A bumetanide-sensitive, potassium carrier-mediated transport system in excitable tissues

The binding of [³H]-bumetanide to rat brain synaptosomes revealed the existence of two binding sites. The high affinity site (R₁ = 46.6 moles/mg protein) binds bumetanide and furosemide with K_d1 of 13 nM and 1.5 μM respectively, while the low affinity site (R₂ = 1.37 nmoles/mg protein) is characterized by K_d2 of 200 μM and 680 μM for bumetanide and furosemide, respectively. Bumetanide sensitive ⁸⁶Rb uptake was 34+14.5, 38.3+1.4, 18.6+1.3 and 29.0+6.1% of total ⁸⁶Rb uptake in synaptic plasma membrane vesicles, rat brain synaptosomes, Neuroblastoma N1E115 cell line and chick chest muscle cells, respectively. Furosemide and bumetanide inhibited ⁸⁶Rb uptake to rat brain SPM- vesicles in a dose dependent fashion. Half maximal inhibition (IC₅₀) was observed at 20 nM and 4 μM for bumetanide and furosemide, respectively. Bumetanide-sensitive transport was dependent on extravesicular sodium and chloride concentrations with a Km of 21 and 25 mM for the two ions, respectively. These results demonstrate the existence of a "loop diuretic" sensitive carrier-mediated K⁺ transport system in brain and other excitable cells.