Agonis
t-binding kine
tics
to
the nico
tinic ace
tylcholine recep
tor (AChR) from
Torpedocalifornica were measured using sequen
tial-mixing s
topped-flow fluorescence me
thods
to de
termine
thecon
tribu
tion of each individual si
te
to agonis
t-induced opening and desensi
tiza
tion. Timed dansyl-C6-choline (DC6C) binding followed by i
ts dissocia
tion upon mixing wi
th high, compe
ting agonis
tconcen
tra
tions revealed four kine
tic componen
ts: an ini
tial, fas
t fluorescence decay, followed by a
transien
tincrease, and
then
two charac
teris
tic decays
tha
t reflec
t dissocia
tion from
the desensi
tized agonis
t si
tes.The
transien
t increase resul
ted from DC6C binding
to
the open-channel based on i
ts preven
tion by proadifen,a noncompe
ti
tive an
tagonis
t. Fur
ther charac
teriza
tion of DC6C channel binding by
the inhibi
tion of [
3H]phencyclidine binding and by equilibrium measuremen
ts of DC6C fluorescence yielded
KD values of2-4
ti
ties/mgr.gif">M for
the desensi
tized AChR and ~600
ti
ties/mgr.gif">M for
the closed s
ta
te. A
t this si
te, DC6C displayed as
trongly blue-shif
ted emission spec
trum, higher in
trinsic fluorescence, and weaker energy
transfer from
tryp
tophans
than when bound
to ei
ther agonis
t si
te. The ini
tial, fas
t fluorescence decay was assigned
toDC6C dissocia
tion from
the
ta.gif" BORDER=0 > si
te of
the AChR in i
ts closed conforma
tion, on
the basis of inhibi
tionwi
th
the si
te-selec
tive an
tagonis
ts
d-
tubocurarine and
-cono
toxin MI. Fas
t decay ampli
tude da
ta indica
tedan apparen
t affini
ty of 0.9
ti
ties/mgr.gif">M for
the closed-s
ta
te
ta.gif" BORDER=0 > si
te;
the closed-s
ta
te
-si
te affini
ty is inferred
tobe near 100
ti
ties/mgr.gif">M. These values and
the known affini
ties for
the desensi
tized conforma
tion show
tha
t the
si
te drives AChR desensi
tiza
tion
to a ~40-fold grea
ter ex
ten
t than
the
ta.gif" BORDER=0 > si
te, undergoes energe
ticallylarger conforma
tional changes, and is
the primary de
terminan
t of agonis
t po
tency.