Rates and Stoichiometries of Metal Ion Probes of Cysteine Residues within Ion Channels

Abstract

Metal ion probes are used to assess the accessibility of cysteine side chains in polypeptides lining the conductive pathways of ion channels and thereby determine the conformations of channel states. Despite the widespread use of this approach, the chemistry of metal ion-thiol interactions has not been fully elucidated. Here, we investigate the modification of cysteine residues within a protein pore by the commonly used Ag(+) and Cd(2+) probes at the single-molecule level, and provide rates and stoichiometries that will be useful for the design and interpretation of accessibility experiments.

Metal Bridges Illuminate Transmembrane Domain Movements during Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

“…Instead, we propose that the relatively small number of double cysteine mutant channels that show strengthened Cd 2ϩ binding results from specific coordination of a single Cd 2ϩ ion interacting simultaneously with both cysteine side chains. This proposal is consistent with recent studies showing that cysteine side chains introduced into ion channel pores bind a single Cd 2ϩ ion and that nearby cysteine side chains then increase the strength of binding of this single Cd 2ϩ ion (29). In effect, those combinations of cysteine pairs that led specifically to a strengthened binding of Cd 2ϩ within the pore (but no other combination of cysteines tested) are proposed to support the formation of Cd 2ϩ bridges between TMs 6 and 12 that impair channel function.…”

“…3). In highly localized regions of ion channel pores, this is generally considered consistent with the formation of a metal bridge between the two cysteine side chains (29). Indeed, the ability of a restricted subset of cysteine pairs specifically to coordinate binding of a single Cd 2ϩ ion in the inner vestibule is consistent with the proposed alignment of TMs 1, 6, and 12 around this region of the pore (Fig.…”

“…3). This suggests that Cd 2ϩ is able to bind to each of these introduced cysteine side chains (29), which is consistent with the proposed pore-lining location of the cysteine residues introduced. The effects of Cd 2ϩ appeared independent of voltage ( Fig.…”

“…2). Similar Cd 2ϩ sensitivity has previously been described for cysteine mutants in other parts of the pore (7,26) and presumably reflects Cd 2ϩ ion binding to these 13 different poreexposed cysteine side chains (29). Channels bearing two cysteine residues, one in each of two different TMs, were also sensitive to cytoplasmic Cd 2ϩ (Figs.…”

Control of the Conductance of Engineered Protein Nanopores through Concerted Loop Motions

“…Engineered protein nanopores have attracted interest for the detection of rare metal ions and neurotransmitters in solution, [1] low-cost sequencing of small numbers of molecules of DNA and RNA, [2] and measurement of the folding and unfolding kinetics of single proteins. [3] In most of these studies, engineered versions of the heptameric a-hemolysin nanopore were used, although other proteins have also been used.…”