Mercury and zinc differentially inhibit shark and human CFTR orthologues: involvement of shark cysteine 102.
The apical membrane is an important site of mercury toxicity in shark rectal gland tubular cells. We compared the effects of mercury and other thiol-reacting agents on shark CFTR (sCFTR) and human CFTR (hCFTR) chloride channels using two-electrode voltage clamping of cRNA microinjected Xenopus laevis oocytes. Chloride conductance was stimulated by perfusing with 10 microM forskolin (FOR) and 1 mM IBMX, and then thio-reactive species were added. In oocytes expressing sCFTR, FOR + IBMX mean stimulated Cl(-) conductance was inhibited 69% by 1 microM mercuric chloride and 78% by 5 microM mercuric chloride (IC(50) of 0.8 microM). Despite comparable stimulation of conductance, hCFTR was insensitive to 1 microM HgCl(2) and maximum inhibition was 15% at the highest concentration used (5 microM). Subsequent exposure to glutathione (GSH) did not reverse the inhibition of sCFTR by mercury, but dithiothreitol (DTT) completely reversed this inhibition. Zinc (50-200 microM) also reversibly inhibited sCFTR (40-75%) but did not significantly inhibit hCFTR. Similar inhibition of sCFTR but not hCFTR was observed with an organic mercurial, p-chloromercuriphenylsulfonic acid (pCMBS). The first membrane spanning domain (MSD1) of sCFTR contains two unique cysteines, C102 and C303. A chimeric construct replacing MSD1 of hCFTR with the corresponding sequence of sCFTR was highly sensitive to mercury. Site-specific mutations introducing the first but not the second shark unique cysteine in hCFTR MSD1 resulted in full sensitivity to mercury. These experiments demonstrate a profound difference in the sensitivity of shark vs. human CFTR to inhibition by three thiol-reactive substances, an effect that involves C102 in the shark orthologue.
Weber
Gerhard J
GJ
Division of Nephrology, Department of Internal Medicine, Yale Univ. School of Medicine, New Haven, CT 06510, USA.
Mehr
Ali Poyan
AP
Sirota
Jeffrey C
JC
Aller
Stephen G
SG
Decker
Sarah E
SE
Dawson
David C
DC
Forrest
John N
JN
D002731:4-Chloromercuribenzenesulfonate; D000818:Animals; D002462:Cell Membrane; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004553:Electric Conductivity; D006801:Humans; D008627:Mercuric Chloride / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes; D012754:Sharks; D013045:Species Specificity; D014982:Xenopus laevis; D019345:Zinc Acetate / Q000494:pharmacology*
D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D008627:Mercuric Chloride; D002731:4-Chloromercuribenzenesulfonate; D019345:Zinc Acetate; D003545:Cysteine