Engineered Ionizable Side Chains 19
pH? What pH? Once the ratio of occupancies of the deprotonated and protonated
forms of an engineered ionizable side chain is obtained, multiplying this value by
the concentration of protons (or hydronium cations, rather) is all that is needed to
calculate the corresponding pKa. However, because the two solutions bathing the
ends of the channel may have different pH values, the question arises as to which
solution’s pH should be used in the calculations. In our experimental work, we
found that ionizable side chains engineered in the transmembrane pore are sensitive
to the pH of both the extracellular and the intracellular solutions with the sensitiv-
ity to either solution’s pH becoming more pronounced as the side chain becomes
closer to either end. To avoid ambiguities, then, whenever we estimated rates and
pKa values from cell-attached recordings, we tried to keep the pH of the pipette
solution at 7.4, a value that is close to that of the cytosol. In a few cases, however,
when lysines or histidines were engineered at sites that strongly favor the deproton-
ated form of the side chains, the pH of the pipette was lowered to 6.0 to increase the
mean number of main-level–sublevel current fluctuations per burst of openings, and
the reported pKas were calculated using (arbitrarily) the pH of the pipette solution.
Fig. 11 pH-buffer concentra-
tion dependence of the rates
and equilibrium constant
of proton transfer. The data
(outside-out configuration;
− 100 mV; pHpipette 7.4;
pHbath 7.4; 1-μM ACh) were
recorded from HEK-293 cells
transiently expressing the
δS12ʹK mutant. The indicated
concentrations of HEPES
are those of the bath and
pipette solutions. Because of
the expected linear depen-
dence of the deprotonation
and protonation rates on
the total concentration of
pH-buffer [that is, the sum
of the concentrations of
the deprotonated (B−) and
protonated (BH) forms of
the buffer; Fig. 9 ], the data
points in (a) and (b) were
fitted with straight lines. The
pKa, however, is expected to
remain the same as the pH-
buffer concentration changes,
and thus, the data points in
(c) were not fitted with any
function