1,721,105 research outputs found
Effects of pH on the uncoupled, coupled and presteady-state currents at the amino acid cotransporter KAAT1
No full textIonic Selectivity of the coupled and uncoupled currents carried by the amino acid cotransporter kaat1
No full textIonic selectivity of the coupled and uncoupled currents carried by the amino acid transporter KAAT1
No full textThe ability of the intestinal amino acid co-transporter KAAT-1 expressed in Xenopus oocytes to transport different cations in either amino acid coupled or uncoupled manner was studied using voltage-clamp conditions. KAAT1-expressing oocytes exhibit a transporter-related current in the absence of organic substrate (uncoupled current). In the presence of various alkali cations the amplitude of this current follows the sequence: ILi > INa > IK approximately equal to IRb approximately equal to ICs. Addition of 1 mM leucine causes large increases in K+ and Na+ currents, while the Li+ current undergoes a more complex change and Rb+ and Cs+ currents are only marginally affected. Pre-steady-state currents in the absence of organic substrate are apparent when Na+, K+, or Li+ are the bathing ions; analysis of these currents in terms of charge movement reveals that Na+, K+, and Li+ interact differently with the transporter. The uncoupled current in mixtures of Na+ and Li+ fails to exhibit anomalous mole-fraction behavior. Kinetic analysis of ion binding and uncoupled permeation argues against a multi-ion single-file mechanism in the KAAT1 cotransporter
Exogenous protein expression in Xenopus Laevis Oocyte, Basic procedure
No full textThe oocytes of the South African clawed frog Xenopus laevis have been widely used as a reliable system for the expression and characterization of different types of proteins, including ion channels and membrane receptors. The large size and resilience of these oocytes make them easy to handle and to microinject with different molecules such as natural mRNAs, cRNAs, and antibodies. A variety of methods can then be used to monitor the expression of the proteins encoded by the microinjected mRNA/cRNA, and to perform a functional characterization of the heterologous polypeptides. In this chapter, after describing the equipment required to maintain X. laevis in the laboratory and to set up a microinjection system, we provide detailed procedures for oocyte isolation, micropipet and cRNA preparation, and oocyte microinjection. A method for the labeling of oocyte-synthesized proteins and for the immunological detection of the heterologous polypeptides is also described
Effects of pH on the uncoupled, coupled and presteady-state currents generated by the amino acid cotransporter KAAT1 expressed in Xenopus laevis oocytes
No full textEffects of pH on the unpoupled, coupled and presteady-state currents generated by the amino acid cotransporter KAAT1 expressed in Xenopus Laevis oocytes
No full textProperties of the Ca(2+)-activated Cl- current of Xenopus oocytes
No full textThe properties of the Ca(2+)-activated Cl- current of Xenopus oocytes have been investigated by voltage-clamp and injections of D-3-deoxy-3-fluoro-myo-inositol 1,4,5-trisphosphate (3-F-lnsP3). Following 3-F-InsP3 injection, a transient phase of Ca(2+)-activated Cl- current occurred, caused by Ca2+ release from internal stores; subsequently, a secondary, long-lasting, current was recorded, signaling Ca2+ influx from the exterior (ICRAC). Changes in external Cl- during the sustained phase produced the expected shifts in reversal potential (Erev), while the conductance varied opposite to the predictions of simple electrodiffusional theory. Application of depolarizing pulses soon (10 s) after 3-F-InsP3 injection elicited membrane currents exhibiting a single exponential rise. During the sustained subsequent phase, the current elicited by depolarizations showed an early peak followed by a prominent decline. During the sustained phase, removal of calcium from the external solution, or its substitution with Ba2-, abolished voltage- and time-dependent components of the depolarization-induced current. Slope conductance analysis of the inactivating records revealed, in addition to the decline of the Ca(2+)-activated Cl- current, the presence of a second, inwardly directed current. This could be identified as a slowly inducible Na+ current already described in Xenopus oocytes
Mutations in specific aminoacid residues confer ph sensitivity to the rat gaba cotrasporter Gat1
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