Cysteine 96 of Ntcp is responsible for NO-mediated inhibition of taurocholate uptake

Abstract

The Na(+) taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the eight cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH-7 cells transiently transfected with cysteine-to-alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) levels that were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in total expression or PM localization, indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) inhibits TC uptake by wild-type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp, but, surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead, we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that wild-type but not C96A Ntcp is S-nitrosylated by NO, suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.

Nitric oxide in liver diseases

“…It has been suggested that the dysregulation of protein S-nitrosylation, mostly mediated by iNOS, is associated with the pathogenesis of various liver diseases, including hepatocellular carcinoma (HCC) [40, 41], hepatic steatosis [9], and cholestasis [42, 43]. …”

“…NO derived from iNOS S-nitrosylated Ntcp at Cys96 and inhibited taurocholic acid uptake in hepatocytes in vitro . This result implies that S-nitrosylation of Ntcp causes sepsis-associated cholestasis by inhibiting taurocholic acid uptake and thus decreasing bile flow [42]. …”

Organic Anion Uptake by Hepatocytes

“…Additional studies suggested that PKC δ also plays a role in ntcp subcellular trafficking, although this did not seem to be dependent on its kinase activity (3, 139). Additionally, S-nitrosylation of ntcp has been described as reducing plasma membrane localization of ntcp (3, 142, 151). This has been attributed to S-nitrosylation of cysteine 96 of ntcp, but elucidation of the mechanism by which this affects subcellular trafficking remains to be determined (142).…”

“…Additionally, S-nitrosylation of ntcp has been described as reducing plasma membrane localization of ntcp (3, 142, 151). This has been attributed to S-nitrosylation of cysteine 96 of ntcp, but elucidation of the mechanism by which this affects subcellular trafficking remains to be determined (142). …”

Loops and layers of post-translational modifications of drug transporters

“…The same research group also demonstrated that replacing cysteine 96 with alanine (C96A) rendered rNtcp insensitive to NO-mediated inhibition of TC uptake. rNtcp/C96A was not S-nitrosylated by NO, suggesting that Cys96 is important in regulating rNtcp function in response to elevated levels of NO [61]. Gonzalez, et al examined the cytoprotective effect of α-Tocopherol against glycochenodeoxycholate-induced cell death in hepatocytes.…”