic pump inNeurospora by ATP withdrawal (with 1mm KCN) depolarizes the membrane to the range of −40 to −70 mV and reduces the slope of theI–V curve by a fixed scaling factor of approximately 0.8. For wild-typeNeurospora, compared under control conditions and during steady-state inhibition by cyanide, theI–V difference curve — presumed to define the current-voltage curve for the electrogenic pump — is a saturation function with maximal current of ≈20 μA/cm2, a half-saturation potential near −300 mV, and a projected reversal potential of ca. −400 mV. This value is close to the maximal free energy available to the pump from ATP hydrolysis, so that pump stoichiometry must be close to 1 H+ extruded:1 ATP split.The time-courses of change in membrane potential and resistance with cyanide are compatible with the steady-stateI–V curves, under the assumption that cyanide has no major effects other than ATP withdrawal. Other inhibitors, uncouplers, and lowered temperature all have more complicated effects.The detailed temporal analysis of voltage-clamp data showed three time-constants in the clamping currents: one of 10 msec, for charging the membrane capacitance (0.9 μF/cm2) a second of 50–75 msec; and a third of 20–30 sec, perhaps representing changes of intracellular composition.

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

SummaryA 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 realisticexperimental 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 driverion selectively to reduce the substrateKm, elevateJmax, jointly raise bothKm andJmax, or lowerKm while raisingJmax. 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.

KCl leakage from microelectrodes and its impact on the membrane parameters of a nonexcitable cell

SummaryMicrocapillary electrodes filled with a variety of salt solutions, including 1m KCl, have been used to measure the membrane potentials and resistances of spherical cells from the mycelial fungusNeurospora (cell diameters 15–25 μm, cell volumes 3–8 pl). During impalements with electrodes containing 0.3–1.0m KCl, membrane potential and resistance decayed over a period of 3–10 min. In contrast, electrodes filled with 0.1m KCl gave stable membrane potentials of −180 mV and membrane resistivities of 40 kΩ cm2, values comparable to earlier results from the fungal hyphae.Salt leakage from 1.0m KCl-filled electrodes (tip diameters 0.2–0.3 μm, resistances 50–75 MΩ) occurred at rates of 4–5 fmol sec−1, as indicated by direct intracellular measurements with ion-sensitive microelectrodes. Depending on cell size, such leakage rates could elevate cytoplasmic KCl content at initial rates of 30–170 mM min−1, and actual values as high as 70mm min−1 were observed. Salt leakage and changes in cytoplasmic KCl concentration were reduced five- to sevenfold when impalements were made with electrodes containing 0.1m KCl.The effects on cell membrane parameters of salt leakage from microelectrodes could be attributed to chloride ions. Substitution of the KCl electrolyte with half-molar K2SO4 or Na2SO4 and molar concentrations of K- and Na-MES [potassium and sodium 2-(N-morpholino)ethanesulfonate] gave stable membrane potentials in excess of −200 mV and membrane resistivities greater than 50 kΩ cm2, while the permeant anions NO3− and SCN− depressed the membrane parameters in a manner similar to that observed with 1m KCl. Furthermore, modest elevation of cytoplasmic chloride concentration (below ca. 50 mM) affected both membrane potential and resistance in direct proportion to the concentration, and could be quantitatively described by the Constant Field Theory with a fixed membrane permeability (PCl∼4×10−8 cm sec−1). Higher cytoplasmic chloride levels produced a collapse of the membrane resistance and drastic depolarization in a fashion requiring large changes of membrane permeability.At least for cells with volumes of 10 pl or less, the standard practice of filling electrodes with 1 or 3m KCl should be abandoned. Half-molar (and lower) concentrations of K2SO4 or Na2SO4 are suggested as satisfactory replacements.

