An activity-based protein profiling probe for the nicotinic acetylcholine receptor

Article abstract of DOI:10.1021/ja805868x

Activity-based protein profiling (ABPP) has been used extensively to characterize the physiological functions of enzymes but has not yet been extended to ion channels. We have synthesized a state-dependent photoaffinity probe for the nicotinic acetylcholine receptor (nAChR) as a proof of concept for the development of ion channel directed ABPP probes. The candidate probe BPyneTEA comprises an nAChR binding moiety, a benzophenone moiety for photolabeling, and an alkyne moiety for biotinylation via click chemistry . Single-molecule current measurements show that BPyneTEA blocks both the closed and open (i.e., nondesensitized) conformations of the nAChR with similar kinetics. In living cells, BPyneTEA photolabels the closed state selectively over the inactive desensitized state. BPyneTEA thus shows promise as a probe for nondesensitized nAChRs and may be useful in studying the molecular physiology of desensitization. The structure and reactivity of ion channel pores in general suggest that they will be a broadly useful target for ABPP probes. Copyright

Full text of DOI:10.1021/ja805868x

Published on Web 11/01/2008
An Activity-Based Protein Profiling Probe for the Nicotinic Acetylcholine
Receptor
Mathew Tantama, Wan-Chen Lin, and Stuart Licht*
Department of Chemistry, Massachusetts Institute of Technology, Building 16, Room 573B,
Cambridge, Massachusetts 02139
Received July 27, 2008; E-mail: lichts@mit.edu
Activity-based protein profiling (ABPP) is rapidly becoming one
of the essential experimental approaches in understanding biological
processes at the systems level.1 ABPP reagents have been prepared
for a number of important enzyme classes,2-4 but ion channels
are one important class of proteins for which ABPP probes have
not been previously reported. Activation and deactivation of ion
channels are central to some of the most important processes in
neurobiology, such as neuronal excitability and synaptic plasticity.5
A probe molecule that selectively labels a subset of the different
activation states of a channel could be used as an activity-based
probe.
The nicotinic acetylcholine receptor (nAChR) is an ion channel
in the Cys-Loop superfamily that becomes cation-permeable upon
binding the neurotransmitter acetylcholine. Like other neurotrans-
mitter-gated channels, nAChRs typically undergo desensitization:
a transition into a long-lived inactive state in response to prolonged
exposure to acetylcholine.6,7 In contrast to the closed states of
nAChRs that predominate in the absence of a neurotransmitter,
desensitized states typically have very high affinities for acetyl-
choline and nicotine8,9 and predominate in the presence of a
neurotransmitter. ABPP probes could be used to help characterize
protein-protein interactions and posttranslational modifications
associated with desensitization and reactivation of nAChRs. Such
Figure 1. Single-channel electrophysiology demonstrates that BPyneTEA
probes would thus be useful for investigating the neurobiology of
desensitization in nicotine addiction10,11 and neuromuscular dis-
orders.12
(A) binds and blocks the open and closed states of the nAChR. Single-
channel activity occurs in clusters in the absence of BPyneTEA (B), but
clusters are terminated early in the presence of 500 µM BPyneTEA (C).
The decrease in the mean open time (D) and the lifetime of the fastest
closed time component (E) with increasing BPyneTEA concentration
indicate that BPyneTEA binding to the open and closed nAChR has an
We synthesized a candidate ABPP probe, named BPyneTEA
(benzophenone-alkyne-triethylammonium), for state-dependent bind-
ing and photolabeling of nAChRs (Figure 1A). This candidate probe
combines features of several “parent” structures that selectively bind
to open or closed nAChRs.13-15 We therefore characterized its
action on nAChRs both electrophysiologically and biochemically
to assess the effect of combining these features in a single structure.
To test the hypothesis that BPyneTEA can block both open and
closed nAChRs, single-channel patch-clamp current recordings were
obtained. To ensure that both open and closed states were
observable, single-channel currents were recorded from a gain-of-
