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Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.

Lambert G, Forster IC, Stange G, Biber J, Murer H - J. Gen. Physiol. (1999)

Bottom Line: Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein.Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway.Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.

View Article: PubMed Central - PubMed

Affiliation: Institute for Physiology, University of Zürich, CH-8057 Zürich, Switzerland.

ABSTRACT
The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P(i) transport. Alkylation in 100 mM Na(+) by either cationic ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA) or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P(i) response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P(i) suppressed the slippage current equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.

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Kinetic Scheme for Type II Na+/Pi Cotransport. States occupied in the cis (outward facing) orientation are 1–5. States occupied in the trans (inward facing) orientation are 6–10. The three modes of operation that involve transmembrane reorientation, as revealed by steady state and pre–steady state studies of WT, are indicated by dark shading: empty carrier (10 ⇔ 1), slippage (2 ⇔ 9), and cotransport (5 ⇔ 6). At least two voltage-dependent transitions have been identified: empty carrier (10 ⇔ 1) and first external Na+ binding/release step (1 ⇔ 2). The voltage dependence of the last Na+ binding/release step on the trans side (9 ⇔ 10) has not been characterized. PFA binding places the system in state 2*, which when occupied prevents the slippage and cotransport modes. The lightly shaded region indicates those transitions and associated states that have been shown to remain intact after alkylation. In addition, the zero voltage rate constants for the empty carrier mode increase after alkylation (see text).
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Figure 9: Kinetic Scheme for Type II Na+/Pi Cotransport. States occupied in the cis (outward facing) orientation are 1–5. States occupied in the trans (inward facing) orientation are 6–10. The three modes of operation that involve transmembrane reorientation, as revealed by steady state and pre–steady state studies of WT, are indicated by dark shading: empty carrier (10 ⇔ 1), slippage (2 ⇔ 9), and cotransport (5 ⇔ 6). At least two voltage-dependent transitions have been identified: empty carrier (10 ⇔ 1) and first external Na+ binding/release step (1 ⇔ 2). The voltage dependence of the last Na+ binding/release step on the trans side (9 ⇔ 10) has not been characterized. PFA binding places the system in state 2*, which when occupied prevents the slippage and cotransport modes. The lightly shaded region indicates those transitions and associated states that have been shown to remain intact after alkylation. In addition, the zero voltage rate constants for the empty carrier mode increase after alkylation (see text).

Mentions: The currently proposed kinetic scheme for type II Na+/Pi cotransport (see Fig. 9) predicts that a voltage step will induce pre–steady state relaxations, contributed by the empty carrier and Na+ binding/debinding, before reaching the final steady state in the slippage mode. Since this mode appeared to be unchanged by alkylation, we would still expect pre–steady state charge movements to be detectable after alkylation. Fig. 7 shows pre–steady state relaxations recorded from a representative oocyte for voltage steps to five test potentials in the presence (A) and absence (B) of external Na+. As before, the endogenous capacitive charging transient was removed by subtracting the response to 3 mM PFA in ND100. Although MTSEA treatment resulted in a significant apparent suppression of relaxations, there was still a charge movement detectable after the endogenous membrane charging was complete (typically after 1–1.5 ms). Moreover, the relaxations were significantly faster in ND0 solution, which indicated that alkylation had altered the kinetics of the empty carrier. The available signal resolution and low expression levels (steady state currents induced by 1 mM Pi were typically 100–150 nA) prevented a full analysis of these relaxations. Nevertheless, single exponential fitting of relaxations induced by large voltage steps indicated that, in 0 mM Na+, alkylation led to an approximately eightfold faster time constant, as shown in Table for three test potentials. Since the relaxations recorded from S460C-expressing oocytes after MTSEA treatment were comparable with the speed of membrane charging, we also confirmed that, under the same perfusion conditions, significant charge movements could not be detected from noninjected oocytes from the same batch once the main capacitive charging was complete.


Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.

