capable of acting on an added substrate of the enzyme to release a fluorescent or
chemiluminescent signal that can readily be detected and quantified. The technique
has been particularly successful in the study of pathways that result in a nuclear signal
since the resulting fluorescent signal will be located in the nucleus. In an alternative
format, EFC can be used to monitor second messengers such as cAMP and hence be
used to study GPCRs. In this case, cAMP is chemically conjugated to the galactosidase
fragment and an antibody that binds to the conjugated cAMP added to the assay. The
antibody sterically prevents the two galactosidase forms combining to form the active
enzyme and hence produce a signal. However, as free cAMP is generated by the
GPCR–adenylyl cyclase system it displaces the conjugated cAMP from the antibody
allowing the two galactosidase fragments to complement and generate a signal thus
allowing the cAMP to be quantified. Similar assays have been developed for tyrosine
kinases and serine threonine kinases and the technique can be used to monitor protein
expression changes in response to receptor activation. The assays are easily
miniaturised and automated to give high throughput precision data.17.4.2 Signal transduction through ligand-gated ion channels
Ligand-gated ion channels constitute one of the mechanisms for the control of the
transmembrane movement of ions down their concentration gradient resulting in a
change in membrane potential. This control of ion movement is exerted on the basis of
ion type (anion or cation), ion charge and ion size. Binding of the ligand to the resting
state of the receptor induces a conformational change in the receptor protein that
results in the opening of the channel and the movement (gating) of ions. The channel
remains open until either the ligand is removed or when, in the continued presence of
the ligand, the receptor protein changes to its desensitised state in which the channel
is closed. Since this mechanism of transduction is independent of any other membrane
component or intracellular molecule, the cellular response to ligand binding is almost
instantaneous. This class of membrane receptors includes numerous receptors that are
involved in signal transmission between neurons, between glia and neurons and
between neurons and muscles.
Four superfamilies of ligand-gated ion channels, classified on the basis of the number
of transmembrane (TM) segments within the subunits (2TM, 3TM, 4TM and 6TM) have
been identified. The 4TM family has been the most thoroughly investigated and all
members shown to consist of five subunits (pentameric). Members of the family include:- nicotinic acetylcholine receptors (nAChR) that are the primary excitatory receptors in
skeletal muscle and the peripheral nervous system; - serotonin (5-hydroxytryptamine) (5HT) receptors located in neurons;
- g-aminobutyric acid receptors (GABAAand GABAB) found in the cortex and which are
inhibitory transmitters; - glycine receptors found in the spinal cord and brainstem and which like the GABA
receptors are inhibitory transmitters.
The five subunits of the nAChR receptor are of four types,a,b,ganddwith a
stoichiometry ofa 2 bgd. All five span the membrane four times mainly witha-helical688 Cell membrane receptors and cell signalling