Metal bridges to probe membrane ion channel structure and function

Abstract

Ion channels are integral membrane proteins that undergo important conformational changes as they open and close to control transmembrane flux of different ions. The molecular underpinnings of these dynamic conformational rearrangements are difficult to ascertain using current structural methods. Several functional approaches have been used to understand two- and three-dimensional dynamic structures of ion channels, based on the reactivity of the cysteine side-chain. Two-dimensional structural rearrangements, such as changes in the accessibility of different parts of the channel protein to the bulk solution on either side of the membrane, are used to define movements within the permeation pathway, such as those that open and close ion channel gates. Three-dimensional rearrangements – in which two different parts of the channel protein change their proximity during conformational changes – are probed by cross-linking or bridging together two cysteine side-chains. Particularly useful in this regard are so-called metal bridges formed when two or more cysteine side-chains form a high-affinity binding site for metal ions such as Cd2+ or Zn2+. This review describes the use of these different techniques for the study of ion channel dynamic structure and function, including a comprehensive review of the different kinds of conformational rearrangements that have been studied in different channel types via the identification of intra-molecular metal bridges. Factors that influence the affinities and conformational sensitivities of these metal bridges, as well as the kinds of structural inferences that can be drawn from these studies, are also discussed.

LRRC8 N termini influence pore properties and gating of volume-regulated anion channels (VRACs)

“…Cd 2+ inhibition of cysteine-substituted channels can demonstrate the close vicinity of the substituted residues because Cd 2+ binding requires multiple cysteines (29, 30). We had to apply Cd 2+ to the N-terminal cysteine mutants from the intracellular side, that is from the pipette.…”

An Additional Ca2+ Binding Site Allosterically Controls TMEM16A Activation

“…To test this hypothesis, we performed double-cysteine substitutions to the third Ca 2+ site and sought to assess whether the transition metal ion Cd 2+ could form a metal bridge between TM2 and TM10 and how such restricting their conformation would impact channel activation. The readily reversible formation of Cd 2+ metal bridge permits temporal probing of the effects from crosslinking TM2 and TM10 on channel activity (Linsdell, 2015). By screening different TM2-TM10 interhelical pairs of cysteines, we found that application of Cd 2+ (0.5, 1, or 10 mM) largely abolishes channel activity of the double mutant E425C/D879C, but not WT or the single-cysteine mutants E425C and D879C, even under saturating 100 mM Ca 2+ (Figures 6A-6E).…”

Positioning of extracellular loop 1 affects pore gating of the cystic fibrosis transmembrane conductance regulator

“…For this reason, it has been suggested that engineered disulfide bonds can "trap" residues into rare and otherwise unnatural conformations (12). To better understand the kinetics of the C110 -C892 linkage, we used acute applications of the soft metal cadmium, as successfully used to probe cysteine proximity in previous studies of CFTR (12,24,42) and many other types of ion channels, to perform metalbridging experiments. To ensure that engineered cysteine residues of these channels were free to coordinate cadmium, the cells were preincubated with 1 mM DTT directly before channel activation.…”