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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02936

Journal of Cell Science 119, 2386-2397 (2006)
Published by The Company of Biologists 2006
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Ryanodine receptor interaction with the SNARE-associated protein snapin

Spyros Zissimopoulos1,*, Duncan J. West2, Alan J. Williams2 and F. Anthony Lai1

1 Wales Heart Research Institute, Department of Cardiology, Cardiff University School of Medicine, Heath Park, Cardiff, CF14 4XN, UK
2 Department of Cardiac Medicine, NHLI, Imperial College London, Dovehouse Street, London, SW3 6LY, UK


Figure 1
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  		Fig. 1. Snapin interaction with the BT1B2 fragment of RyR2. (A) Schematic diagram of BT1B2 sub-overlapping fragments. (B) Protein expression of RyR2 fragments in yeast: protein extracts (50 µg per sample) of yeast Y190 transformed with plasmids as indicated were analysed by western blotting using AbMyc (12% SDS-PAGE gel; pGBKT7: empty vector). (C) Yeast two-hybrid liquid ß-galactosidase assay of yeast Y190 transformed with the plasmids as indicated. Mean values of ß-galactosidase units obtained from five individual colonies per sample, with normalisation against the positive control pVA3 + pTD1, are shown (pLAM5 + pTD1, negative control). (D) Snapin was tested for an interaction with BT1B2 using co-immunoprecipitation (IP) assays of proteins synthesised and radiolabelled in vitro. BT1B2 and snapin were co-expressed in the TNT system in the presence of canine pancreatic microsomal membranes (PMM), BT1B2 was immunoprecipitated by AbMyc, and the presence of co-precipitated snapin was analysed by autoradiography (15% SDS-PAGE gel). An aliquot of the TNT reaction (10% of the volume processed in co-IP) was included in the autoradiogram, as well as individual TNT reactions for BT1B2 and snapin to serve as molecular weight standards.

 

Figure 2
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  		Fig. 2. Mapping the RyR2-interacting region of snapin. (A) Schematic diagram of snapin sub-overlapping fragments. (B) Protein expression of snapin fragments in yeast: protein extracts (50 µg per sample) of yeast Y190 transformed with plasmids as indicated were analysed by western blotting using AbHA (12% SDS-PAGE gel; pACT2: empty vector). (C) Yeast two-hybrid liquid ß-galactosidase assay of yeast Y190 transformed with the plasmids as indicated. Mean values of ß-galactosidase units obtained from five individual colonies per sample, with normalisation against the positive control, are shown.

 

Figure 3
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  		Fig. 3. Snapin interaction with the BT1B2 fragment of RyR1 and RyR3. (A) Protein expression of the corresponding BT1B2 fragments from the three mammalian RyRs and SNAP25 in yeast: protein extracts (50 µg per sample) of yeast Y190 transformed with plasmids as indicated were analysed by western blotting using AbMyc (12% SDS-PAGE gel). (B) Yeast two-hybrid liquid ß-galactosidase assay of yeast Y190 transformed with the plasmids as indicated. Mean values of ß-galactosidase units obtained from five individual colonies per sample, with normalisation against the positive control, are shown. (C) Snapin was tested for an interaction with the corresponding BT1B2 fragment of RyR1 and RyR3 using co-immunoprecipitation (IP) assays of proteins synthesised and radiolabelled in vitro. BT1B2R1/R3 and snapin were co-expressed in the TNT system in the presence of canine pancreatic microsomal membranes (PMM), the RyR fragment was immunoprecipitated by AbMyc, and the presence of co-precipitated snapin was analysed by autoradiography (15% SDS-PAGE gel). An aliquot of the TNT reaction (10% of the volume processed in co-IP) was included in the autoradiogram, as well as individual TNT reactions for BT1B2R1/R3 and snapin.

