Oligomeric forms of Aβ peptides are implicated in Alzheimer’s disease (AD) and disrupt membrane integrity, leading to cytosolic calcium (Ca²⁺) elevation. Proposed mechanisms by which Aβ mediates its effects include lipid destabilization, activation of native membrane channels, and aggregation of Aβ into Ca²⁺-permeable pores. We distinguished between these using total internal reflection fluorescence (TIRF) microscopy to image Ca²⁺ influx in Xenopus laevis oocytes. Aβ1–42 oligomers evoked single-channel Ca²⁺ fluorescence transients (SCCaFTs), which resembled those from classical ion channels but which were not attributable to endogenous oocyte channels. SCCaFTs displayed widely variable open probabilities (P_o) and stepwise transitions among multiple amplitude levels reminiscent of subconductance levels of ion channels. The proportion of high P_o, large amplitude SCCaFTs grew with time, suggesting that continued oligomer aggregation results in the formation of highly toxic pores. We conclude that formation of intrinsic Ca²⁺-permeable membrane pores is a major pathological mechanism in AD and introduce TIRF imaging for massively parallel single-channel studies of the incorporation, assembly, and properties of amyloidogenic oligomers.

Oligomeric forms of β-amyloid (Aβ) peptides associated with Alzheimer's disease (AD) disrupt cellular Ca²⁺regulation by liberating Ca²⁺into the cytosol from both extracellular and intracellular sources. We elucidated the actions of intracellular Aβ₄₂by imaging Ca²⁺responses to injections of Aβ oligomers intoXenopusoocytes. Two types of signal were observed: (1) local, “channel-like” transients dependent on extracellular Ca²⁺influx, which resembled signals from amlyoid pores formed by extracellular application of oligomers; and (2) local transients and global Ca²⁺waves, resembling Ca²⁺puffs and waves mediated by inositol trisphosphate (IP₃). The latter responses were suppressed by antagonists of the IP₃receptor (caffeine and heparin), pretreatment with the G_i/o-protein inhibitor pertussis toxin, and pretreatment with lithium to deplete membrane inositol lipids. We show that G-protein-mediated stimulation of IP₃production and consequent liberation of Ca²⁺from the endoplasmic reticulum by intracellular Aβ oligomers is cytotoxic, potentially representing a novel pathological mechanism in AD which may be further exacerbated by AD-linked mutations in presenilins to promote opening of IP₃receptor/channels.

We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in mammalian cells. Orai1 was detected predominantly as dimers under resting conditions and as tetramers when coexpressed with C-STIM1 to activate Ca ²⁺ influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca ²⁺ , redistribution of STIM1 to the plasma membrane, and STIM1/Orai1 puncta formation, and may cause the channel to be in the activated state. Consistent with this possibility, Orai1 was found predominantly as a dimer when coexpressed with STIM1 in living cells under resting conditions. We further show that Orai3, like Orai1, is dimeric under resting conditions and is predominantly tetrameric when activated by C-STIM1. Interestingly, a dimeric Orai3 stoichiometry was found both before and during application of 2-aminoethyldiphenyl borate (2-APB) to activate a nonselective cation conductance in its STIM1-independent mode. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca ²⁺ -selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.

Neurotransmitter release from presynaptic terminals is primarily regulated by rapid Ca²⁺ influx through membrane-resident voltage-gated Ca²⁺ channels (VGCCs). Moreover, accumulating evidence indicates that the endoplasmic reticulum (ER) is extensively present in axonal terminals of neurons and plays a modulatory role in synaptic transmission by regulating Ca²⁺ levels. Familial Alzheimer’s disease (FAD) is marked by enhanced Ca²⁺ release from the ER and downregulation of Ca²⁺ buffering proteins. However, the precise consequence of impaired Ca²⁺ signaling within the vicinity of VGCCs (active zone (AZ)) on exocytosis is poorly understood. Here, we perform in silico experiments of intracellular Ca²⁺ signaling and exocytosis in a detailed biophysical model of hippocampal synapses to investigate the effect of aberrant Ca²⁺ signaling on neurotransmitter release in FAD. Our model predicts that enhanced Ca²⁺ release from the ER increases the probability of neurotransmitter release in FAD. Moreover, over very short timescales (30–60 ms), the model exhibits activity-dependent and enhanced short-term plasticity in FAD, indicating neuronal hyperactivity—a hallmark of the disease. Similar to previous observations in AD animal models, our model reveals that during prolonged stimulation (~450 ms), pathological Ca²⁺ signaling increases depression and desynchronization with stimulus, causing affected synapses to operate unreliably. Overall, our work provides direct evidence in support of a crucial role played by altered Ca²⁺ homeostasis mediated by intracellular stores in FAD.