Transthyretin oligomers induce calcium influx via voltage-gated calcium channels Xu Hou,* Helena C. Parkington, Harold A. Coleman, Adam Mechler,à Lisandra L. Martin,à Marie-Isabel Aguilar* and David H. Small* *Department of Biochemistry and Molecular Biology,  Department of Physiology and àSchool of Chemistry, Monash University, Clayton, Victoria 3800, Australia Abstract The deposition of transthyretin (TTR) amyloid in the PNS is a major pathological feature of familial amyloidotic polyneurop- athy. The aim of the present study was to examine whether TTR could disrupt cytoplasmic Ca 2+ homeostasis and to determine the role of TTR aggregation in this process. The aggregation of amyloidogenic TTR was examined by solution turbidity, dynamic light scattering and atomic force microsco- py. A nucleation-dependent polymerization process was observed in which TTR formed low molecular weight aggre- gates (oligomers < 100 nm in diameter) before the appear- ance of mature fibrils. TTR rapidly induced an increase in the concentration of intracellular Ca 2+ ([Ca 2+ ] i ) when applied to SH-SY5Y human neuroblastoma cells. The greatest effect on [Ca 2+ ] i was induced by a preparation that contained the highest concentration of TTR oligomers. The TTR-induced increase in [Ca 2+ ] i was due to an influx of extracellular Ca 2+ , mainly via L- and N-type voltage-gated calcium channels (VGCCs). These results suggest that increasing [Ca 2+ ] i via VGCCs may be an important early event which contributes to TTR-induced cytotoxicity, and that TTR oligomers, rather than mature fibrils, may be the major cytotoxic form of TTR. Keywords: amyloid, atomic force microscopy, fura-2, oligo- mers, transthyretin, voltage-gated calcium channels. J. Neurochem. (2007) 100, 446–457. Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant neurodegenerative disease that occurs in the PNS. It is characterized by the extracellular deposition of amyloid fibrils that are predominantly composed of misfolded trans- thyretin (TTR) (Andrade 1952; Costa et al. 1978). TTR is produced in the liver and the choroid plexus as a carrier protein for thyroxine (Schreiber and Richardson 1997). The native structure of TTR consists of four identical subunits that form an extensive b-sheet structure (Blake et al. 1974). More than 80 pathogenic mutations have been identified in TTR which lead to misfolding and formation of amyloid deposits (Connors et al. 2003). Although a few studies have investigated how amyloid- ogenic TTR induces its cytotoxic effects (Sousa et al. 2001; Monteiro et al. 2006; Teixeira et al. 2006), the mechanisms of TTR-induced neurotoxicity remain poorly understood. We have recently demonstrated that binding of TTR to the plasma membrane correlates with TTR-induced cytotoxicity, and increased membrane fluidity was implicated as an initial event in the mechanism (Hou et al. 2005). However, the downstream cellular events that lead to neurotoxicity remain obscure. It has been reported that a b-amyloid protein (Ab)-induced increase in the concentration of intracellular Ca 2+ ([Ca 2+ ] i ) is involved in the neurotoxicity in Alzheimer’s disease (AD) (Mattson et al. 1992; Mattson Received June 7, 2006; revised manuscript received July 16, 2006; accepted August 14, 2006. Address correspondence and reprint requests to Associate Professor David H. Small, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. E-mail: David.Small@med.monash.edu.au Abbreviations used:Ab, b-amyloid protein; AD, Alzheimer’s disease; AFM, atomic force microscopy; AgTX TK, x-agatoxin TK; [Ca 2+ ] i , concentration of intracellular calcium; CL, cholesterol; Ctrl-N, negative control; Ctrl-P, positive control; CTX GVIA, x-conotoxin GVIA; DLS, dynamic light scattering; DMEM, Dulbecco’s modified Eagle’s medium; DMPC, dimyristoyl-L-a-phosphatidylcholine; DMPE, dimyristoyl-L-a- phosphatidylethanolamine; DMPS, dimyristoyl-L-a-phosphatidylserine; ER, endoplasmic reticulum; FAP, familial amyloidotic polyneuropathy; fura-2 AM, fura-2 acetoxymethyl ester; HBSS, Hanks’ balanced salt solution; HiK, high potassium; NFP, nifedipine; PrP, prion proteins; SM, sphingomyelin; TSG, thapsigargin; TTR, transthyretin; VGCC, voltage- gated calcium channel; WT, wild type. Journal of Neurochemistry , 2007, 100, 446–457 doi:10.1111/j.1471-4159.2006.04210.x 446 Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 446–457 Ó 2006 The Authors