Limits...
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.

Show MeSH

Related in: MedlinePlus

Graphical representation of accessibility of three putative extracellular linkers based on effective second order reaction rate k* plotted on a logarithmic scale (filled squares). A larger k* indicates that the site has a greater accessibility from the external aqueous medium. Sites that were labeled with MTSEA-Biotin, for which the respective mutants showed no detectable change in activity, are indicated (empty squares). Data for ECL-1 and ECL-4 were obtained from this study (Table I); data for ECL-3 were replotted from a previous study (Lambert et al., 2001).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233999&req=5

fig8: Graphical representation of accessibility of three putative extracellular linkers based on effective second order reaction rate k* plotted on a logarithmic scale (filled squares). A larger k* indicates that the site has a greater accessibility from the external aqueous medium. Sites that were labeled with MTSEA-Biotin, for which the respective mutants showed no detectable change in activity, are indicated (empty squares). Data for ECL-1 and ECL-4 were obtained from this study (Table I); data for ECL-3 were replotted from a previous study (Lambert et al., 2001).

Mentions: Although we used MTSEA as the standard probe reagent, exposure to impermeant MTSET gave qualitatively similar results. This confirmed that the sites were accessible from the external medium only. Small differences in reactivity between MTSEA and MTSET were observed in the rates of modification (i.e., the rate of change of the electrogenic activity) at different sites in ECL-1 and ECL- 4 (Table I). The faster reaction rates of MTSET for most mutants most likely reflect the higher reactivity of this reagent with small thiols compared with MTSEA (Karlin and Akabas, 1998). For G134C and A538C, MTSEA was more reactive than MTSET, perhaps because the greater bulk of MTSET restricts its access to these sites. As summarized in Fig. 8, the effective second order reaction rate constants lay within the range of values that we have previously reported for Cys mutants in ECL-3 (Lambert et al., 2001) and establish conclusively that these three regions are accessible from the external aqueous environment. Rates of modification for extracellularly accessible sites in other transport proteins such as the excitatory amino acid transporter (EAAT1) (e.g., Leighton et al., 2002), the mitochondrial citrate transporter (Ma et al., 2004), and the serotonin transporter (SERT) (Chen et al., 1997) have been reported to lie in the same range as we found for NaPi-IIa. In the present study, the modification rates for mutants in ECL-1 were consistently faster than those in ECL-4. This would suggest that ECL-4 was less accessible from the extracellular medium. At one site in ECL-1 (Asp-135) and one site in ECL-4 (Trp-536) we were unable to detect the novel cysteines using the biotinylation assay, which indicated that these cysteines were inaccessible, either because of the bulk of MTSEA-Biotin or the specific folding of the linkers at these sites.


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)

Graphical representation of accessibility of three putative extracellular linkers based on effective second order reaction rate k* plotted on a logarithmic scale (filled squares). A larger k* indicates that the site has a greater accessibility from the external aqueous medium. Sites that were labeled with MTSEA-Biotin, for which the respective mutants showed no detectable change in activity, are indicated (empty squares). Data for ECL-1 and ECL-4 were obtained from this study (Table I); data for ECL-3 were replotted from a previous study (Lambert et al., 2001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233999&req=5

fig8: Graphical representation of accessibility of three putative extracellular linkers based on effective second order reaction rate k* plotted on a logarithmic scale (filled squares). A larger k* indicates that the site has a greater accessibility from the external aqueous medium. Sites that were labeled with MTSEA-Biotin, for which the respective mutants showed no detectable change in activity, are indicated (empty squares). Data for ECL-1 and ECL-4 were obtained from this study (Table I); data for ECL-3 were replotted from a previous study (Lambert et al., 2001).
Mentions: Although we used MTSEA as the standard probe reagent, exposure to impermeant MTSET gave qualitatively similar results. This confirmed that the sites were accessible from the external medium only. Small differences in reactivity between MTSEA and MTSET were observed in the rates of modification (i.e., the rate of change of the electrogenic activity) at different sites in ECL-1 and ECL- 4 (Table I). The faster reaction rates of MTSET for most mutants most likely reflect the higher reactivity of this reagent with small thiols compared with MTSEA (Karlin and Akabas, 1998). For G134C and A538C, MTSEA was more reactive than MTSET, perhaps because the greater bulk of MTSET restricts its access to these sites. As summarized in Fig. 8, the effective second order reaction rate constants lay within the range of values that we have previously reported for Cys mutants in ECL-3 (Lambert et al., 2001) and establish conclusively that these three regions are accessible from the external aqueous environment. Rates of modification for extracellularly accessible sites in other transport proteins such as the excitatory amino acid transporter (EAAT1) (e.g., Leighton et al., 2002), the mitochondrial citrate transporter (Ma et al., 2004), and the serotonin transporter (SERT) (Chen et al., 1997) have been reported to lie in the same range as we found for NaPi-IIa. In the present study, the modification rates for mutants in ECL-1 were consistently faster than those in ECL-4. This would suggest that ECL-4 was less accessible from the extracellular medium. At one site in ECL-1 (Asp-135) and one site in ECL-4 (Trp-536) we were unable to detect the novel cysteines using the biotinylation assay, which indicated that these cysteines were inaccessible, either because of the bulk of MTSEA-Biotin or the specific folding of the linkers at these sites.

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