16 G. D. Cymes and C. Grosman
and/or deprotonated open-channel dwell times may follow exponential distributions
with more than a single component even within an individual opening, which would
suggest that proton transfer to and from the engineered amino-acid side chain does
not proceed as a two-state, one-step chemical reaction.
In the particular case of our application of this method to the muscle AChR,
channel openings elicited in the presence of 1-μM ACh occurred either as “bursts”
of long openings in quick succession (usually displaying many main-level–sub-
level or main-level–superlevel interconversions) or as isolated short openings that
were too short for the interconversions to be clearly detected. Thus, the pKa values
Fig. 9 Kinetic interpretation of open-channel current-level fluctuations. The channel intercon-
verts among closed, desensitized (collectively referred to as “shut states”) and open conformations
with or without an extra proton bound to the pore domain. The association and dissociation of a
single proton to and from the pore domain of the open channel may manifest as a discrete change
in the rate of ion flow. Kinetic analysis of these fluctuations, and of the open-to-shut transitions,
yields the rates of protonation and deprotonation; these rates, along with the pH of the solution,
can be combined to calculate the pKa of the engineered side chain. The three proton donors and
three proton acceptors present in the solutions are indicated. BH and B− denote the protonated and
deprotonated forms of the pH-buffer, respectively. Note that the kinetics of both proton transfer
and channel shutting affect the duration of sojourns in the two levels of open-channel current.
(Reproduced from Cymes et al. 2005 )