scholarly journals Structure–Function Relations of the First and Fourth Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 489-503 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Andrea Bacconi ◽  
Katja Kohler ◽  
Jürg Biber ◽  
...  
Keyword(s):  
Steady State ◽  
Conformational Changes ◽  
Underlying Mechanism ◽  
Transport Kinetics ◽  
State Behavior ◽  
Voltage Dependent ◽  
Cysteine Scanning Mutagenesis ◽  
Charge Movements ◽  
Kinetics Of ◽  
State Kinetic

Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

scholarly journals Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 475-488 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Katja Kohler ◽  
Andrea Bacconi ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Reaction Rates ◽  
Transport Pathway ◽  
Extracellular Medium ◽  
Voltage Range ◽  
Substituted Cysteine Accessibility ◽  
Voltage Dependent ◽  
Opposite Behavior ◽  
Rate Limiting ◽  
Kinetics Of

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

scholarly journals Kinetics of Steady-state Currents and Charge Movements Associated with the Rat Na+/Glucose Cotransporter

1995 ◽  
Vol 270 (45) ◽  
pp. 27099-27105 ◽  
Author(s):  
Mariana Panayotova-Heiermann ◽  
Donald D. F. Loo ◽  
Ernest M. Wright
Keyword(s):  
Steady State ◽  
Charge Movements ◽  
Kinetics Of

scholarly journals Modulation of voltage-dependent sodium and potassium currents by charged amphiphiles in cardiac ventricular myocytes. Effects via modification of surface potential.

1993 ◽  
Vol 101 (3) ◽  
pp. 355-375 ◽  
Author(s):  
S Ji ◽  
J N Weiss ◽  
G A Langer
Keyword(s):  
Steady State ◽  
Surface Potential ◽  
Ventricular Myocytes ◽  
Potassium Currents ◽  
Surface Charges ◽  
Negative Direction ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
Sodium And Potassium ◽  
Cardiac Ventricular Myocytes

Modulation of voltage-dependent sodium and potassium currents by charged amphiphiles was investigated in cardiac ventricular myocytes using the patch-clamp technique. Negatively charged sodium dodecylsulfate (SDS) increased amplitude of INa, whereas positively charged dodecyltrimethylammonium (DDTMA) decreased INa. Furthermore, SDS shifted the steady-state activation and inactivation of INa in the negative direction, whereas DDTMA shifted the curves in the opposite direction. These shifts provided an explanation for the changes in current amplitude. Activation and inactivation kinetics of INa were accelerated by SDS but slowed by DDTMA. These changes in both steady-state gating and kinetics of INa are consistent with a decrease of the intramembrane field by SDS and an increase of the field by DDTMA due to an alteration of surface potential after their insertion into the outer monolayer of the sarcolemma. The effect of SDS on the steady-state inactivation of INa was concentration dependent and partially reversed by screening surface charges with increased extracellular [Ca2+]. These amphiphiles also altered the activation of the delayed rectifier K+ current (IK,del), producing a shift in the negative direction by SDS but in the positive direction by DDTMA. These results suggest that the insertion of charged amphiphiles into the cell membrane alters the behavior of voltage-dependent INa and IK,del by changing the surface charge density, and consequently the surface potential and implies, although indirectly, that the lipid surface charges are important to the voltage-dependent gating of these channels.

Nonlinear steady-state kinetics of chloramphenicol acetyltransferase

1991 ◽  
Vol 69 (9) ◽  
pp. 630-634
Author(s):  
M. James C. Crabbe ◽  
Derek Goode
Keyword(s):  
Steady State ◽  
Chloramphenicol Acetyltransferase ◽  
Binding Region ◽  
Medium Effects ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Kinetics Of ◽  
State Kinetic

Steady-state kinetic analysis of chloramphenicol acetyltransferase showed that medium effects (higher temperatures or pH, higher ionic strengths, or lower values for dielectric constant) altered the kinetic behaviour of the enzyme with acetyl-CoA as substrate, but did not significantly affect behaviour with chloramphenicol. This was manifest as an increase in the degree of the rate equation to a 2:2 function. This is interpreted in terms of perturbations to the enzyme at or near the acetyl-CoA binding region of the enzyme.Key words: acetyl coenzyme A, chloramphenicol, antibiotics, enzyme kinetics.

scholarly journals On the mechanism of the amiloride-sodium entry site interaction in anuran skin epithelia.

