Novel Chemical Tools to Study Ion Channel Biology (Advances in Experimental Medicine and Biology)

25

M. H. Akabas ()
Departments of Physiology & Biophysics, Neuroscience and Medicine, Albert Einstein College
of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
e-mail: [email protected]

Cysteine Modification: Probing Channel

Structure, Function and Conformational

Change

Myles H. Akabas

Abstract Cysteine substitution has been a powerful tool to investigate the
structure and function of proteins. It has been particularly useful for studies of
membrane proteins in their native environment, embedded in phospholipid mem-
branes. Among the 20 amino acids, cysteine is uniquely reactive. This reactivity
has motivated the synthesis of a wide array of sulfhydryl reactive chemicals. The
commercially available array of sulfhydryl reactive reagents has allowed investi-
gators to probe the local steric and electrostatic environment around engineered
cysteines and to position fluorescent, paramagnetic and mass probes at specific
sites within proteins and for distance measurements between pairs of sites. Prob-
ing the reactivity and accessibility of engineered cysteines has been extensively
used in Substituted Cysteine Accessibility Method (SCAM) investigations of ion
channels, membrane transporters and receptors. These studies have successfully
identified the residues lining ion channels, agonist/antagonist and allosteric mod-
ulator binding sites, and regions whose conformation changes as proteins transi-
tion between different functional states. The thousands of cysteine-substitution
mutants reported in the literature demonstrate that, in general, mutation to cyste-
ine is well tolerated. This has allowed systematic studies of residues in transmem-
brane segments and in other parts of membrane proteins. Finally, by inserting
pairs of cysteines and assaying their ability to form disulfide bonds, changes in
proximity and mobility relationships between specific positions within a protein
can be inferred. Thus, cysteine mutagenesis has provided a wealth of data on
the structure of membrane proteins in their functional environment. This data
can complement the structural insights obtained from the burgeoning number of
crystal structures of detergent solubilized membrane proteins whose functional
state is often uncertain. This article will review the use of cysteine mutagenesis
to probe structure-function relationships in ion channels focusing mainly on Cys-
loop receptors.

© Springer Science+Business Media New York 2015
C. Ahern, S. Pless (eds.), Novel Chemical Tools to Study Ion Channel Biology,
Advances in Experimental Medicine and Biology 869,
DOI 10.1007/978-1-4939-2845-3_3