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 = 147 microM) or intracellularly (IC50 = 117 microM). 4. Inclusion of the first twenty amino acids of the N-terminus sequence of the Shaker B K+ channel ('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 inactivation peptide, and under these conditions extracellular 4-AP had a reduced potency (IC50 values of 471 and 537 microM for 0.5 and 2 mg ml-1 inactivation peptide, respectively). 5. A permanently charged analogue of 4-AP, 4-aminopyridine methiodide (4-APMI), was found to block mKv1.1 when applied inside the cell, but was without effect when administered externally. 6. Decreasing the intracellular pH (pHi) to 6.4 caused an increase in 4-AP potency (IC50 = 76 microM), whereas at pHi 9.0, the 4-AP potency fell (IC50 = 295 microM). Conversely, increasing extracellular pH (pHo) to 9.0 caused an increase in 4-AP potency (IC50 = 93 microM), whereas at pHo 6.4, 4-AP potency decreased (IC50 = 398 microM). 7. Taken together, these findings support the hypotheses that the uncharged form of 4-AP crosses the membrane, and that it is predominantly the cationic form which acts on mKv1.1 channels intracellularly, possibly at or near to the binding site for the inactivation peptide.
Stephens
G J
GJ
Electrophysiology Laboratory, Wyeth Research, UK.
Garratt
J C
JC
Robertson
B
B
Owen
D G
DG
D015761:4-Aminopyridine / Q000378:metabolism / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000631:Aminopyridines / Q000494:pharmacology; D000818:Animals; D001923:Brain Chemistry; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000494:pharmacology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000378:metabolism; D014162:Transfection
D000631:Aminopyridines; D010446:Peptide Fragments; D026902:Potassium Channel Blockers; D015221:Potassium Channels; C084482:4-aminopyridine methiodide; D015761:4-Aminopyridine