Mercury and zinc differentially inhibit shark and human CFTR orthologues: involvement of shark cysteine 102

Abstract

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.

The human CFTR protein expressed in CHO cells activates an aquaporin 3 in a cAMP dependent pathway: study by Digital Holographic Microscopy

“…4), suggesting that HgCl 2 decreased the water transport and not the Cl 2 flux. This result is in agreement with the study of Weber et al in which it was shown that the human CFTR protein was weakly sensitive to mercury (Weber et al, 2006).…”

Divergent CFTR orthologs respond differently to the channel inhibitors CFTRinh-172, glibenclamide, and GlyH-101

“…Ovarian lobules of mature female Xenopus laevis (Xenopus I, Dexter, MI) were obtained, and mature stage V and VI oocytes were selected for two-electrode voltage clamping as described previously (5,63). After 12 h the oocytes were injected with 10 ng cRNA/50 nl or an equivalent volume of water and then stored for 1-2 days in modified Barth solution at 18°C as described previously (5).…”

Decryption of tissue factor

“…However, no data (experimental or theoretical) are provided supporting the re-formation of the disulfide bridge hypothetically present in a reduced form on untreated cells. Existing publications related to the protein treatment with mercuric chloride show that it oxidizes only a single thiol group[39, 40] and that a similar effect could be achieved by treating tissue factor-bearing cells with other metal compounds[41]. …”