Pharmacology of a cloned potassium channel from mouse brain (MK-1) expressed in CHO cells: effects of blockers and an 'inactivation peptide'.
1. Chinese hamster ovary cells (CHO), maintained in cell culture, were stably transfected with DNA for the MK-1 voltage-activated potassium channel, previously cloned from a mouse brain library. 2. Voltage-activated currents were recorded by the whole cell patch clamp method. In CHO cells transfected with the vector only, there were no significant outward voltage activated currents. However, large outward voltage-activated potassium currents were always observed in those cells which had been transfected with the vector containing the DNA encoding for MK-1. 3. These potassium currents activated from -40 mV, and reversed at the potassium equilibrium potential. The half-maximal conductance of MK-1 was at -10 mV and had a slope factor of 11 mV when fitted with a Boltzmann function. There was only very slight (< 10%) inactivation of MK-1 even at very large positive voltages. 4. MK-1 was reversibly blocked by: 4-aminopyridine (4-AP, 0.1-4 mM), Toxin I 10-100 nM), mast cell degranulating peptide (1 microM), tetraethylammonium (TEA, 4-10 mM), tedisamil (100 microM), quinine (100 microM) and ciclazindol (100 microM); all applied to the outside of the cell from a 'U tube' rapid perfusion system. 4-AP may block closed as well as open MK-1 potassium channels. 5. A synthetic 20 amino acid peptide derived from the N-terminus sequence of the Shaker B potassium channel (the 'inactivation peptide') produced dramatic inactivation of MK-1 when applied to the inside, but not the outside of the cell. Reducing peptide concentration or 'degrading' the peptide produced less inactivation. 6. The block of MK-1 by the synthetic inactivation peptide was quite different in time dependence from block by internal TEA (0.4-4 mM), which probably blocks much more quickly but less potently than the peptide.
Robertson
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Electrophysiology Laboratory, Wyeth Research, Taplow, Maidenhead.
Owen
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DG
D015761:4-Aminopyridine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry / Q000187:drug effects* / Q000235:genetics; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004594:Electrophysiology; D047908:Intracellular Signaling Peptides and Proteins; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008818:Mice, Neurologic Mutants; D008839:Microelectrodes; D008969:Molecular Sequence Data; D009431:Neural Conduction / Q000187:drug effects; D010455:Peptides / Q000494:pharmacology*; D015221:Potassium Channels / Q000187:drug effects* / Q000235:genetics; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D015221:Potassium Channels; D013757:Tetraethylammonium Compounds; C076103:Shaker B inactivating peptide; D015761:4-Aminopyridine