Bacterial Expression of the Ryanodine Receptor Pore Forming Region and a Potassium Channel Chimera

Abstract

Several lines of evidence exist to support the proposal that RyR2 contains a pore composed of structural elements analogous to the pore forming regions (PFR) of K+ channels. Our analogy model, constructed using the bacterial potassium channel KcsA as the template, encompasses transmembrane helices 8-10 and provides an excellent description for the pore domain of RyR2 with the overall structure and arrangement of key structural elements of the model closely resembling those of KcsA (Biophysical Journal 2004, 87, 2335-2351).Although good progress has been made in understanding ion handling capabilities in RyR2 the exact mechanisms remain elusive and controversial. To test and define the analogy model we have constructed and expressed both the PFR of RyR2 alone and a KcsA_RyR2_PFR chimera whereby the 22 residue N-terminal helix and 41 residue C-terminal domain of KcsA have been added by primer extension to the RyR2 PFR region and cloned into a modified pET expression vector containing an N-terminal hexa-His tag. Just as voltage sensor modules are transferable among potassium channels, our chimera will give us information on the functionality and transferability of the RyR2 PFR. Preliminary results indicate that both the chimera and PFR constructs express in large amounts in rosetta bacterial cells and are targeted to the membrane with no detectable protein in the soluble fraction. Detergent trials have identified LDAO as the best candidate for solubilisation from the membrane although other commonly used detergents are also capable of solubilisation, albeit to a lesser degree. Both constructs were purified as tetramers following nickel-affinity and gel filtration chromatography. This suggests strongly that both the RyR2 PFR and chimera are functional proteins capable of tetramerisation.Supported by the British Heart Foundation.