1979 ◽  
Vol 73 (3) ◽  
pp. 307-326 ◽  
Author(s):  
D J Benos ◽  
L J Mandel ◽  
R S Balaban
Keyword(s):  
Steady State ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Transport Kinetics ◽  
Entry Site ◽  
Interaction Sites ◽  
Noncompetitive Inhibitor ◽  
Kinetics Of ◽  
Entry Mechanism

The steady-state transport kinetics of the interaction between external sodium and the diuretic drug, amiloride, was studied in isolated anuran skin epithelia. We also investigated the effect of calcium on the amiloride-induced inhibition of short-circuit current (Isc) in these epithelial preparations. The major conclusions of this study are: (a) amiloride is a noncompetitive inhibitor of Na entry in bullfrog and grassfrog skin, but displays mixed inhibition in R. temporaria and the toad. A hypothesis which states that the interaction sites for amiloride and Na on the putative entry protein are spatially distinct in all of these species is proposed. (b) The stoichiometry of interaction between amiloride and the Na entry mechanism is not necessarily one-to-one. (c) The external Ca requirement for the inhibitory effect of amiloride is not absolute. Amiloride, at all concentrations, is equally effective in inhibiting Isc of bullfrog skin independently from the presence or absence of external Ca.

Substrate interactions in the human type IIa sodium-phosphate cotransporter (NaPi-IIa)

2005 ◽  
Vol 288 (5) ◽  
pp. F969-F981 ◽  
Author(s):  
Leila V. Virkki ◽  
Ian C. Forster ◽  
Jürg Biber ◽  
Heini Murer
Keyword(s):  
Steady State ◽  
Sodium Phosphate ◽  
Complete Removal ◽  
Xenopus Laevis Oocytes ◽  
Charge Movement ◽  
Steady State Current ◽  
Transport Cycle ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
State Charge

We have characterized the kinetics of substrate transport in the renal type IIa human sodium-phosphate cotransporter (NaPi-IIa). The transporter was expressed in Xenopus laevis oocytes, and steady-state and pre-steady-state currents and substrate uptakes were characterized by voltage-clamp and isotope flux. First, by measuring simultaneous uptake of a substrate (32Pi, 22Na) and charge in voltage-clamped oocytes, we established that the human NaPi-IIa isoform operates with a Na:Pi:charge stoichiometry of 3:1:1 and that the preferred transported Pi species is HPO42−. We then probed the complex interrelationship of substrates, pH, and voltage in the NaPi-IIa transport cycle by analyzing both steady-state and pre-steady-state currents. Steady-state current measurements show that the apparent HPO42− affinity is voltage dependent and that this voltage dependency is abrogated by lowering the pH or the Na+ concentration. In contrast, the voltage dependency of the apparent Na+ affinity increased when pH was lowered. Pre-steady-state current analysis shows that Na+ ions bind first and influence the preferred orientation of the transporter in the absence of Pi. Pre-steady-state charge movement was partially suppressed by complete removal of Na+ from the bath, by reducing extracellular pH (both in the presence and absence of Na+), or by adding Pi (in the presence of 100 mM Na). None of these conditions suppressed charge movement completely. The results allowed us to modify previous models for the transport cycle of NaPi-II transporters by including voltage dependency of HPO42− binding and proton modulation of the first Na+ binding step.

Using lithium to probe sequential cation interactions with GAT1

2012 ◽  
Vol 302 (11) ◽  
pp. C1661-C1675 ◽  
Author(s):  
Anne-Kristine Meinild ◽  
Ian C. Forster
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Voltage Dependence ◽  
Cation Binding ◽  
Experimental Conditions ◽  
Net Charge ◽  
Cation Binding Site ◽  
Voltage Dependent ◽  
Experimental Findings ◽  
Kinetics Of

Li+ interacts with the Na+/Cl−-dependent GABA transporter, GAT1, under two conditions: in the absence of Na+ it induces a voltage-dependent leak current; in the presence of Na+ and GABA, Li+ stimulates GABA-induced steady-state currents. The amino acids directly involved in the interaction with the Na+ and Li+ ions at the so-called “ Na2” binding site have been identified, but how Li+ affects the kinetics of GABA cotransport has not been fully explored. We expressed GAT1 in Xenopus oocytes and applied the two-electrode voltage clamp and 22Na uptake assays to determine coupling ratios and steady-state and presteady-state kinetics under experimental conditions in which extracellular Na+ was partially substituted by Li+. Three novel findings are: 1) Li+ reduced the coupling ratio between Na+ and net charge translocated during GABA cotransport; 2) Li+ increased the apparent Na+ affinity without changing its voltage dependence; 3) Li+ altered the voltage dependence of presteady-state relaxations in the absence of GABA. We propose an ordered binding scheme for cotransport in which either a Na+ or Li+ ion can bind at the putative first cation binding site ( Na2). This is followed by the cooperative binding of the second Na+ ion at the second cation binding site ( Na1) and then binding of GABA. With Li+ bound to Na2, the second Na+ ion binds more readily GAT1, and despite a lower apparent GABA affinity, the translocation rate of the fully loaded carrier is not reduced. Numerical simulations using a nonrapid equilibrium model fully recapitulated our experimental findings.

