TABLE 2

Dissociation rates koff and calculated KD values

Macroscopic currents were recorded from outside-out macropatches at − 100 mV. The solution was switched from one containing amiloride or benzamil at a high concentration (1−100 μM) to a solution without drug. The resulting current increase was fitted to a single exponential τ and the off-rate koff was calculated as 1/τ.n = 2 to 4 experiments per condition. The equilibrium dissociation constant KD was calculated from on- and off-rates taken from Tables 1 and 2 as indicated.

koffKD = koff/kon
Mutant Amiloride Benzamil Amiloride Benzamil
S−1 μM
wt(αβγ) 2.09 ± 0.40 0.11 ± 0.03 0.026 ± 0.008 0.002 ± 0.001
αS583C 27.6 ± 2.96 2.66 ± 0.65 0.258 ± 0.028 N.D.
βG525C N.D. 20.6 ± 5.9 N.D. 1.212 ± 0.071
αβ 17.0 ± 3.0 3.19 ± 0.73 0.250 ± 0.099 0.054 ± 0.020
  • N.D., not determined.