We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).
Phosphate is an essential component of life and must be actively transported into cells against its electrochemical gradient. In vertebrates, two unrelated families of Na+-dependent Pitransporters carry out this task. Remarkably, the two families transport different Pispecies: whereas type II Na+/Picotransporters (SCL34) prefer divalent HPO42−, type III Na+/Picotransporters (SLC20) transport monovalent H2PO4−. The SCL34 family comprises both electrogenic and electroneutral members that are expressed in various epithelia and other polarized cells. Through regulated activity in apical membranes of the gut and kidney, they maintain body Pihomeostasis, and in salivary and mammary glands, liver, and testes they play a role in modulating the Picontent of luminal fluids. The two SLC20 family members PiT-1 and PiT-2 are electrogenic and ubiquitously expressed and may serve a housekeeping role for cell Pihomeostasis; however, also more specific roles are emerging for these transporters in, for example, bone mineralization. In this review, we focus on recent advances in the characterization of the transport kinetics, structure-function relationships, and physiological implications of having two distinct Na+/Picotransporter families.
Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements Ravera, S; Virkki, L V; Murer, H; Forster, I C Ravera, S; Virkki, L V; Murer, H; Forster, I C (2007). Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements. American Journal of Physiology -Cell Physiology , 293(2):C606-C620. AbstractMembers of the SLC20 family or type III Na(+) -coupled P(i) cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular P(i) homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic P(i) cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na(+)-dependent (32)P(i) uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li(+) substituted for Na(+). The dependence of the P(i)-induced current on P(i) concentration was Michaelian, and the dependence on Na(+) concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent P(i) affinity constant showed a minimum in the pH range 6.2-6.8 of approximately 0.05 mM and increased to approximately 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual (22)Na-(32)P(i) uptake and suggested a 2:1 Na(+):P(i) stoichiometry. A correlation of (32)P(i) uptake and net charge movement indicated one charge translocation per P(i). Changes in oocyte surface pH were consistent with transport of monovalent P(i). On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na(+):H(2)PO(4)(-):Na(+). Significantly, in contrast to type II Na(+)-P(i) cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity.Ravera S, Virkki LV, Murer H, Forster IC. Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements. bers of the SLC20 family or type III Na ϩ -coupled Pi cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular Pi homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic Pi cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na ϩ -dependent 32 Pi uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization...
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