The effect of deep pore mutations on the action of phenylalkylamines on the Kv1.3 potassium channel.
We investigated the action of the phenylalkylamines verapamil and N-methyl-verapamil on the Kv1.3 potassium channel using the whole-cell configuration of the patch-clamp technique. Our goal was to identify their binding as a prerequisite for using the phenylalkylamines as small, well-defined molecular probes, not only to expand the structural findings made with peptide toxins or by crystallization, but also to use them as lead compounds for the generation of more potent and therefore more specific K+ channel modulators. Competition experiments with charybdotoxin, known to interact with external residues of Kv1.3, showed no interaction with verapamil. The internal application of quarternary N-methyl-verapamil in combination with verapamil suggested competition for the same internal binding site. Verapamil affinity was decreased 6 fold by a mutation (M395V) in a region of the internal pore which forms part of the internal tetraethylammonium (TEA+) binding site, although mutations at neighbouring residues (T396 and T397) were without effect. Modification of C-type inactivation by mutations in the internal pore suggest that this region participates in the inactivation process. The action of phenylalkylamines and local anaesthetics on L-type Ca2+ channels and Na channels, respectively, and verapamil on Kv1.3 indicate very similar blocking mechanisms. This might allow the use of these compounds as molecular probes to map the internal vestibule of all three channel types.
Rauer
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Department of Physiology and Biophysics, University of California, Irvine 92697-4560, USA.
Grissmer
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S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000187:drug effects; D020746:Calcium Channels, L-Type; D003460:Crystallization; D051664:Kv1.3 Potassium Channel; D008961:Models, Structural; D008969:Molecular Sequence Data; D009154:Mutation; D015221:Potassium Channels / Q000737:chemistry / Q000187:drug effects*; D024642:Potassium Channels, Voltage-Gated; D051381:Rats; D015222:Sodium Channels / Q000187:drug effects; D013329:Structure-Activity Relationship; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000378:metabolism / Q000494:pharmacology*
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; C496975:Kcna3 protein, rat; D051664:Kv1.3 Potassium Channel; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D015222:Sodium Channels; D013757:Tetraethylammonium Compounds; D014700:Verapamil