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  • The interactions of apamin and tetraethylammonium are differentially affected by single mutations in the pore mouth of small conductance calcium-activated potassium (SK) channels.

The interactions of apamin and tetraethylammonium are differentially affected by single mutations in the pore mouth of small conductance calcium-activated potassium (SK) channels.

Biochemical pharmacology (2012-12-29)
Sébastien Dilly, Fabian Philippart, Cédric Lamy, Sylvie Poncin, Dirk Snyders, Vincent Seutin, Jean-François Liégeois
ABSTRACT

Valine residues in the pore region of SK2 (V366) and SK3 (V520) were replaced by either an alanine or a phenylalanine to evaluate the impact on the interactions with the allosteric blocker apamin. Unlike TEA which showed high sensitivity to phenylalanine mutated channels, the binding affinity of apamin to the phenylalanine mutants was strongly reduced. In addition, currents from phenylalanine mutants were largely resistant to block by apamin. On the other hand, when the valine residue was replaced by an alanine residue, an increase of the binding affinity and the amount of block by apamin was observed for alanine mutated SK2 channels, but not for mutated SK3 channels. Interestingly, the VA mutation reduced the sensitivity to TEA. In silico data confirmed these experimental results. Therefore, such mutations in the pore region of SK channels show that the three-dimensional structure of the SK tetramers can be disorganized in the outer pore region leading to reduced interaction of apamin with its target.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Tetraethylammonium chloride hydrate
Sigma-Aldrich
Tetraethylammonium iodide, 98%
Sigma-Aldrich
Tetraethylammonium hydroxide solution, 35 wt. % in H2O
Sigma-Aldrich
Tetraethylammonium bromide, reagent grade, 98%
Supelco
Tetraethylammonium chloride, for electrochemical analysis, ≥99.0%
Sigma-Aldrich
Tetraethylammonium chloride, BioUltra, for molecular biology, ≥99.0% (AT)
Sigma-Aldrich
Tetraethylammonium hydroxide solution, ~25% in methanol (~1.5 M)
Sigma-Aldrich
Tetraethylammonium bromide, ReagentPlus®, 99%
Supelco
Tetraethylammonium hydroxide solution, ~1.0 M (CH3CH2)4NOH in H2O, electrochemical grade
Sigma-Aldrich
Tetraethylammonium hydroxide solution, 20 wt. % in H2O
Sigma-Aldrich
Apamin, synthetic, ≥97% (HPLC)
Sigma-Aldrich
Apamin, from bee venom, ≥95% (HPLC)
Sigma-Aldrich
Tetraethylammonium chloride, ≥98% (titration)