scholarly journals Voltage Clamp Fluorometric Measurements on a Type II Na+-coupled Pi Cotransporter: Shedding Light on Substrate Binding Order

2006 ◽  
Vol 127 (5) ◽  
pp. 539-555 ◽  
Author(s):  
Leila V. Virkki ◽  
Heini Murer ◽  
Ian C. Forster
Keyword(s):  
Voltage Clamp ◽  
Conformational Changes ◽  
Substrate Binding ◽  
Type Ii ◽  
New Model ◽  
Fluorescence Measurements ◽  
Voltage Dependent ◽  
Electrophysiological Studies ◽  
Ion Substitution ◽  
Charge Movements

Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637–651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (ΔF), which suggested that this site lies in an environment that is affected by conformational change in the protein. ΔF was substrate dependent (no ΔF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1–18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes.

scholarly journals Interpretation of the Kinetics of Consecutive Enzyme-catalysed Reactions: Studies on the Arginase-Ornithine Carbamoyltransferase System

1982 ◽  
Vol 35 (2) ◽  
pp. 137 ◽  
Author(s):  
RD Teasdale ◽  
PD Jeffrey ◽  
PW Kuchel ◽  
LW Nichol
Keyword(s):  
Steady State ◽  
Ornithine Carbamoyltransferase ◽  
Enzyme Substrate ◽  
Steady State Kinetic ◽  
Kinetic Mechanisms ◽  
Heterogeneous Association ◽  
Kinetics Of ◽  
State Kinetic ◽  
Enzymic Assay

A method that permits the use of measurements on the concentration of the intermediate in a coupled enzymic assay in determining the presence or absence of an interaction between the enzymes is presented. The method is shown to be closely analogous to a previously formulated procedure involving the determination of the rate of production of the final product of such a sequence and is shown to be applicable regardless of the complexity of the operative kinetic mechanisms, provided it may be assumed that all enzyme-substrate complexes are in the steady-state. Kinetic results obtained with the arginase--ornithine carbamoyltransferase couple, in which the intermediate ornithine is monitored, are examined in these terms to conclude that no heterogeneous association is operative between the enzymes.

scholarly journals Slow charge movement in mammalian skeletal muscle.

1985 ◽  
Vol 85 (1) ◽  
pp. 1-19 ◽  
Author(s):  
B J Simon ◽  
K G Beam
Keyword(s):  
Steady State ◽  
State Dependence ◽  
Charge Movement ◽  
Mammalian Skeletal Muscle ◽  
Maximum Charge ◽  
A Value ◽  
Voltage Dependent ◽  
Charge Movements ◽  
State Charge ◽  
Omohyoid Muscle

Voltage-dependent charge movements were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Contraction was abolished with hypertonic sucrose. The standard (ON-OFF) protocol for eliciting charge movements was to depolarize the fiber from -90 mV to a variable test potential (V) and then repolarize the fiber to -90 mV. The quantity of charge moved saturated at test potentials of approximately 0 mV. The steady state dependence of the amount of charge that moves as a function of test potential could be well fitted by the Boltzmann relation: Q = Qmax/(1 + exp[-(V - V)/k]), where Qmax is the maximum charge that can be moved, V is the potential at which half the charge moves, and k is a constant. At 15 degrees C, these values were Qmax = 28.5 nC/microF, V = -34.2 mV, and k = 8.7 mV. Qmax, k, and V exhibited little temperature dependence over the range 7-25 degrees C. "Stepped OFF" charge movements were elicited by depolarizing the fiber from -90 mV to a fixed conditioning level that moved nearly all the mobile charge (0 mV), and then repolarizing the fiber to varying test potentials. The sum of the charge that moved when the fiber was depolarized directly from -90 mV to a given test potential and the stepped OFF charge that moved when the fiber was repolarized to the same test potential had at all test potentials a value close to Qmax for that fiber. In nearly all cases, the decay phase of ON, OFF, and stepped OFF charge movements could be well fitted with a single exponential. The time constant, tau decay, for an ON charge movement at a given test potential was comparable to tau decay for a stepped OFF charge movement at the same test potential. Tau decay had a bell-shaped dependence on membrane potential: it was slowest at a potential near V (the midpoint of the steady state charge distribution) and became symmetrically faster on either side of this potential. Raising the temperature from 7 to 15 degrees C caused tau decay to become faster by about the same proportion at all potentials, with a Q10 averaging 2.16. Raising the temperature from 15 to 25 degrees C caused tau decay to become faster at potentials near V, but not at potentials farther away.(ABSTRACT TRUNCATED AT 400 WORDS)

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