Synthesis and characterization of methylbromoamiloride, a potential biochemical probe of epithelial Na+ channels.
We report the synthesis of a radioactive, methylated analog of bromoamiloride which inhibits the amiloride-sensitive, epithelial Na+ channel reversibly and with high affinity. This synthesis was achieved by methylation of a nitrogen in the acylguanidinium moiety with tritiated methyliodide of high specific activity. This methylated bromoamiloride molecule (CH3BrA) was purified by both thin layer and high performance liquid chromatography. Proton nuclear magnetic resonance and mass spectroscopy techniques were used to determine the structure of this analog. This compound inhibited both short-circuit current of in vitro frog skin and 22Na+ influx into apical plasma membrane vesicles made from cultured toad kidney cells (line A6) with the same or lower apparent inhibitory dissociation constant as bromoamiloride. Irradiation with ultraviolet light rendered this inhibition irreversible in both A6 vesicles and frog skin. Preparation of radioactive CH3BrA yielded specific activities in excess of 1 Ci/mmol. We suggest that this compound will be useful in the isolation and purification of this ubiquitous Na+ channel.
Lazorick
K
K
Miller
C
C
Sariban-Sohraby
S
S
Benos
D
D
D000584:Amiloride / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004848:Epithelium / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008745:Methylation; D010782:Photolysis; D011892:Rana catesbeiana; D012867:Skin / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D007473:Ion Channels; C029229:6-bromoamiloride; D000584:Amiloride; C046793:3,5-diamino-N-(aminoiminomethyl)-6-bromopyrazine-N-methylcarboxamide; D012964:Sodium