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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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Cysteine engineering at sites in ECL-1 and ECL-4 induces deviations from WT voltage dependency. (A) Voltage dependency of G134C (squares), I136C (triangles), F137C (inverted triangles), and M533C (diamonds), before (filled symbols) and after (open symbols) incubation in MTSEA (1 mM, 3 min). Pi-dependent currents, corrected for leak, were normalized to the response at 0 mV. The ordinate scale represents the relative change in IPi as a function of membrane potential. Data pooled from n > 4 cells. WT data is represented by continuous line. (B) Voltage dependency index for all functional mutants given by ratio of response to 1 mM Pi at −100 mV to that at 0 mV (100 mM Na+) before (filled squares) and after (open squares) MTS treatment (1 mM MTSEA, 3 min). Continuous reference line indicates WT index. Dotted reference line indicates that G134C-MTS, S532C ± MTS, and M533C + MTS have the same index. Arrows indicate direction of voltage dependency change for selected mutants after MTS incubation.
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fig7: Cysteine engineering at sites in ECL-1 and ECL-4 induces deviations from WT voltage dependency. (A) Voltage dependency of G134C (squares), I136C (triangles), F137C (inverted triangles), and M533C (diamonds), before (filled symbols) and after (open symbols) incubation in MTSEA (1 mM, 3 min). Pi-dependent currents, corrected for leak, were normalized to the response at 0 mV. The ordinate scale represents the relative change in IPi as a function of membrane potential. Data pooled from n > 4 cells. WT data is represented by continuous line. (B) Voltage dependency index for all functional mutants given by ratio of response to 1 mM Pi at −100 mV to that at 0 mV (100 mM Na+) before (filled squares) and after (open squares) MTS treatment (1 mM MTSEA, 3 min). Continuous reference line indicates WT index. Dotted reference line indicates that G134C-MTS, S532C ± MTS, and M533C + MTS have the same index. Arrows indicate direction of voltage dependency change for selected mutants after MTS incubation.

Mentions: From the I–V data of Fig. 6 A, it was obvious that the effect of cys substitution at Gly-134 and modification of Cys-136, Cys-137C, and Cys-533 was to reduce the transport activity induced by 1 mM Pi over a wide voltage range. By normalizing the leak-corrected I–V data to V = 0, the relative effect of hyperpolarizing membrane potentials on the cotransport activity before and after MTS exposure was determined (Fig. 7 A). These data showed that cys substitution at Ile-136 and Met-533 resulted in constructs with a voltage dependency that was indistinguishable from the WT. Cys modification at these sites increased (I136C + MTS) and decreased (M533 + MTS), respectively, the response to changes in membrane potential in the hyperpolarizing direction relative to the WT. Cys substitution at Phe-137 gave an increased voltage dependency compared with the WT that was further augmented after MTS exposure, like I136C + MTS. Finally, cys substitution at Gly-134 gave a reduced voltage dependency compared with the WT that was comparable to M533C + MTS. After MTS exposure, the voltage dependency of G134C was similar to that of I136C + MTS.


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)

Cysteine engineering at sites in ECL-1 and ECL-4 induces deviations from WT voltage dependency. (A) Voltage dependency of G134C (squares), I136C (triangles), F137C (inverted triangles), and M533C (diamonds), before (filled symbols) and after (open symbols) incubation in MTSEA (1 mM, 3 min). Pi-dependent currents, corrected for leak, were normalized to the response at 0 mV. The ordinate scale represents the relative change in IPi as a function of membrane potential. Data pooled from n > 4 cells. WT data is represented by continuous line. (B) Voltage dependency index for all functional mutants given by ratio of response to 1 mM Pi at −100 mV to that at 0 mV (100 mM Na+) before (filled squares) and after (open squares) MTS treatment (1 mM MTSEA, 3 min). Continuous reference line indicates WT index. Dotted reference line indicates that G134C-MTS, S532C ± MTS, and M533C + MTS have the same index. Arrows indicate direction of voltage dependency change for selected mutants after MTS incubation.
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Related In: Results  -  Collection

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

fig7: Cysteine engineering at sites in ECL-1 and ECL-4 induces deviations from WT voltage dependency. (A) Voltage dependency of G134C (squares), I136C (triangles), F137C (inverted triangles), and M533C (diamonds), before (filled symbols) and after (open symbols) incubation in MTSEA (1 mM, 3 min). Pi-dependent currents, corrected for leak, were normalized to the response at 0 mV. The ordinate scale represents the relative change in IPi as a function of membrane potential. Data pooled from n > 4 cells. WT data is represented by continuous line. (B) Voltage dependency index for all functional mutants given by ratio of response to 1 mM Pi at −100 mV to that at 0 mV (100 mM Na+) before (filled squares) and after (open squares) MTS treatment (1 mM MTSEA, 3 min). Continuous reference line indicates WT index. Dotted reference line indicates that G134C-MTS, S532C ± MTS, and M533C + MTS have the same index. Arrows indicate direction of voltage dependency change for selected mutants after MTS incubation.
Mentions: From the I–V data of Fig. 6 A, it was obvious that the effect of cys substitution at Gly-134 and modification of Cys-136, Cys-137C, and Cys-533 was to reduce the transport activity induced by 1 mM Pi over a wide voltage range. By normalizing the leak-corrected I–V data to V = 0, the relative effect of hyperpolarizing membrane potentials on the cotransport activity before and after MTS exposure was determined (Fig. 7 A). These data showed that cys substitution at Ile-136 and Met-533 resulted in constructs with a voltage dependency that was indistinguishable from the WT. Cys modification at these sites increased (I136C + MTS) and decreased (M533 + MTS), respectively, the response to changes in membrane potential in the hyperpolarizing direction relative to the WT. Cys substitution at Phe-137 gave an increased voltage dependency compared with the WT that was further augmented after MTS exposure, like I136C + MTS. Finally, cys substitution at Gly-134 gave a reduced voltage dependency compared with the WT that was comparable to M533C + MTS. After MTS exposure, the voltage dependency of G134C was similar to that of I136C + MTS.

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