Potassium-proton symport inNeurospora: kinetic control by pH and membrane potential

SummaryActive transport of potassium in K+-starvedNeurospora 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. ForNeurospora, 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 ofNeurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in theNeurospora plasma membrane. Addition of 5-200 μM K+ to the bathing medium causes 10–150 mV depolarization of the unclamped membrane, and yields a sigmoidI–V curve with a steep slope (maximal conductance of 10–30 μS/cm2) for voltages of −300 to −100 mV, i.e., in the normal physiologic range. Outside that range the apparentI–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 theI–V difference method under reversing conditions.In the absence of voltage clamping, inhibitors—such as cyanide or vanadate—which block the primary proton pump inNeurospora 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 on the cotransport system itself.Detailed study of the effects of [K+]o and pHo on theI–V curve for K+-H+ symport has revealed that increasing membrane potential systematicallydecreases the apparent affinity of the transporter for K+, butincreases affinity for protons (Km range: for [K+]o, 15–45 μM; 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.

Gating and conductance in an outward-rectifying K+ channel from the plasma membrane of Saccharomyces cerevisiae.

SummaryThe plasma membrane of the yeast Saccharomyces cerevisiae has been investigated by patch-clamp techniques, focusing upon the most conspicuous ion channel in that membrane, a K+-selective channel. In simple observations on inside-out patches, the channel is predominantly closed at negative membrane voltages, but opens upon polarization towards positive voltages, typically displaying long flickery openings of several hundred milliseconds, separated by long gaps (G). Elevating cytoplasmic calcium shortens the gaps but also introduces brief blocks (B, closures of 2–3 msec duration). On the assumption that the flickery open intervals constitute bursts of very brief openings and closings, below the time resolution of the recording system, analysis via the beta distribution revealed typical closed durations (interrupts, I) near 0.3 msec, and similar open durations. Overall behavior of the channel is most simply described by a kinetic model with a single open state (O), and three parallel closed states with significantly different lifetimes: long (G), short (B) and very short (I). Detailed kinetic analysis of the three open/closed transitions, particularly with varied membrane voltage and cytoplasmic calcium concentration, yielded the following stability constants for channel closure: KI=3.3 · e−zu in which u=eVm/kT is the reduced membrane voltage, and z is the charge number; KG = 1.9 · 10−4([Ca2+] · ezu )−1; and KB =2.7 · 103([Ca2+] · ezu )2. Because of the antagonistic effects of both membrane voltage (Vm ) and cytoplasmic calcium concentration ([Ca2+]cyt) on channel opening from the B state, compared with openings from the G state, plots of net open probability (P0) vs. either Vm or [Ca2+] are bell-shaped, approaching unity at low calcium (μm) and high voltage (+150 mV), and approaching 0.25 at high calcium (10 mm) and zero voltage. Current-voltage curves of the open channel are sigmoid vs. membrane voltage, saturating at large positive or large negative voltages; but time-averaged currents, along the rising limb of P0 (in the range 0 to +150 mV, for 10 μm [Ca2+]) make this channel a strong outward rectifier. The overall properties of the channel suggest that it functions in balancing charge movements during secondary active transport in Saccharomyces.

Functional comparison of plant inward-rectifier channels expressed in yeast

Functional expression of plant ion channels in the yeast Saccharomyces cerevisiae is readily demonstrated by the successful screening of plant cDNA libraries for complementation of transport defects in especially constructed strains of yeast. The first experiments of this sort identified two potassium-channel genes from Arabidopsis thaliana, designated KAT1 and AKT1 (Anderson et al., 1992; Sentenac et al., 1992), both of which code for proteins resembling the Shaker superfamily of K(+) channels in animal cells. Patch-clamp analysis, directly in yeast, of the two channel proteins (Kat1 and Akt1) reveals both functional similarities and functional differences: similarities in selectivity and in normal gating kinetics; and differences in time-dependent effects of ion replacement, in the affinities of blocking ions, and in dependence of gating kinetics on extracellular K(+). Kat1, previously described in yeast (Bertl et al., 1995), is about 20-fold more permeable to K(+) than to Na(+) or NH(+)(4), shows K(+)-independent gating kinetics, and is blocked with moderate effectiveness (30-50% at 10 mM) by barium and tetraethylammonium (TEA(+)) ions. Akt1, by contrast, is weakly inhibited by TEA(+), more strongly inhibited by Ba(2+), and very strongly inhibited by Cs(+). Furthermore Na(+) and NH(+)(4), while having about the same permeance to Akt1 as to Kat1, have delayed effects on Akt1: brief replacement of extracellular K(+) by Na(+) enhances by nearly 100% the subsequent K(+) currents after sodium removal; and brief replacement of K(+) by NH(+)(4) reduces subsequent K(+) currents by nearly 75%. Furthermore, lowering of extracellular K(+) concentration, by replacement with osmotically equivalent sorbitol, significantly retards the opening of Akt1 channels; that is, the gating kinetics for Akt1 are clearly influenced by the concentration of permeant ions. In this respect, Akt1 resembles the native yeast outward rectifier, Ypk1 (Duk1; Reid et al., 1996). The data suggest that all of the ions tested bind within the open channels, such that the weakly permeant species (Na(+), NH(+)(4)) are easily displaced by K(+), but the blocking species (Cs(+), Ba(2+), TEA(+)) are not easily displaced. With Akt1, furthermore, the permeant ions bind to a modulator site where they persist after removal from the medium, and through which they can alter the channel conductance. Extracellular K(+) itself also binds to a modulator site, thereby enhancing the rate of opening of Akt1.

