Summary
In order to permit future characterization and possible isolation of the Na+−H+ exchanger from the apical membrane of proximal tubular cells, studies were performed to solubilize and reconstitute this transporter. Rabbit brush border membranes were prepared by a magnesium aggregation method, solubilized with the detergent octyl glucoside, and reconstituted into artificial phospholipid vesicles. In the presence of a pH gradient (pHin 6.0, pHout 8.0), the uptake of 1mm 22Na+ into the proteoliposomes was five- to sevenfold higher than into liposomes. Amiloride (2mm) inhibited proton gradient-stimulated uptake of sodium by 50%. As compared to proton gradient conditions, the uptake of sodium was lower in the absence of a pH gradient but was significantly higher when the outside and inside pH was 6.0 than 8.0. TheK a for sodium in reconstituted proteoliposomes studied under pH gradient conditions was 4mm. The uptake of sodium in proteoliposomes prepared from heat-denatured membrane proteins was significantly decreased. These studies demonstrate that proteoliposomes prepared from octyl glucoside-solubilized brush border membrane proteins and asolectin exhibit proton gradient-stimulated, amiloride-inhibitable, electroneutral uptake of sodium. The ability to solubilize and reconstitute the Na+−H+ exchanger from the apical membrane of the proximal tubule will be of value in isolating and characterizing this transporter.
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Aronson, P.S., Nee, J., Suhm, M.A. 1982. Modifier role of internal H+ in activating the Na+−H+ exchanger in renal microvillus membrane vesicles.Nature (London) 299:161–163
Besterman, J.M., May, W.S., Levine, H., Cragoe, E.J., Cuatrecasas, P. 1985. Amiloride inhibits phorbol ester stimulated Na+/H+ exchange and protein kinase C.J. Biol. Chem. 260:1155–1159
Dubinsky, W.P., Frizzell, R.A. 1983. A novel effect of amiloride on H+-dependent Na+ transport.Am. J. Physiol. 245:C157-C159
Friedrich, T., Sablotni, J., Burckhardt, G. 1986. Identification of the renal Na+/H+ exchanger with N,N′-dicyclohexylcarbodiimide (DCCD) and amiloride analogues.J. Membrane Biol. 94:253–266
Grinstein, S., Rothstein, A. 1986. Mechanisms of regulation of Na+−H+ exchanger.J. Membrane Biol. 90:1–12
Harper, J.F. 1984. Peritz F test: Basic program of a robust multiple comparison test for statistical analysis of all differences among group means.Comput. Biol. Med. 14:437–445
Kahn, A.M., Branham, S., Weinman, E.J. 1983. Mechanism of urate andp-aminohippurate transport in rat renal microvillus membrane vesicles.Am. J. Physiol. 245:F151-F158
Kahn, A.M., Dolson, G.M., Hise, M.K., Bennett, S.C., Weinman, E.J. 1985. Parathyroid hormone and dibutyryl cAMP inhibit Na+/H+ exchange in renal brush border vesicles.Am. J. Physiol. 248:F212-F218
Kinsella, J.L., Aronson, P.S. 1980. Properties of the Na+−H+ exchanger in renal microvillus membrane vesicles.Am. J. Physiol. 238:F461-F469
LaBelle, E.F. 1984. Reconstituted amiloride-inhibited transporter from rabbit kidney medulla is responsible for Na+−H+ exchange.Biochim. Biophys. Acta 770:79–92
LaBelle, E.F. 1986. Amiloride-inhibited Na+−H+ exchange in human kidney medulla microsomes.Am. J. Physiol. 251:F232-F237
LaBelle, E.F., Lee, S.O. 1982. Solubilization and reconstitution of an amiloride-inhibited sodium transporter from rabbit kidney medulla.Biochemistry 21:2693–2697
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurements with the Folin-reagent.J. Biol. Chem. 193:265–275
Mahnensmith, R.L., Aronson, P.S. 1985. The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathophysiological processes.Circ. Res. 57:773–788
Murer, H., Hopfer, U., Kinne, R. 1976. Sodium/proton antiport in brush border membrane vesicles isolated from small intestine and kidney.Biochem. J. 154:597–604
Nakamura, T., Hsu, C., Rosen, B.P. 1986. Cation/proton antiport systems inEscherichia coli.J. Biol. Chem. 261:678–683
Simchowitz, L., Cragoe, E.J., Jr. 1986. Inhibition of chemotactic factor-activated Na+−H+ exchange in human neutrophils by analogues of amiloride: Structure-activity relationships in the amiloride series.Mol. Pharmacol. 30:112–120
Vigne, P., Frelin, C., Ardinot, M., Borosotto, M., Cragoe, E.J., Jr., Lazdunski, M. 1984. [3H] ethylpropylamiloride, a radio-labelled diuretic for the analysis of the Na+/H+ exchange system. Its use with kidney cell membranes.EMBO J. 3:2647–2651
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Weinman, E.J., Shenolikar, S., Cragoe, E.J. et al. Solubilization and reconsitution of renal brush border Na+−H+ exchanger. J. Membrain Biol. 101, 1–9 (1988). https://doi.org/10.1007/BF01872814
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DOI: https://doi.org/10.1007/BF01872814