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.
We report the cloning, sequence analysis, tissue distribution, functional expression, and chromosomal localization of the human pancreatic sodium bicarbonate cotransport protein (pancreatic NBC (pNBC)). The transporter was identified by searching the human expressed sequence tag data base. An I.M.A.G.E. clone W39298 was identified, and a polymerase chain reaction probe was generated to screen a human pancreas cDNA library. pNBC encodes a 1079-residue polypeptide that differs at the N terminus from the recently cloned human sodium bicarbonate cotransporter isolated from kidney (kNBC) (Burnham, C. E., Amlal, H., Wang, Z., Shull, G. E., and Soleimani, M. (1997) J. Biol. Chem. 272, 19111-19114). Northern blot analysis using a probe specific for the N terminus of pNBC revealed an ϳ7.7-kilobase transcript expressed predominantly in pancreas, with less expression in kidney, brain, liver, prostate, colon, stomach, thyroid, and spinal chord. In contrast, a probe to the unique 5 region of kNBC detected an ϳ7.6-kilobase transcript only in the kidney. In situ hybridization studies in pancreas revealed expression in the acini and ductal cells. The gene was mapped to chromosome 4q21 using fluorescent in situ hybridization. Expression of pNBC in Xenopus laevis oocytes induced sodium bicarbonate cotransport. These data demonstrate that pNBC encodes the sodium bicarbonate cotransporter in the mammalian pancreas. pNBC is also expressed at a lower level in several other organs, whereas kNBC is expressed uniquely in kidney.
SummaryAmides and acidic amino acids represent the major long distance transport forms of organic nitrogen. Six amino acid permeases (AAPs) from Arabidopsis mediating transport of a wide spectrum of amino acids were isolated. AAPs are distantly related to plasma membrane amino acid transport systems N and A and to vesicular transporters such as VGAT from mammals. A detailed comparison of the properties by electrophysiology after heterologous expression in Xenopus oocytes shows that, although capable of recognizing and transporting a wide spectrum of amino acids, individual AAPs differ with respect to speci®city. Apparent substrate af®nities are in¯uenced by structure and net charge and vary by three orders of magnitude. AAPs mediate cotransport of neutral amino acids with one proton. Uncharged forms of acidic and basic amino acids are cotransported with one proton. Since all AAPs are differentially expressed, different tissues may be supplied with a different spectrum of amino acids. AAP3 and AAP5 are the only transporters mediating ef®cient transport of the basic amino acids. In vivo competition shows that the capability to transport basic amino acids in planta might be overruled by excess amides and acidic amino acids in the apoplasm. With the exception of AAP6, AAPs do not recognize aspartate; only AAP6 has an af®nity for aspartate in the physiologically relevant range. This property is due to an overall higher af®nity of AAP6 for neutral and acidic amino acids. Thus AAP6 may serve a different role either in cooperating with the lower af®nity systems to acquire amino acids in the low concentration range, as a system responsible for aspartate transport or as an uptake system from the xylem. In agreement, a yeast mutant de®cient in acidic amino acid uptake at low aspartate concentrations was complemented only by AAP6. Taken together, the AAPs transport neutral, acidic and cationic amino acids, including the major transport forms, i.e. glutamine, asparagine and glutamate. Increasing proton concentrations strongly activate transport of amino acids. Thus the actual apoplasmic concentration of amino acids and the pH will determine what is transported in vivo, i.e. major amino acids such as glutamine, asparagine, and glutamate will be mobilized preferentially.
We have investigated the functional role of Cl؊ in the human Na ؉ /Cl؊ /␥-aminobutyric acid (GABA) and Na ؉ / glucose cotransporters (GAT1 and SGLT1, respectively) expressed in Xenopus laevis oocytes. Substrate-evoked steady-state inward currents were examined in the presence and absence of external Cl ؊ . Replacement of Cl ؊ by gluconate or 2-(N-morpholino)ethanesulfonic acid decreased the apparent affinity of GAT1 and SGLT1 for Na ؉ and the organic substrate. In the absence of substrate, GAT1 and SGLT1 exhibited charge movements that manifested as pre-steady-state current transients. Removal of Cl؊ shifted the voltage dependence of charge movements to more negative potentials, with apparent affinity constants (K 0.5 ) for Cl ؊ of 21 and 115 mM for SGLT1 and GAT1, respectively. The maximum charge moved and the apparent valence were not altered. GAT1 stoichiometry was determined by measuring GABAevoked currents and the unidirectional influx of 36 Cl ؊ , 22
The transport mechanism of the potato StSUT1 H ؉ / sucrose cotransporter expressed in Xenopus oocytes was investigated using the 2-electrode voltage clamp and radiotracer flux methods. Sucrose induced inward currents through the transporter that were dependent on the extracellular sucrose and H ؉ concentrations and the membrane voltage. Sucrose is the major mobile carbohydrate in the majority of higher plants. Sucrose is loaded into the phloem against a large concentration gradient and is transported to heterotrophic tissues where it is used for metabolism or storage. Phloem loading is catalyzed by specific transport proteins, which couple the uptake of sucrose to the electrochemical potential gradient for protons generated by the H ϩ -ATPase (Buckhout, 1989(Buckhout, , 1994Bush, 1988Bush, , 1990Bush, , 1993Williams et al., 1990Williams et al., , 1992. Recently, cDNAs encoding putative H ϩ /sucrose cotransporters have been isolated from spinach (SoSUT1; Riesmeier et al., 1992), potato (StSUT1; Riesmeier et al., 1993a), Arabidopsis thaliana (SUC1 and SUC2; Sauer and Stolz, 1994), and Plantago major (PmSUC2; Gahrtz et al., 1994). They are all expressed in the phloem with PmSUC2 and StSUT1 localized specifically in companion cells (Riesmeier et al., 1993a;Stadler et al., 1995).1 Antisense inhibition of the potato StSUT1 transporter showed that it plays a vital role in phloem loading, assimilates partitioning, and is essential for the growth and development of potato plants (Riesmeier et al. 1993b).Little is known about the molecular mechanisms of sucrose transport. Previous studies of cloned sucrose transporters utilized transfected Saccharomyces cerevisiae cells where sucrose transport was stimulated by increasing the extracellular proton concentration [H ϩ ] o and reduced by protonophores (Riesmeier et al., 1992(Riesmeier et al., , 1993aSauer and Stolz, 1994). These observations indicate that the uptake of sucrose is dependent on a proton gradient. To understand the molecular mechanisms of sucrose transport in more detail, we expressed the potato StSUT1 transporter in Xenopus oocytes and used a combination of electrophysiological and radiotracer flux methods to determine the effects of membrane potential and external sucrose and H ϩ concentrations on the transport kinetics of StSUT1. We show that StSUT1 is electrogenic with membrane potential affecting the maximal rate of transport and apparent affinities for H ϩ and sucrose. The kinetic properties of StSUT1 can be explained by an 8-state ordered simultaneous model with H ϩ binding to the transporter before sucrose. EXPERIMENTAL PROCEDURESMolecular Biology Methods-To obtain sucrose transport activity in oocytes, the NotI fragment of StSUT1 was inserted into the NotI site of pKJB1, a vector with a poly(A) tail of 70 adenosine residues (Boorer et al., 1996). The resulting plasmid pKStSUT1 was linearized with KpnI, and capped cRNA was transcribed in vitro using T7 RNA polymerase and an RNA transcription kit (Ambion, Austin, TX).Preparation of Oocytes-Xenopus oocytes...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.