A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes

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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.