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

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/microns2, mean, SD) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/microns2) 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 In-tramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a "gating charge" of approximately 3.5 electronic charges for SGLT1 and approximately 9 for the mutant Shaker K+ channel from the ratio of Qmax 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 (pfs) of 1.4 x 10(-14) cm3/sec for CHIP and of 1.5 x 10(-16) cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (Pf) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit approximately 100-fold less effective than CHIP.

Employing Escherichia coli to functionally express, purify, and characterize a human transporter

“…However, although Western blot analysis of whole-oocyte lysates indicates that hSGLT1⌬N is expressed at similar levels as hSGLT1 wild type, the protein is not inserted into the oocyte plasma membrane as determined by steady-state and presteadystate measurements (20,24). This finding demonstrates that this variation causes improper trafficking or missorting in the eukaryotic host cell.…”

“…The catalytic turnover number for the reconstituted transporter in proteoliposomes was determined by the ratio of V max :number of transporters, whereas that for the transporter in oocytes was based on electrophysiological experiments using the maximum current:maximum charge (I max :Q max ) ratio (24).…”

Transport model of the human Na+-coupled l-ascorbic acid (vitamin C) transporter SVCT1

“…Transporter-mediated pre-steady-state currents can be used to estimate transporter density (37). We estimated the number (per oocyte) of functional units (N T) of the SVCT1 transporter expressed in the plasma membrane using Eq.…”

Phosphorylation of Soybean Nodulin 26 on Serine 262 Enhances Water Permeability and Is Regulated Developmentally and by Osmotic Signals

“…Based on morphological estimates, an S real /S sphere value of 9 was used to correct for the increase in the surface area of the oocyte plasma membrane caused by the presence of folds and microvilli (Zampighi et al, 1995).…”