Abstract
The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212±48 particles/μm2, mean, sd) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/μm2) only on the P face. The utility and generality of the method were demonstrated by estimating the “gating charge” per particle of the Na+/ glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K+ channel proteins, and the single molecule water permeability of CHIP (Channel-like Intramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a “gating charge” of ∼3.5 electronic charges for SGLT1 and ∼9 for the mutant Shaker K+ channel from the ratio of Q max to density of particles measured on the same oocytes. The “gating charges” were 3-fold larger than the “effective valences” calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (p f s) of 1.4 × 10−14 cm3/ sec for CHIP and of 1.5 × 10−16 cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (P f ) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit ∼100-fold less effective than CHIP.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Anderson, C.S., MacKinnon, R., Smith, C., Miller, C. 1988. Charybdotoxin block of single Ca2+ activated K+ channels. Effect of channel gating, voltage, and ionic strength. J. Gen. Physiol. 91:317–333
Barnard, E.A., Miledi, R., Sumikawa, K. 1982. Translation of exogeneous messenger RNA encoding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes. Proc. R. Soc. Lond. Ser. B. 215:241–246
Bezanilla, F., Armstrong, C.M. 1977. Inactivation of the sodium channel I. Sodium current experiments. J. Gen. Physiol. 70:549–566
Bezanilla, F., Perozo, E., Stefani, E. 1994. Gating of Shaker K channels. II. The components of gating currents and a model of channel activation. Biophys. J. 66:1011–1021
Bezanilla, F., Stefani, E. 1994. Voltage-dependent gating of ionic channels. Ann. Rev. Biomol. Struct. 23:819–846
Bluemink, J.G., Hage, W.J., van den Hoef, M.H.F., Dictus, W.J.A.G. 1983. Freeze-fracture electron microscopy of membrane changes in progesterone-induced oocytes and eggs of Xenopus laevis. Eur. J. Cell Biol 31:85–93
Deutsch, C., Price, M., Lee, S., King, V.F., Garcia, M.L. 1991. Characterization of high binding sites for charybdotoxin in human T lymphocytes. Evidence for association with the voltage-gated K+ channel. J. Biol. Chem. 266:3668–3674
Dick, E.G., Dick, D.A.T. 1970. The effect of microvilli on the water permeability of single toad oocytes. J. Cell Sci. 6:451–476
Dunia, I., Manenti, S., Rousselet A., Benedetti, E.L. 1987. Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic protein of the eye lens fibers. J. Cell Biol. 105:1679–1689
Ehring, G.R., Zampighi, G.A., Horwitz, J., Bok, D., Hall, J.E. 1990. Properties of channels reconstituted from major intrinsic protein of lens fiber membranes. J. Gen. Physiol. 96:631–664
Gorin, M.B., Yancey, S.B., Cline, J., Revel, J.P., Horwitz, J. 1984. The major intrinsic protein of lens fiber membrane: Characterization and structure based on cDNA cloning. Cell 39:49–56
Li, M., Unwin, N., Stauffer, K.A., Jan, Y-N, Jan, L.Y. 1994. Images of purified Shaker potassium channels. Curr. Biol. 4:110–115
Loo, D.D.F., Hazama, A., Supplisson, S., Turk, E., Wright E.M. 1993. Relaxation kinetics of the Na+ glucose cotransporter. Proc Natl. Acad. Sci. USA 90:5767–5771
MacKinnon, R. 1991. Determination of the subunit stoichiometry of a voltage-activated potassium channel. Nature 350:232–235
Mulders, M.S., Preston, G.M., Deen, M.T., Guggino, W.B., van Os, C.H., Agre, P. Water channel properties of Major Intrinsic Protein (MIP) of lens. J. Biol. Chem. (in press)
Parent, L., Supplisson, S., Loo, D.F., Wright, E.M. 1992. Electrogenic properties of the cloned Na+/glucose cotransporter. I. Voltageclamp studies. J. Membrane Biol. 125:49–62
Perozo, E., MacKinnon, R, Bezanilla, F., Stefani, E. 1993. Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels. Neuron 11:353–358
Preston, G.M., Agre, P. 1991. Isolation of the cRNA for erythrocyte integral membrane protein of 28 KD member of an ancient channel family. Proc. Natl. Acad. Sci. USA 88:11110–11114
Preston, G.M., Jung, J.S., Guggino, W.B., Agre, P. 1992. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256:385–387
Schoppa, N.E., McCormack, K., Tanouye, M.A., Sigworth, F.J. 1992. The size of gating charge in wild-type and mutant Shaker potassium channels. Science 255:1712–1715
Stefani, E., Toro, L., Perozo, E., Bezanilla, F. 1994. Gating of Shaker K+ channels: I. Ionic and gating currents. Biophys. J. 66:996–1010
Taglialatela, M., Toro, L., Stefani, E. 1992. Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes. Biophys. J. 61:78–82
Toggenburger, G., Kessler, M., Rothstein, A., Semenza, G., Tannenbaum, C. 1978. Similarity in effects of Na gradients and membrane potentials on D-glucose transport by, and phlorizin binding to, vesicles derived from brush border of rabbit intestinal mucosal cells. J. Membrane Biol. 40:269–290
Verbavatz, J.M., Brown, D., Sabolic, L., Valenti, G., Ausiello, D.A., Vanhoek, A.N., Ma, T., Verkman, A.S. 1993. Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes. A freeze-fracture study. J. Cell Biol 123:605–618
Zampighi, G.A., Kreman, M., Ramon, F., Moreno, A.L., Simon, S.A. 1988. Structural characteristics of gap junctions. I. Channel number in coupled and uncoupled conditions. J. Cell Biol. 106:1667–1678
Zampighi, G.A., Hall, J.E., Ehring, G.R., Simon, S.A. 1989. The structural organization and protein composition of lens fiber junctions. J. Cell Biol 108:2255–2275
Zeidel, M.L., Nielsen, S., Smith, B.L., Ambudkar, S.V., Maunsbach, A.B., Agre, P. 1994. Ultrastructure, pharmacological inhibition and transport selectivity of aquaporin channel-forming integral protein in proteoliposomes. Biochemistry 33:1606–1615
Zhang, R., Verkman, A.S. 1991. Water and urea permeability properties of Xenopus oocytes: expression of mRNA from toad urinary bladder. American J. Physiol. 260(Cell Physiol. 29) C26-C34
Author information
Authors and Affiliations
Additional information
We thank Manoli Contreras for preparation and injection of oocytes. This work was supported by NIH Grants EY-04110 (to GAZ), DK-19567 (to EW), GM30376 (to F.B.) and EY-05661 (to J.E.H.).
Rights and permissions
About this article
Cite this article
Zampighi, G.A., Kreman, M., Boorer, K.J. et al. A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes. J. Membarin Biol. 148, 65–78 (1995). https://doi.org/10.1007/BF00234157
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00234157