Allosteric block of KCa2 channels by apamin.
Activation of small conductance calcium-activated potassium (K(Ca)2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K(Ca)2.2 and K(Ca)2.3 with the same high affinity (K(D) approximately 5 pM for both subtypes) but requires significantly higher concentrations to block functional current (IC(50) values of approximately 100 pM and approximately 5 nM, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K(Ca)2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.
Lamy
Cédric
C
Laboratory of Pharmacology and Groupe Interdisciplinaire de Génoprotéomique Appliquée Neurosciences, University of Liège, 4000 Liège, Belgium.
Goodchild
Samuel J
SJ
Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Weatherall
Kate L
KL
Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Jane
David E
DE
Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Liégeois
Jean-François
JF
Laboratory of Medicinal Chemistry and Centre Interfacultaire de Recherche du Medicament, University of Liège, 4000 Liège, Belgium.
Seutin
Vincent
V
Laboratory of Pharmacology and Groupe Interdisciplinaire de Génoprotéomique Appliquée Neurosciences, University of Liège, 4000 Liège, Belgium.
Marrion
Neil V
NV
Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom. Electronic address: N.V.Marrion@bris.ac.uk.
D000494:Allosteric Regulation / Q000187:drug effects / Q000235:genetics; D000495:Allosteric Site / Q000235:genetics; D000818:Animals; D001030:Apamin / Q000737:chemistry / Q000494:pharmacology*; D001516:Bees / Q000737:chemistry; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008958:Models, Molecular; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019789:Tetraethylammonium / Q000494:pharmacology
C496899:Kcnn2 protein, rat; D026902:Potassium Channel Blockers; D051640:Small-Conductance Calcium-Activated Potassium Channels; D001030:Apamin; D019789:Tetraethylammonium