ON THE MECHANISM OF 4-AMINOPYRIDINE ACTION ON THE CLONED MOUSE-BRAIN POTASSIUM CHANNEL MKV1.1

1. This study used the whole-cell patch clamp technique to investigate the mechanism of action of the K+ channel blocker 4-aminopyridine (4- AP) on the cloned K+ channel mouse Kv1.1 (mKv1.1) expressed in Chinese hamster ovary cells. 2. Cells transfected with mKv1.1 expressed a non -inactivating, delayed rectifier-type K+ current. 4-AP induced a dose- , voltage- and use-dependent block of mKv1.1. 3. 4-AP blockade of mKv1 .1 was similar whether 4-AP was administered extracellularly (IC50 = 1 47 mu M) or intracellularly (IC50 = 117 mu M). 4. Inclusion of the fir st twenty amino acids of the N-terminus sequence of the Shaker B K+ ch annel ('inactivation peptide') in the patch electrode transformed mKv1 .1 into a rapidly inactivating current. The time constant of decay for the modified current was dependent on the concentration of inactivati on peptide, and under these conditions extracellular 4-AP had a reduce d potency (IC50 values of 471 and 537 mu M for 0.5 and 2 mg ml(-1) ina ctivation peptide, respectively) 5. A permanently charged analogue of 4-AP, 4-aminopyridine methiodide (4-APMI), was found to block mKv1.1 w hen applied inside the cell, but was without effect when administered externally. 6. Decreasing the intracellular pH (pH(i)) to 6.4 caused a n increase in 4-AP potency (IC50 = 76 mu M), whereas at pH(i) 9.0, the 4-AP potency fell (IC50 = 295 mu M). Conversely, increasing extracell ular pH (pH(o)) to 9.0 caused an increase in 4-AP potency (IC50 = 93 m u M), whereas at pH(o) 6.4, 4-AP potency decreased (IC50 = 398 mu M). 7. Taken together, these findings support the hypotheses that the unch arged form of 4-AP crosses the membrane, and that it is predominantly the cationic form which acts on mKv1.1 channels intracellularly, possi bly at or near to the binding site for the inactivation peptide.