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Structure-function relations of the first and fourth predicted extracellular linkers of the type IIa Na+/Pi cotransporter: I. Cysteine scanning mutagenesis.

Ehnes C, Forster IC, Kohler K, Bacconi A, Stange G, Biber J, Murer H - J. Gen. Physiol. (2004)

Bottom Line: For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V < or = -80 mV, whereas the WT exhibited a linear voltage dependency.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.

View Article: PubMed Central - PubMed

Affiliation: Physiologisches Institut, Universität Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

ABSTRACT
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 < or = -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.

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(A) Topological representation of the rat type IIa Na+/Pi cotransporter comprises a backbone of eight putative membrane-spanning domains (TMD-1 to -8) and corresponding linker regions. A functionally essential cysteine bridge is formed between Cys-306 and Cys-334 in the large second extracellular linker (ECL-2) and an additional bridge between Cys-225 and either Cys-520 or Cys-597 has also been recently proposed (Kohler et al., 2003). The cluster of functionally important (MTS-accessible) sites previously identified by SCAM in the third extracellular linker (ECL-3) (Lambert et al., 2001) and two sites in the first intracellular linker (ICL-1) (Kohler et al., 2002a) are indicated (filled squares). Sites 130–140 in the putative first extracellular linker (ECL-1) and 532–538 in the predicted fourth extracellular linker (ECL-4) were mutated to cysteines in this study (gray-filled squares) (B) Comparison of the amino acid sequences for the predicted transmembrane domains flanking ECL-1 (TMD-1 and -2) and ECL-4 (TMD-7 and -8) show a high degree of homology between different isoforms of the type II Na+/Pi cotransporter family (SLC34). Bold lettered amino acids in ECL-1 and ECL-4 for the rat isoform were mutated to cysteines for the present study. Amino acids are named using the single letter code.
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fig1: (A) Topological representation of the rat type IIa Na+/Pi cotransporter comprises a backbone of eight putative membrane-spanning domains (TMD-1 to -8) and corresponding linker regions. A functionally essential cysteine bridge is formed between Cys-306 and Cys-334 in the large second extracellular linker (ECL-2) and an additional bridge between Cys-225 and either Cys-520 or Cys-597 has also been recently proposed (Kohler et al., 2003). The cluster of functionally important (MTS-accessible) sites previously identified by SCAM in the third extracellular linker (ECL-3) (Lambert et al., 2001) and two sites in the first intracellular linker (ICL-1) (Kohler et al., 2002a) are indicated (filled squares). Sites 130–140 in the putative first extracellular linker (ECL-1) and 532–538 in the predicted fourth extracellular linker (ECL-4) were mutated to cysteines in this study (gray-filled squares) (B) Comparison of the amino acid sequences for the predicted transmembrane domains flanking ECL-1 (TMD-1 and -2) and ECL-4 (TMD-7 and -8) show a high degree of homology between different isoforms of the type II Na+/Pi cotransporter family (SLC34). Bold lettered amino acids in ECL-1 and ECL-4 for the rat isoform were mutated to cysteines for the present study. Amino acids are named using the single letter code.

Mentions: The predicted topology of NaPi-IIa, derived from studies on the rat isoform, suggests a protein with eight transmembrane domains, a large extracellular loop with two N-glycosylation sites and intracellular NH2 and COOH termini (Magagnin et al., 1993; Murer et al., 2000) (Fig. 1 A). Antibody accessibility studies (Lambert et al., 1999b) and cysteine scanning approaches (Lambert et al., 1999a, 2000; Kohler et al., 2002a,b) also support this model. Using SCAM we have identified sites in the putative third extracellular linker (ECL-3) (Lambert et al., 1999a, 2001) and the putative first intracellular linker (ICL-1) (Kohler et al., 2002a,b) that when modified by methanethiosulfonate (MTS) reagents result in block of cotransport mode activity. Given that NaPi-IIa is a functional monomer (Kohler et al., 2000), and taking into account our previous SCAM findings, we currently postulate that these two topologically opposed linker regions associate with one another to constitute the transport pathway (Kohler et al., 2002a,b). To examine if other external linker regions may also define NaPi-IIa transport properties, we performed SCAM on sites in the first and fourth predicted linker regions, designated ECL-1 and ECL-4, respectively (Fig. 1 A). As shown in Fig. 1 B, these regions are very well conserved among several isoforms of the SLC34A family and therefore we might expect them to play common functional roles.


