Determinants of 4-aminopyridine sensitivity in a human brain kv1.4 k(+) channel: phenylalanine substitutions in leucine heptad repeat region stabilize channel closed state.

The biophysical and pharmacological effects of individual phenylalanine-for-leucine (Phe-for-Leu) substitutions in the leucine heptad repeat region located at the cytosolic surface of the channel pore, on whole-cell K(+) currents, were studied in cloned and mutated human brain Kvl.4 K(+) channels (hKvl.4) transiently transfected into HeLa cells. Although L2 and L5 are not considered part of the 4-aminopyridine (4-AP) binding site, unlike the L4 heptad leucine, Phe substitutions at L2 (L464) or L5 (L485) increase 4-AP sensitivity by 400-fold, as seen previously in the L4F mutant channel. Greater depolarizing shifts manifest in the voltage dependence of activation and inactivation in L2F (20 mV) and L5F (30 mV) than in L4F (10 mV) relative to hKv1.4. L1F (L457) and L3F (L471) increase 4-AP sensitivity by 8- and 150-fold, respectively, and produce depolarizing shifts in activation of approximately 5 mV without affecting inactivation. The apparent free energy differences of 4-AP binding in each mutant suggest enhanced drug-channel interactions (L2F > or = L4F > or = L5F > L3F > L1F). Deactivation kinetics are accelerated in L2F (11-fold), L5F (8-fold), L1F (5-fold), and L3F (2-fold), at -50 mV. All Phe-for-heptad-Leu substitutions produce gating changes suggesting variable stabilization of the channel closed state conformation, with L1F, L2F, and L5F exhibiting the strongest correlations between altered gating and increased 4-AP sensitivity. If 4-AP blocks the open channel by promoting closure of the activation gate (recent Armstrong-Loboda model), then changes in the leucine heptad repeat that stabilize the channel closed state may contribute to increased 4-AP sensitivity by amplifying the mechanism of 4-AP block.