The light-gated cation channel channelrhodopsin-2 (ChR2) has rapidly become a principal tool in neuroscience and its use is currently considered in multiple therapeutic
Phosphorylation of Na + /K + -ATPase by cGMP-dependent protein kinase (PKG) has been studied in enzymes purified from pig, dog, sheep and rat kidneys, and in Xenopus oocytes. PKG phosphorylates the a-subunits of all animal species investigated. Phosphorylation of the b-subunit was not observed. The stoichiometry of phosphorylation estimated for pig, sheep and dog renal Na + /K + -ATPase is 3.5, 2.2 and 2.1 mol P i per mol a-subunit, respectively. Proteolytic fingerprinting of the pig a1-subunits phosphorylated by PKG using specific antibodies raised against N-terminus or C-terminus reveals that phosphorylation sites are located within the intracellular loop of the a-subunit between the 35 kDa N-terminal and 27 kDa C-terminal fragments. Phosphorylation sites within the a1-subunit of the purified Na + /K + -ATPase do not appear to be easily accessible for PKG since incorporation of P i requires 0.2% of Triton X-100. Administration of cGMP and PKG in the presence of 5 mm ATP, which prevents inactivation of the Na + /K + -ATPase by detergent, leads to stimulation of hydrolytic activity by 61%. Administration of 50 mm of cGMP or dbcGMP in yolk-free homogenates of Xenopus oocytes leads to stimulation of ouabain-dependent ATPase activity by 130±198% and to incorporation of 33 P into the a-subunit without the detergent. Hence, PKG plays regulatory role in active transmembraneous transport of Na + and K + via phosphorylation of the catalytic subunit of the Na + /K + -ATPase.
The major excitatory neurotransmitter transporter EAAC1 in the mammalian central nervous system is considered a possible target for Chinese herbal medicine. Extracts of Acorus tatarinowii (Schott) were tested for their effects on EAAC1 activity. XENOPUS oocytes with heterologously expressed EAAC1 were used as the model system. Rate of glutamate uptake was determined by means of the isotopic tracer technique. Glutamate-induced current was recorded under a two-electrode voltage clamp. As a highly effective component, alpha-asarone was identified. The rate of glutamate uptake was stimulated by 200 microM of alpha-asarone by about 15 %. In contrast, the same concentration reduced the EAAC1-mediated current by about 35 % at a holding potential of - 60 mV; half maximum inhibition was obtained at about 60 microM. Our experimental data suggest that both stimulation of glutamate uptake and inhibition of EAAC1-mediated current by alpha-asarone could contribute to reduced excitatory activity.
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