20 G. D. Cymes and C. Grosman
Although recording from excised patches would have allowed us to use solutions
of the same pH on either side of the membrane, the requirement for long and stable
patch-clamp recordings seemed to justify the use of the cell-attached configura-
tion. In some cases, however, we did record single-channel currents from excised
patches, and in these cases, the pipette and bath solutions had the same pH, unless
of course, the effect of pH-sidedness was being studied. Excised-patch recordings
were preferred over cell-attached recordings only in two situations: (1) when the
ionizable side chains were engineered at the intracellular end of the pore, where
the protonatable groups would “feel” the pH of the cytosol much more than the pH
of the pipette solution; and (2) when a quantitative analysis of the effect of differ-
ent pH values on the rates and equilibrium constant of proton transfer was desired
(Fig. 10 ).
The pH values we are referring to, here, are bulk values, that is, the pH values we
measure when we prepare the solutions. Clearly, these values may differ from those
in the microenvironment of the ionizable moieties, but the definition of an equilib-
rium constant calls for the bulk concentrations of reactants and products. Indeed,
when we estimate the dissociation equilibrium constant ( KD) of a ligand from its
Fig. 12 Voltage dependence
of the rates and equilibrium
constant of proton transfer.
The data (cell-attached
configuration; pHpipette 7.4;
10-mM pH-buffer; 1-μM
ACh) were recorded from
HEK-293 cells transiently
expressing the δS12ʹK
mutant. The data points in
(a) and (b) were fitted with
exponential functions, and
thus, the data points in (c)
(remember that pKa = − log
Ka) were fitted with a straight
line