Structure–Function Relations of the First and Fourth Extracellular Linkers of the Type IIa Na /P i Cotransporter: II. Substrate Interaction and Voltage Dependency of Two Functionally Important Sites

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 steadystate 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 Cys533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages. DOI: https://doi.org/10.1085/jgp.200409061 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-1302 Published Version Originally published at: Ehnes, C; Forster, I C; Bacconi, A; Kohler, K; Biber, J; Murer, H (2004). Structure-function relations of the first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter: II. Substrate interaction and voltage dependency of two functionally important sites. Journal of General Physiology, 124(5):489503. DOI: https://doi.org/10.1085/jgp.200409061 T h e Jo u rn al o f G en er al P h ys io lo g y 489 J. Gen. Physiol. © The Rockefeller University Press • 0022-1295/2004/11/489/15 $8.00 Volume 124 November 2004 489–503 http://www.jgp.org/cgi/doi/10.1085/jgp.200409061 Structure–Function Relations of the First and Fourth Extracellular Linkers of the Type IIa Na /P i Cotransporter: II. Substrate Interaction and Voltage Dependency of Two Functionally Important Sites Colin Ehnes, Ian C. Forster, Andrea Bacconi, Katja Kohler, Jürg Biber, and Heini Murer Institute of Physiology, University of Zurich, CH-8057 Zurich, Switzerland abstract Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na /P i 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 P i , 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 P i 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 P i , which suggested that voltagedependent 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.Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na /P i 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 P i , 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 P i 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 P i , which suggested that voltagedependent 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. key words: phosphate transport proteins • mutagenesis site directed • cysteine • electrophysiology • transport model I N T R O D U C T I O N Structure–function studies on membrane transport proteins are used to identify functionally important sites and confirm or establish secondary topological features. The application of the substituted cysteine accessibility method (SCAM) to the renal type IIa Na /P i cotransporter (NaPi-IIa) has enabled us to identify functionally important sites that may constitute part of the transport pathway through the protein (Lambert et al., 2001; Kohler et al., 2002a,b). This involves the first intracellular loop (ICL-1) in the NH 2 -terminal half of the protein and the third extracellular loop located in the COOHterminal half of the protein. The modification of cysteines substituted at these sites in almost all cases led to complete block of the cotransport mode, and in some cases increased the leak mode activity (Kohler et al., 2002b). To continue our study of the functional contribution of predicted transmembrane domain linker regions of NaPi-IIa, we have applied SCAM to the two putative extracellular loops ECL-1 and ECL-4 (Ehnes et al., 2004). Cys substitution at each of nine sites in ECL-1 and six sites in ECL-4 was well tolerated, and 32 P i uptake and electrogenic behavior were documented for these mutants (Ehnes et al., 2004). In contrast to our previous studies (Lambert et al., 1999, 2001; Kohler et al., 2002a,b), at the completion of the methanethiosulfonate (MTS) modification reaction, these mutants still exhibited a finite cotransport activity that we attributed to altered steady-state voltage-dependent kinetics (Ehnes et al., 2004). Two mutants were selected for more detailed investigation in this study because their voltage dependency changed in a reciprocal manner depending on whether the novel cysteine was modified or not. C. Ehnes and I.C. Forster contributed equally to this work. Address correspondence to Ian C. Forster, Physiologisches Institut, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Fax: 41-1-635 5715; email: [email protected] K. Kohler’s present address is Laboratory of Morphogenesis and Cell Signaling, UMR144, Institut Curie, Paris, France. Abbreviations used in this paper: MTS, methanethiosulfonate; MTSEA, 2-aminoethyl MTS hydrobromide; NaPi-IIa, type IIa Na /P i cotransporter; WT, wild type. on A uust 2, 2006 w w w .jg.org D ow nladed fom