Figure 12.
Electrostatic interactions determine the channel activation rate. (A) Inside-out current recordings illustrating the effect of charge neutralization and/or charge reversal on KCa3.1 activation kinetics. Measurements were performed in symmetrical 200-mM K2SO4 conditions at a pipette potential of 60 mV. The current records presented in this figure demonstrate that the mutation E363R resulted in a 45-fold increase of the channel activation time constant ton, whereas charge reversal at 362 with the R362E mutant caused a fourfold decreased in ton. (B) First latency analysis for the E363C and E363R mutants. The vertical spike marks the time at which the internal solution was switched from 0 (EGTA; thick line) to 25 µM Ca2+. Double arrow lines refer to latency period before channel opening. Unitary current inside-out recordings were performed in symmetrical 200-mM K2SO4 conditions at 100 mV (E363C) or 60 mV (E363R) of applied pipette potential. Latency of 22 ± 4 s (n = 3) and 1.1 ± 0.2 s (n = 3) was estimated for E363R and E363C, respectively, compared with 0.22 ± 0.07 s (n = 8) for KCa3.1 WT. The channel steady-state open probability once activated was estimated at 0.15 ± 0.08 (n = 4) and 0.12 ± 0.02 (n = 3) for E363R and E363C channels, respectively, compared with 0.22 ± 0.07 (n = 8) for KCa3.1 WT. (C) Gradual decrease in ton resulting from charge modification at position 362 from one positive charge (R362) to one negative charge (R362E). As observed with E363, the presence of a net positive charge at either 362 or 363 tends to increase ton, with the most prominent effect observed with the E363R mutant. Asterisks indicate a significant difference from WT (*, P < 0.05). (D) Horizontal bar graph summarizing the results on the contribution of electrostatic interactions to the action of either E363 or R362 to the channel activation time constant. The double mutation R362E/E363R obtained by swapping the charges on 362 and 363 led to a ton of 1,700 ± 200 ms (n = 6) compared with 306 ± 47 ms (n = 8) for WT. The increase in activation time with the E363R mutation could also be partly reversed by neutralizing the polar and/or charged residues predicted to interact with E363. The most important effects were seen with the mutation S367C with a ton of 4,000 ± 100 ms (n = 5) for the E363R/S367C double mutant, a value significantly different from the single E363R channel. We concluded that there was a strong functional coupling between E363 and S367 during channel activation. Asterisks indicate a significant difference from WT (**, P < 0.001). (E) Plot of the relative channel activation energy as a function of the total charge for the residues at positions 362 and 363. The activation energy relative to WT was taken as KT ln(ton_mutant/ton_wt), where K and T have their usual meanings. The linear fit corresponds to a correlation coefficient of 0.9 and a difference in energy of 0.7 kcal/mol per elementary charge. This analysis supports a model whereby electrostatic interactions involving R362 and E363 play a prominent role in the KCa3.1 activation process.