The functional unit of the renal type IIa Na+/Pi cotransporter is a monomer.
The composition of the functional unit of the rat renal type IIa Na(+)/P(i) cotransporter (NaPi-IIa) was investigated by using two approaches based on the differential sensitivities of the wild type (WT) and mutant S460C proteins to 2-aminoethylmethanethiosulfonate hydrobromide (MTSEA), a charged cysteine modifier. Transport activity of S460C is completely blocked after incubation in MTSEA, whereas that of the WT remains unaffected. First, Xenopus laevis oocytes were coinjected with cRNAs coding for the WT and S460C in different proportions, and the transport inhibition after MTSEA incubation was assayed by electrophysiology. The relationship between MTSEA inhibition and proportion of cRNA was consistent with that for a functional monomer. Second, concatameric proteins were constructed that either comprised two WT proteins (WT-WT), two S460C mutants (S460C-S460C), or one of each (WT-S460C). Western blots of oocytes injected with fusion protein cRNA showed bands at approximately 200 kDa, whereas a main band at approximately 90 kDa was obtained for the WT cRNA alone. The kinetic properties of concatamers were the same as for the single proteins. Transport activity of the WT-WT concatamer was unaffected by MTSEA incubation, fully inhibited for S460C-S460C, but 50% inhibited for WT-S460C. This behavior was also consistent with NaPi-IIa being a functional monomer.
Köhler
K
K
Physiologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Forster
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IC
Lambert
G
G
Biber
J
J
Murer
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D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D008970:Molecular Weight; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D050608:Sodium-Phosphate Cotransporter Proteins; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D013329:Structure-Activity Relationship; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D007202:Indicators and Reagents; C493556:Slc34a1 protein, rat; D050608:Sodium-Phosphate Cotransporter Proteins; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D027981:Symporters; C093171:methanethiosulfonate ethylammonium; D005020:Ethyl Methanesulfonate