The TRK1 Potassium Transporter Is the Critical Effector for Killing of Candida albicans by the Cationic Protein, Histatin 5

The principal feature of killing of Candida albicans and other pathogenic fungi by the catonic protein Histatin 5 (Hst 5) is loss of cytoplasmic small molecules and ions, including ATP and K+, which can be blocked by the anion channel inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. We constructed C. albicans strains expressing one, two, or three copies of the TRK1 gene in order to investigate possible roles of Trk1p (the organisms principal K+ transporter) in the actions of Hst 5. All measured parameters (Hst 5 killing, Hst 5-stimulated ATP efflux, normal Trk1p-mediated K+ (86Rb+) influx, and Trk1p-mediated chloride conductance) were similarly reduced (5–7-fold) by removal of a single copy of the TRK1 gene from this diploid organism and were fully restored by complementation of the missing allele. A TRK1 overexpression strain of C. albicans, constructed by integrating an additional TRK1 gene into wild-type cells, demonstrated cytoplasmic sequestration of Trk1 protein, along with somewhat diminished toxicity of Hst 5. These results could be produced either by depletion of intracellular free Hst 5 due to sequestered binding, or to cooperativity in Hst 5-protein interactions at the plasma membrane. Furthermore, Trk1p-mediated chloride conductance was blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid in all of the tested strains, strongly suggesting that the TRK1 protein provides the essential pathway for ATP loss and is the critical effector for Hst 5 toxicity in C. albicans.

Low-affinity potassium uptake by Saccharomyces cerevisiae is mediated by NSC1, a calcium-blocked non-specific cation channel

Previous descriptions by whole-cell patch clamping of the calcium-inhibited non-selective cation channel (NSC1) in the plasma membrane of Saccharomyces cerevisiae (H. Bihler, C.L. Slayman, A. Bertl, FEBS Lett. 432 (1998); S.K. Roberts, M. Fischer, G.K. Dixon, D.Sanders, J. Bacteriol. 181 (1999)) suggested that this inwardly rectifying pathway could relieve the growth inhibition normally imposed on yeast by disruption of its potassium transporters, Trk1p and Trk2p. Now, demonstration of multiple parallel effects produced by various agonists and antagonists on both NSC1 currents and growth (of trk1 Delta trk2 Delta strains), has identified this non-selective cation pathway as the primary low-affinity uptake route for potassium ions in yeast. Factors which suppress NSC1-mediated inward currents and inhibit growth of trk1 Delta trk2 Delta cells include (i) elevating extracellular calcium over the range of 10 microM-10 mM, (ii) lowering extracellular pH over the range 7.5-4, (iii) blockade of NSC1 by hygromycin B, and (iv) to a lesser extent by TEA(+). Growth of trk1 Delta trk2 Delta cells is also inhibited by lithium and ammonium; however, these ions do not inhibit NSC1, but instead enter yeast cells via NSC1. Growth inhibition by lithium ions is probably a toxic effect, whereas growth inhibition by ammonium ions probably results from competitive inhibition, i.e. displacement of intracellular potassium by entering ammonium.