Abstract
Isoform 2 of the ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. In the present study, two kinds of RyR2 cDNA were constructed, one encoding the wild type mouse RyR2 (RyR2(wt)) and the other encoding modified RyR2, into which was inserted a cDNA encoding green fluorescent protein (GFP). GFP was inserted into the divergent region 1 (DR1) of RyR2, after the Asp-4365 (RyR2(D4365-GFP)). HEK293 cells expressing both RyR2(wt) and RyR2(D4365-GFP) cDNAs showed caffeine- and ryanodine-sensitive calcium release, demonstrating that both wild type and modified RyR2s form functional calcium release channels. Cells expressing the fusion protein, RyR2(D4365-GFP), were readily identified by their fluorescence due to the presence of GFP, indicating that the inserted GFP folded properly. Both expressed RyR2s were purified from cell lysates in a single step by affinity chromatography using a GST-FKBP12.6 as the affinity ligand. Cryoelectron microscopy of purified RyR2s showed structurally intact receptors, and three-dimensional reconstructions were obtained by single particle image processing. The three-dimensional reconstruction of RyR2(wt) appeared very similar to that of the native RyR2 purified from dog heart. The location of the inserted GFP, and consequently of DR1, was mapped on the three-dimensional structure of RyR2 to one of the subunit's characteristic domains, domain 3, also known as the "handle" domain. This study describes the first internal fusion of a protein into a ryanodine receptor, and it demonstrates the potential of this technology for localizing functional and structural domains on the three-dimensional structure of RyR.
Highlights
Isoform 2 of the ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle
1 The abbreviations used are: RyR2, type 2 RyR or cardiac RyR; RyR, ryanodine receptor; RyR1 and -3, RyR isoform 1 and 3, respectively; DR1, -2, and -3, divergent region 1, 2, and 3, respectively; EM, electron microscopy; FKBP12.6, 12.6-kDa FK506-binding protein; GFP, green fluorescent protein; GST, glutathione S-transferase; PIPES, 1,4-piperain the heart, where it plays a crucial role in excitation-contraction coupling [1]
HEK293 cells were transfected with 12 g of RyR2 cDNAs
Summary
RyR2, type 2 RyR or cardiac RyR; RyR, ryanodine receptor; RyR1 and -3, RyR isoform 1 and 3, respectively; DR1, -2, and -3, divergent region 1, 2, and 3, respectively; EM, electron microscopy; FKBP12.6, 12.6-kDa FK506-binding protein; GFP, green fluorescent protein; GST, glutathione S-transferase; PIPES, 1,4-piperain the heart, where it plays a crucial role in excitation-contraction coupling [1]. Reporter protein for monitoring gene expression and protein targeting [16], and it appears to be a good choice as an insertional fusion protein, because its characteristic green fluorescence depends on the proper folding of the protein [17, 18] This approach has been shown to be feasible for structural analysis of surface-exposed regions of icosahedral viruses by cryo-EM [19]. A modified RyR2, containing GFP inserted after Asp-4365 within DR1, and termed RyR2D4365-GFP, was characterized by cryo-EM and three-dimensional reconstruction This approach should be generally useful for mapping the RyR2 linear sequence onto the three-dimensional structure of the receptor, thereby providing insights into the structural basis of RyR2 functions
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