Activation of an ATP-dependent K+ conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules

AbstractIn rabbit proximal convoluted tubules, an ATP-sensitive K+ (KATP) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na+ transport and basolateral K+ conductance. This K+ conductance is inhibited by taurine. We sought to isolate this K+ channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K+ conductance, largely inhibited by extracellular Ba2+ and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K+ current which was Ba2+-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K+ channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K+ current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K+ current. The possible involvement of AK in the KATP channel’s regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations

Electrogenic amino acid exchange via the rBAT transporter

AbstractA cDNA clone was isolated from rabbit renal cortex using DNA-mediated expression cloning, which caused alanine-dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na-independent amino acid transporter previously cloned elsewhere. Exposure of cDNA-injected oocytes to neutral amino acids led to voltage-dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut-open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells