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

26 M. H. Akabas

Keywords Sulfhydryl · Thiol · Thiolate · SCAM · GABAA receptor · Acetylcholine
receptor · Potassium channels · Methanethiosulfonate · Membrane transporter

1 Introduction

In the beginning there was sulfur. Sulfur is present in two amino acids, methionine
and cysteine, that have very different properties. Methionine contains a relatively
unreactive thioether (–S–CH 3 ) group. In contrast, cysteine contains a highly reac-
tive sulfhydryl group (-SH).The reactivity of cysteine and the variety of sulfhydryl
reactive reagents has made the substitution of engineered cysteines into proteins an
extremely productive approach to studying protein structure and function.
Cysteine’s reactivity is a function of the sulfur atom. Sulfur has a 3s^2 3p^4 outer
shell electron configuration. As a result, in compounds, sulfur can exist in oxida-
tion states ranging from − 2 to + 6 (Table. 1 ). Biologically, the most common oxida-
tion states are − 2 for cysteine thiols (–SH) and − 1 for disulfide linked cysteines
(–S–S–). However, application of oxidizing reagents, such as H 2 O 2 and copper
phenanthroline (Cu:phen), can result in oxidation of cysteine sulfur atoms to higher
oxidation states, including sulfinic and sulfonic acids (Table 1 ). Biologically, oxida-
tion to these states cannot be reversed but may play a role in regulatory processes
(Lo Conte and Carroll 2013 ). In the cytoplasmic reducing environment, cysteines
rarely form disulfide bonds. However, in the oxidizing extracellular environment
disulfide bonds from readily between cysteines in close proximity (Hwang et al.
1992 ; Depuydt et al. 2011 ; Feige and Hendershot 2011 ; Fass 2012 ). Thus, most
disulfide bonds are found in the extracellular domain of integral membrane proteins
or in secreted proteins (Katzen and Beckwith 2003 ; Feige and Hendershot 2011 ;
Winther and Thorpe 2014 ).
In free solution, the pKa for the ionization of the cysteine thiol (– SH) to the thio-
late anion (– S− + H+) is 8.5. Thus, at a physiological pH ~ 7.4 in free solution cys-
teine will be ionized approximately 10 % of the time. However, within proteins, the

Compound name Structure Oxidation

state

Thiol R–S–H − 2

Thiolate R–S– − 2

Disulfide R–S–S–R’ − 1

Sulfenic acid R–S–OH 0

Sulfinic acid R-S-OH

O



+ 2

Sulfonic acid

R-S-OH

O

O

+ 4

Table 1 Sulfur oxidation
states