 

Figure 4
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  		Fig. 4. Co-immunoprecipitation of native RyR with snapin. Native RyR was first immunoprecipitated (IP) with an appropriate antibody from solubilised (A) cardiac (2 mg) or skeletal muscle heavy SR (200 µg), or (B) synaptosomes (2 mg), and was subsequently incubated with radiolabelled, TNT-expressed snapin. Co-precipitated snapin was then analysed by autoradiography (15% SDS-PAGE gels). The first two lanes, showing 10% and 1% respectively, of the TNT reaction processed in the co-IPs provide a molecular weight standard and also enable estimation of co-immunoprecipitated snapin.

 

Figure 5
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  		Fig. 5. GST affinity chromatography of snapin with native RyR. GST pull-down experiments of native RyR incubated with purified GST fusion proteins and detection by western blotting. Solubilised (A) skeletal muscle (200 µg) or (B) cardiac heavy SR (1 mg) were incubated with purified GST fusion proteins (2 nmol) as indicated, proteins were precipitated with glutathione-Sepharose 4B beads, and co-precipitated RyR was analysed by western blotting using Ab2149 (4% SDS-PAGE/agarose gels). The first two lanes show loading of (A) skeletal muscle or (B) cardiac heavy SR.

 

Figure 6
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  		Fig. 6. Effect of snapin on [3H]ryanodine binding to skeletal muscle RyR. Skeletal muscle heavy SR (50 µg) was incubated at a range of buffered free Ca2+ concentrations with 10 nM [3H]ryanodine in the presence or absence of 1 µM GST-{Delta}NSnp. Non-specific binding was determined by the addition of 10 µM unlabelled ryanodine. A mean value of specific binding was determined from at least three separate experiments. For each Ca2+ concentration, the left column in the pair is the control and the right column is in the presence of snapin.

 

Figure 7
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  		Fig. 7. SNAP25 and native RyR compete for snapin binding. Co-immunoprecipitation (co-IP) experiments of native RyR incubated with snapin radiolabelled and synthesised in vitro, in the presence of increasing amounts of SNAP25. RyR was first immunoprecipitated (A) with Ab2142 from solubilised skeletal muscle heavy SR (200 µg), or (B) with Ab1093 from solubilised cardiac heavy SR (2 mg), and subsequently incubated with radiolabelled, TNT-expressed snapin together with increasing amounts of purified GST-SNAP25 as indicated (total GST content constant to 1 µM made up with GST only), and co-precipitated snapin was analysed by autoradiography (15% SDS-PAGE gels). An aliquot of the TNT reaction, 10% of the volume processed in co-IP, was included in the first lane of the autoradiogram. Densitometry analysis was carried out from three separate experiments for (C) skeletal muscle or (D) cardiac heavy SR, followed by normalisation against the control sample (with no GST-SNAP25 but in the presence of 1 µM GST only).

 

Figure 8
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  		Fig. 8. Potential role for RyR and snapin in neurotransmitter release. (A) The arrival of an action potential at the presynaptic terminal causes plasma membrane depolarisation and Ca2+ entry through voltage-operated Ca2+ channels. Ca2+ entry induces fusion of synaptic vesicles (SV) with the plasma membrane through a process involving the SNARE core complex and synaptotagmin, and this process is reinforced through the interaction of snapin with SNAP25. In addition, Ca2+ entry activates RyR channels that are mobilising Ca2+ from internal stores. RyR channel activation and/or the rise in intracellular Ca2+ might cause snapin dissociation from the RyR and translocation to SNAP25, resulting in further enhancement of neurotransmitter release. (B) Ca2+ entry through voltage-operated Ca2+ channels induces fusion of synaptic vesicles with the plasma membrane and consequent neurotransmitter release. Furthermore, RyR channels located on the endoplasmic reticulum (ER) are activated by the Ca2+-induced Ca2+-release mechanism and provide an additional source of Ca2+. Snapin interaction with the RyR might increase channel open probability and result in augmented Ca2+ release. (C) In the absence of plasma membrane depolarisation, some membrane fusion events still occur, most probably as a result of spontaneous localised Ca2+-release events from ryanodine-sensitive Ca2+ stores. Snapin interaction with the RyR might increase Ca2+ spark frequency and/or amplitude, accounting for spontaneous neurotransmitter release.

 

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© The Company of Biologists Ltd 2006