A Method for Determining the Unitary Functional Capacity of Cloned Channels and Transporters Expressed in Xenopus Laevis Oocytes

摘要: The Xenopus laevis oocyte is widely used to express exogenous channels and transporters well suited for functional measurements including currents, electrolyte nonelectrolyte fluxes, water permeability even enzymatic activity. It difficult, however, transform recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here method estimating exogenously expressed inserted plasma membrane oocytes. based on facts that P (protoplasmic) face water-injected control exhibit an extremely low density endogenous particles (212±48 particles/μm2, mean, sd) increased (up 5,000/μm2) only face. utility generality were demonstrated by “gating charge” per particle Na+/ glucose cotransporter (SGLT1) nonconducting mutant Shaker K+ proteins, molecule CHIP (Channel-like Intramembrane Protein) MIP (Major Intrinsic Protein). ∼3.5 electronic charges SGLT1 ∼9 from ratio Q max measured same charges” 3-fold larger than “effective valences” calculated fitting Boltzmann equation charge transfer data suggesting movement occur several steps. Single permeabilities (p f s) 1.4 × 10−14 cm3/ sec 1.5 10−16 cm3/sec whole-oocyte (P ) particles. Therefore, oocytes, albeit ∼100-fold less effective CHIP.