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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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Mutant S460C and wild type show comparable voltage dependence. (A) Steady state current–voltage curves for representative cells expressing wild type (□) and S460C (▵) for the cotransport (left) and slippage (right) modes. For cotransport mode, the current (Ip) represents difference between current induced by 1 mM Pi and current in absence of Pi normalized to the value at −100 mV (n = 4). For slippage mode, the current (Is) represents difference between the holding current and current induced by 3 mM PFA, normalized to the value at −100 mV (n = 4). SEMs smaller than symbol size are not shown. (B) Pre–steady state relaxations induced by voltage steps from −60 mV holding potential to voltages in the range −140 to +80 mV in ND100 solution. Inset shows original records before (left) and after (right) application of 3 mM PFA. Q–V curve found by integrating the transient current after subtraction of the PFA response. (▪) ON charge movement, (□) OFF charge movement. Continuous line is fit of  to mean of ON and OFF charges, which gave fit parameters: Qmax = 5.7 nC, z = 0.7; V0.5 = −51 mV.
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Figure 5: Mutant S460C and wild type show comparable voltage dependence. (A) Steady state current–voltage curves for representative cells expressing wild type (□) and S460C (▵) for the cotransport (left) and slippage (right) modes. For cotransport mode, the current (Ip) represents difference between current induced by 1 mM Pi and current in absence of Pi normalized to the value at −100 mV (n = 4). For slippage mode, the current (Is) represents difference between the holding current and current induced by 3 mM PFA, normalized to the value at −100 mV (n = 4). SEMs smaller than symbol size are not shown. (B) Pre–steady state relaxations induced by voltage steps from −60 mV holding potential to voltages in the range −140 to +80 mV in ND100 solution. Inset shows original records before (left) and after (right) application of 3 mM PFA. Q–V curve found by integrating the transient current after subtraction of the PFA response. (▪) ON charge movement, (□) OFF charge movement. Continuous line is fit of to mean of ON and OFF charges, which gave fit parameters: Qmax = 5.7 nC, z = 0.7; V0.5 = −51 mV.

Mentions: Voltage dependence was the final property of type IIa Na+/Pi cotransport investigated for the S460C mutant, normally characterized in terms of steady state and pre–steady state behavior. Fig. 5 A shows the steady state voltage dependence of the Pi-induced current (left current–voltage plot), which was obtained by subtracting the holding current in the absence of Pi from that under saturating Pi (1 mM) and 100 mM Na+ (pH 7.4). These data indicate that the voltage dependence of the WT and S460C were indistinguishable for V < 0 mV. Moreover, the voltage dependence of the normalized slippage current (right current–voltage plot) for the WT and S460C, using 3 mM PFA as the blocking agent, was essentially unchanged.


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)

Mutant S460C and wild type show comparable voltage dependence. (A) Steady state current–voltage curves for representative cells expressing wild type (□) and S460C (▵) for the cotransport (left) and slippage (right) modes. For cotransport mode, the current (Ip) represents difference between current induced by 1 mM Pi and current in absence of Pi normalized to the value at −100 mV (n = 4). For slippage mode, the current (Is) represents difference between the holding current and current induced by 3 mM PFA, normalized to the value at −100 mV (n = 4). SEMs smaller than symbol size are not shown. (B) Pre–steady state relaxations induced by voltage steps from −60 mV holding potential to voltages in the range −140 to +80 mV in ND100 solution. Inset shows original records before (left) and after (right) application of 3 mM PFA. Q–V curve found by integrating the transient current after subtraction of the PFA response. (▪) ON charge movement, (□) OFF charge movement. Continuous line is fit of  to mean of ON and OFF charges, which gave fit parameters: Qmax = 5.7 nC, z = 0.7; V0.5 = −51 mV.
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Related In: Results  -  Collection

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Figure 5: Mutant S460C and wild type show comparable voltage dependence. (A) Steady state current–voltage curves for representative cells expressing wild type (□) and S460C (▵) for the cotransport (left) and slippage (right) modes. For cotransport mode, the current (Ip) represents difference between current induced by 1 mM Pi and current in absence of Pi normalized to the value at −100 mV (n = 4). For slippage mode, the current (Is) represents difference between the holding current and current induced by 3 mM PFA, normalized to the value at −100 mV (n = 4). SEMs smaller than symbol size are not shown. (B) Pre–steady state relaxations induced by voltage steps from −60 mV holding potential to voltages in the range −140 to +80 mV in ND100 solution. Inset shows original records before (left) and after (right) application of 3 mM PFA. Q–V curve found by integrating the transient current after subtraction of the PFA response. (▪) ON charge movement, (□) OFF charge movement. Continuous line is fit of to mean of ON and OFF charges, which gave fit parameters: Qmax = 5.7 nC, z = 0.7; V0.5 = −51 mV.
Mentions: Voltage dependence was the final property of type IIa Na+/Pi cotransport investigated for the S460C mutant, normally characterized in terms of steady state and pre–steady state behavior. Fig. 5 A shows the steady state voltage dependence of the Pi-induced current (left current–voltage plot), which was obtained by subtracting the holding current in the absence of Pi from that under saturating Pi (1 mM) and 100 mM Na+ (pH 7.4). These data indicate that the voltage dependence of the WT and S460C were indistinguishable for V < 0 mV. Moreover, the voltage dependence of the normalized slippage current (right current–voltage plot) for the WT and S460C, using 3 mM PFA as the blocking agent, was essentially unchanged.

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