Structure-function relations of the first and fourth predicted extracellular linkers of the type IIa Na+/Pi cotransporter: I. Cysteine scanning mutagenesis.

Ehnes C, Forster IC, Kohler K, Bacconi A, Stange G, Biber J, Murer H - J. Gen. Physiol. (2004)

(A) Topological representation of the rat type IIa Na+/Pi cotransporter comprises a backbone of eight putative membrane-spanning domains (TMD-1 to -8) and corresponding linker regions. A functionally essential cysteine bridge is formed between Cys-306 and Cys-334 in the large second extracellular linker (ECL-2) and an additional bridge between Cys-225 and either Cys-520 or Cys-597 has also been recently proposed (Kohler et al., 2003). The cluster of functionally important (MTS-accessible) sites previously identified by SCAM in the third extracellular linker (ECL-3) (Lambert et al., 2001) and two sites in the first intracellular linker (ICL-1) (Kohler et al., 2002a) are indicated (filled squares). Sites 130–140 in the putative first extracellular linker (ECL-1) and 532–538 in the predicted fourth extracellular linker (ECL-4) were mutated to cysteines in this study (gray-filled squares) (B) Comparison of the amino acid sequences for the predicted transmembrane domains flanking ECL-1 (TMD-1 and -2) and ECL-4 (TMD-7 and -8) show a high degree of homology between different isoforms of the type II Na+/Pi cotransporter family (SLC34). Bold lettered amino acids in ECL-1 and ECL-4 for the rat isoform were mutated to cysteines for the present study. Amino acids are named using the single letter code.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233999&req=5

fig1: (A) Topological representation of the rat type IIa Na+/Pi cotransporter comprises a backbone of eight putative membrane-spanning domains (TMD-1 to -8) and corresponding linker regions. A functionally essential cysteine bridge is formed between Cys-306 and Cys-334 in the large second extracellular linker (ECL-2) and an additional bridge between Cys-225 and either Cys-520 or Cys-597 has also been recently proposed (Kohler et al., 2003). The cluster of functionally important (MTS-accessible) sites previously identified by SCAM in the third extracellular linker (ECL-3) (Lambert et al., 2001) and two sites in the first intracellular linker (ICL-1) (Kohler et al., 2002a) are indicated (filled squares). Sites 130–140 in the putative first extracellular linker (ECL-1) and 532–538 in the predicted fourth extracellular linker (ECL-4) were mutated to cysteines in this study (gray-filled squares) (B) Comparison of the amino acid sequences for the predicted transmembrane domains flanking ECL-1 (TMD-1 and -2) and ECL-4 (TMD-7 and -8) show a high degree of homology between different isoforms of the type II Na+/Pi cotransporter family (SLC34). Bold lettered amino acids in ECL-1 and ECL-4 for the rat isoform were mutated to cysteines for the present study. Amino acids are named using the single letter code.
Mentions: The predicted topology of NaPi-IIa, derived from studies on the rat isoform, suggests a protein with eight transmembrane domains, a large extracellular loop with two N-glycosylation sites and intracellular NH2 and COOH termini (Magagnin et al., 1993; Murer et al., 2000) (Fig. 1 A). Antibody accessibility studies (Lambert et al., 1999b) and cysteine scanning approaches (Lambert et al., 1999a, 2000; Kohler et al., 2002a,b) also support this model. Using SCAM we have identified sites in the putative third extracellular linker (ECL-3) (Lambert et al., 1999a, 2001) and the putative first intracellular linker (ICL-1) (Kohler et al., 2002a,b) that when modified by methanethiosulfonate (MTS) reagents result in block of cotransport mode activity. Given that NaPi-IIa is a functional monomer (Kohler et al., 2000), and taking into account our previous SCAM findings, we currently postulate that these two topologically opposed linker regions associate with one another to constitute the transport pathway (Kohler et al., 2002a,b). To examine if other external linker regions may also define NaPi-IIa transport properties, we performed SCAM on sites in the first and fourth predicted linker regions, designated ECL-1 and ECL-4, respectively (Fig. 1 A). As shown in Fig. 1 B, these regions are very well conserved among several isoforms of the SLC34A family and therefore we might expect them to play common functional roles.

Bottom Line: For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V < or = -80 mV, whereas the WT exhibited a linear voltage dependency.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.

View Article: PubMed Central - PubMed

Affiliation: Physiologisches Institut, Universität Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

ABSTRACT
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 < or = -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.

Show MeSH
Related in: MedlinePlus