Lambert G, Forster IC, Stange G, Biber J, Murer H - J. Gen. Physiol. (1999)

Kinetic Scheme for Type II Na+/Pi Cotransport. States occupied in the cis (outward facing) orientation are 1–5. States occupied in the trans (inward facing) orientation are 6–10. The three modes of operation that involve transmembrane reorientation, as revealed by steady state and pre–steady state studies of WT, are indicated by dark shading: empty carrier (10 ⇔ 1), slippage (2 ⇔ 9), and cotransport (5 ⇔ 6). At least two voltage-dependent transitions have been identified: empty carrier (10 ⇔ 1) and first external Na+ binding/release step (1 ⇔ 2). The voltage dependence of the last Na+ binding/release step on the trans side (9 ⇔ 10) has not been characterized. PFA binding places the system in state 2*, which when occupied prevents the slippage and cotransport modes. The lightly shaded region indicates those transitions and associated states that have been shown to remain intact after alkylation. In addition, the zero voltage rate constants for the empty carrier mode increase after alkylation (see text).
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Related In: Results  -  Collection

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Figure 9: Kinetic Scheme for Type II Na+/Pi Cotransport. States occupied in the cis (outward facing) orientation are 1–5. States occupied in the trans (inward facing) orientation are 6–10. The three modes of operation that involve transmembrane reorientation, as revealed by steady state and pre–steady state studies of WT, are indicated by dark shading: empty carrier (10 ⇔ 1), slippage (2 ⇔ 9), and cotransport (5 ⇔ 6). At least two voltage-dependent transitions have been identified: empty carrier (10 ⇔ 1) and first external Na+ binding/release step (1 ⇔ 2). The voltage dependence of the last Na+ binding/release step on the trans side (9 ⇔ 10) has not been characterized. PFA binding places the system in state 2*, which when occupied prevents the slippage and cotransport modes. The lightly shaded region indicates those transitions and associated states that have been shown to remain intact after alkylation. In addition, the zero voltage rate constants for the empty carrier mode increase after alkylation (see text).
Mentions: The currently proposed kinetic scheme for type II Na+/Pi cotransport (see Fig. 9) predicts that a voltage step will induce pre–steady state relaxations, contributed by the empty carrier and Na+ binding/debinding, before reaching the final steady state in the slippage mode. Since this mode appeared to be unchanged by alkylation, we would still expect pre–steady state charge movements to be detectable after alkylation. Fig. 7 shows pre–steady state relaxations recorded from a representative oocyte for voltage steps to five test potentials in the presence (A) and absence (B) of external Na+. As before, the endogenous capacitive charging transient was removed by subtracting the response to 3 mM PFA in ND100. Although MTSEA treatment resulted in a significant apparent suppression of relaxations, there was still a charge movement detectable after the endogenous membrane charging was complete (typically after 1–1.5 ms). Moreover, the relaxations were significantly faster in ND0 solution, which indicated that alkylation had altered the kinetics of the empty carrier. The available signal resolution and low expression levels (steady state currents induced by 1 mM Pi were typically 100–150 nA) prevented a full analysis of these relaxations. Nevertheless, single exponential fitting of relaxations induced by large voltage steps indicated that, in 0 mM Na+, alkylation led to an approximately eightfold faster time constant, as shown in Table for three test potentials. Since the relaxations recorded from S460C-expressing oocytes after MTSEA treatment were comparable with the speed of membrane charging, we also confirmed that, under the same perfusion conditions, significant charge movements could not be detected from noninjected oocytes from the same batch once the main capacitive charging was complete.

Bottom Line: Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein.Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway.Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.

View Article: PubMed Central - PubMed

Affiliation: Institute for Physiology, University of Zürich, CH-8057 Zürich, Switzerland.

ABSTRACT
The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P(i) transport. Alkylation in 100 mM Na(+) by either cationic ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA) or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P(i) response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P(i) suppressed the slippage current equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.

Show MeSH
Related in: MedlinePlus