Allosteric Block of KCa2 Channels by Apamin

Cédric Lamy,Samuel J Goodchild,Kate L Weatherall,David E Jane,Jean-François Liégeois,Vincent Seutin,Neil V Marrion

doi:10.1074/jbc.m110.110072

Cédric Lamy, Samuel J Goodchild + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110.110072

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Abstract

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.

Highlights

Point mutations within the outer pore region of KCa2 channels have been shown to affect the sensitivity of block by apamin [16, 29], which has led to the assumption that the bee venom toxin acts as a pore blocker
It has been recently reported that a point mutation in the S3-S4 extracellular loop had a major impact on the sensitivity of hKCa2.1 current to block by apamin [33]
This information places doubt on whether apamin can act as a pore blocker, as it is unlikely that apamin is large enough to bind to an extracellular loop and traverse deep into the pore to cause block

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Cell and Membrane Preparation—Wildtype rat KCa2.2 (GenBankTM accession number NM_019314) and human KCa2.3 (GenBank accession number AF031815) channel DNAs were subcloned into the mammalian plasmid expression vectors pcDNA3 (Invitrogen, Paisley, UK) and pFLAGCMV2 (Sigma, Poole, UK), respectively. Allosteric Block of KCa2 Channels by Apamin (Invitrogen), supplemented with 10% fetal calf serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) at 37 °C They were plated onto 35-mm dishes (Falcon) 48 h before transfection. HEK293 cells were plated on a 100-mm dish for 2 days and transfected with the corresponding plasmid using the polyethyleneimine method without enhanced green fluorescent protein. Cells were bathed in a control external solution that consisted of KCl (120 mM), HEPES (10 mM), EGTA (10 mM), CaCl2 (6.19 mM, calculated free [Ca2ϩ]i 60 nM), MgCl2 (1.44 mM, calculated free [Mg2ϩ]i 1 mM), pH 7.4, with ϳ40 mM KOH. Apamin and D-TC solutions were prepared on the day of experiments from a frozen stock of 100 ␮M and 1 mM in water, respectively.

The abbreviations used are

RESULTS

Findings

DISCUSSION