Cloning and characterization of two K+ transporters of Debaryomyces hansenii.

Two genes from the halotolerant yeast Debaryomyces hansenii were cloned, DhTRK1 and DhHAK1. These genes encode K(+) transporters with sequence similarities to the TRK and HAK transporters from Debaryomyces occidentalis and Candida albicans. The DhHAK1p transporter was only expressed in K(+)-starved cells, as shown by Northern blot analysis. Both DhTRK1p and DhHAK1p were expressed in a trk1Delta trk2Delta mutant of Saccharomyces cerevisiae, unable to grow at low K(+). This expression resulted in partial recovery of growth and ability to retain K(+) at low concentrations. In liquid media, 0.5 M NaCl affected growth of these S. cerevisiae transformants as it does in D. hansenii, resulting in a much less deleterious effect than in wild-type S. cerevisiae. Kinetics of Rb(+) uptake in the transformants suggest that DhTRK1p and DhHAK1p code for moderate-affinity K(+) transporters exhibiting a sigmoid response against Rb(+) concentration and presenting a deviation from classic Michaelis-Menten kinetics at low substrate concentrations. Rb(+) uptake by the DhTRK1p transporter was stimulated by millimolar concentrations of Na(+) at pH 4.5. The good performance of DhTRK1p in the presence of NaCl may be a key feature in the halotolerance of D. hansenii.

Sodium and potassium transport in the halophilic yeast Debaryomyces hansenii.

Debaryomyces hansenii, a halophile yeast found in shallow sea waters and salty food products grows optimally in 0.6 M of either NaCl or KCl, accumulating high concentrations of Na(+) or K(+). After growth in NaCl or KCl, a rapid efflux of either accumulated cation was observed if the cells were incubated in the presence of KCl or NaCl, respectively, accompanied by a slower accumulation of the cation present in the incubation medium. However, a similar, rapid efflux was observed if cells were incubated in buffer, in the absence of external cations. This yeast shows a cation uptake activity of both (86)Rb(+) and (22)Na(+) with saturation kinetics, and much higher affinity for (86)Rb(+) than for (22)Na(+). The pH dependence of the kinetics constants was similar for both cations, and although K(m) values were higher at pH 8.0, there was also an increase in the V(max) values. The accumulation of (22)Na(+) was found to be increased in cells grown in the presence of 0.6 M NaCl. (86)Rb(+) was also accumulated more in these cells, but to a slightly greater extent. The inhibition kinetics of the uptake of (22)Na(+) by K(+), and that of (86)Rb(+) by Na(+) was found to be non-competitive. It can be concluded that Na(+) in D. hansenii is not excluded but instead, its metabolic systems must be resistant to high salt concentrations.

Enzyme kinetics of the prime K+ channel in the tonoplast of Chara: selectivity and inhibition.

The prime potassium channel from the tonoplast of Chara corallina has been analyzed in terms of an enzymatic kinetic model (Gradmann, Klieber & Hansen 1987, Biophys. J. 53:287) with respect to its selectivity for K+ over Rb+ and to its blockage by Cs+ and by Ca2+. The channel was investigated by patch-clamp techniques over a range of membrane voltages (Vm, referred to an extracytoplasmic electrical potential of zero) from -200 mV to +200 mV under various ionic conditions (0 to 300 mM K+, Rb+, Cs+, Ca2+, and Cl-) on the two sides of isolated patches. The experimental data are apparent steady-state current-voltage relationships under all experimental conditions used and amplitude histograms of the seemingly noisy open-channel currents in the presence of Cs+. The used model for K+ uniport comprises a reaction cycle of one binding site through four states, i.e., (1) K(+)-loaded and charged, facing the cytoplasm, (2) K(+)-loaded and charged facing the vacuole, (3) empty, facing the vacuole, and (4) empty, facing the cytoplasm. Vm enters the system in the form of a symmetric Eyring barrier between state 1 and 2. The numerical results for the individual rate constants are (in 10(6)s-1 for zero voltage and 1 M substrate concentration): k12: 1,410, k21: 3,370, k23: 105,000, k32: 10,600, k34: 194, k43: 270, k41: 5,290, k14: 15,800. For the additional presence of an alternate transportee (here Rb+), the model can be extended in an analog way by another two states ((5) Rb(+)-loaded and charged, facing cytoplasm, and (6) Rb(+)-loaded and charged, facing vacuole) and six more rate constants (k45: 300, k54: 240, k56: 498, k65: 4,510, k63: 4,070, k36: 403). This six-state model with its unique set of fourteen parameters satisfies the complete set of experimental data. If the competing substrate can be bound but not translocated (here Cs+ and Ca2+). k56 and k65 of the model are zero, and the stability constants Kcyt (= k36/k63) and Kvac (= k45/k54) turn out to be Kcyt(Ca2+): 250 M-1 x exp(Vm/(64 mV)), kvac(Ca2+): 10 M-1 x exp(-Vm/(66 mV)), Kcyt(Cs+): 0, and Kvac(Cs+): 46 M-2 x exp(-Vm/(12.25 mV)).(ABSTRACT TRUNCATED AT 400 WORDS)

Generalized kinetic analysis of ion-driven cotransport systems: II. Random ligand binding as a simple explanation for non-michaelian kinetics.

Solute uptake in many cells is characterized by a series of additive Michaelis-Menten functions. Several explanations for these kinetics have been advanced: unstirred layers, transport across more than one membrane, effects of solute concentration on membrane potential, numerous carrier systems. Although each of these explanations might suffice for individual cases, none provides a comprehensive basis for interpretation of the kinetics. The most common mechanism of solute absorption involves cotransport of solute with a driver ion. A model is developed in which solute and driver ion bind randomly to a membrane-bound carrier which provides a single transmembrane pathway for transport. The kinetic properties of the model are explored with particular reference to its capacity to generate additive Michaelian functions for initial rate measurements of isotopic solute influx. In accord with previous analysis of ordered binding models (Sanders, D., Hansen, U.-P., Gradmann, D., Slayman, C.L. (1984) J. Membrane Biol. 77:123), the conventional assumption that transmembrane transit rate-limits transport has not been applied. Random binding carriers can exhibit single or multiple Michaelian kinetics in response to changing substrate concentration. These kinetics include high affinity/low velocity and low affinity/high velocity phases (so-called "dual isotherms") which are commonly observed in plant cells. Other combinations of the Michaelis parameters can result in cis-(substrate) inhibition. Despite the generality of the random binding scheme and the complexity of the underlying rate equation, a number of predictive and testable features emerge. If external driver ion concentration is saturating, single Michaelian functions always result and increasing internal substrate concentration causes uncompetitive inhibition of transport. Numerical analysis of the model in conditions thought to resemble those in many experiments demonstrates that small relative differences in a few key component rate constants of the carrier reaction cycle are instrumental in generation of dual isotherms. The random binding model makes the important prediction that the contributions of the two isotherms show opposing dependence on external concentration of driver ion as this approaches saturation. In the one case in which this dependence has been examined experimentally, the model provides a good description of the data. Charge translocation characteristics of the carrier can be determined from steady-state kinetic data on the basis of the response of substrate flux to modulation of internal driver ion concentration.(ABSTRACT TRUNCATED AT 400 WORDS)