function muscle-type nAChR mutant, RG153S,16 activated using
the weak agonist choline. Single-channel activity occurs as clusters
of openings and closings that represent the conformational transi-
tions of exactly one nAChR. Clusters are normally terminated by
entry into long-lived desensitized states. In the presence of
BPyneTEA, however, a cluster of activity may be terminated early
by blockade of either closed or open states (Supporting Figure 3A).
Direct observation of individual BPyneTEA blockade events at
the single-molecule level supports the hypothesis that this molecule
binds both the open and closed states (Figure 1B and C). Blockade
of the open state truncates open intervals within a cluster,15,17
decreasing the mean open time with increasing BPyneTEA
concentration (Figure 1D and Supporting Figure 3B).
association rate constant of ∼106 M-1 ·s-1
.
From the concentration dependence, the association rate constant
for open-state blockade is (1.3 ( 0.7) × 106 M-1 ·s-1 (best fit
value ( standard error). This association rate constant depends on
transmembrane voltage; an analysis of the voltage dependence
suggests that BPyneTEA binds the nAChR 20 ( 10% into the
transmembrane electrical field relative to the cell surface (Support-
ing Figure 4).
Blockade of the closed state truncates closed intervals within a
cluster, decreasing the observed mean closed times with increasing
BPyneTEA concentration (Figure 1E and Supporting Figure 3B).
In this case, there are multiple kinetic components in the closed
time distribution, but there is only one that decreases in a
BPyneTEA-dependent fashion; this component represents the
lifetime of the closed (and blocker-free) state. The association rate
constant for binding to the closed state was determined from the
BPyneTEA concentration-dependent decrease of the fastest closed
time component and is (5 ( 2) × 106 M-1 ·s-1 (best fit value (
standard error). Both open and closed states thus bind BPyneTEA
with an association rate constant of ∼1 × 106 M-1 ·s-1. The
difference in blockade rate constants between the open and closed
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10.1021/ja805868x CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
be detected.22 Optimization of the blocker and benzophenone
moieties may allow improved selectivity for the closed state; the
modular design of the molecule is expected to enable facile
synthesis of second-generation probes.
The potential utility of a channel-targeted ABPP strategy depends
on whether it will be generalizable to a large number of structurally
distinct channels. Large changes in pore structure (as judged by
accessibility to reactive probes in solution) have been observed for
other Cys-Loop receptors such as the serotonin receptor,23 as well
as glutamate receptors24 and potassium channels.25 In addition, the
many characterized state-selective channel blockers and inhibitors
offer a rich set of potential pore-binding groups for ion channel
targeted ABPP probes. Ion channels as a class thus share many of
the advantages of enzyme active sites as ABPP targets and appear
likely to be a generally useful target for ABPP techniques.
Acknowledgment. This work was supported by the Beckman
Foundation and the MIT Department of Chemistry.
Figure 2. Photolabeling nAChRs expressed in live cells in the absence
(closed) or presence (desensitized) of acetylcholine (ACh) demonstrates
that BPyneTEA preferentially binds closed receptors. Closed receptors are
labeled ∼2-fold more efficiently than desensitized receptors at concentrations
of 250 µM and 50 µM. At 10 µM BPyneTEA, differential labeling was not
statistically significant.
Supporting Information Available: Synthetic, electrophysiology,
and biochemical methods; discussion of blockade models and voltage-
dependence of blockade; statistical analysis of live-cell labeling; labeling
results in the absence of light or BPyneTEA. This material is available
free of charge via the Internet at https://pubs.acs.org.
states is not statistically significant (unpaired, two-sided t test, p )
0.068). The kinetic studies also allow estimation of upper limits
for dissociation constants of BPyneTEA binding to closed and open
states: <∼20 µM for the closed state and <∼80 µM for the open
state (Supporting Figure 3B).
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