Autophagy and cancer drug resistance in dialogue: Pre-clinical and clinical evidence.

The emergence of drug resistance is a major challenge for oncologists. Resistance can be categorized as acquired or intrinsic; the alteration of several biological mechanisms contributes to both intrinsic and acquired resistance. Macroautophagy/autophagy is the primary process in eukaryotes for the degradation of macromolecules and organelles. This process is critical in maintaining cellular homeostasis. Given its function as either a pro-survival or a pro-death phenomenon, autophagy has a complex physio-pathological role. In some circumstances, autophagy can confer chemoresistance and promote cell survival, whereas in others it can promote chemosensitivity and contribute to cell death. The role of autophagy in the modulation of cancer drug resistance reflects its impact on apoptosis and metastasis. The regulation of autophagy in cancer is mediated by various factors including AMP-activated protein kinase (AMPK), MAPK, phosphoinositide 3-kinase (PI3K)-AKT, BECN1 and ATG proteins. Non-coding RNAs are among the main regulators of autophagy, e.g., via the modulation of chemoresistance pathways. Due to the significant contribution of autophagy in cancer drug resistance, small molecule modulators and natural compounds targeting autophagy have been introduced to alter the response of cancer cells to chemotherapy. Furthermore, nanotherapeutic approaches based on autophagy regulation have been introduced in pre-clinical cancer therapy. In this review we consider the potential for using autophagy regulators for the clinical treatment of malignancies.

Cardiac pathology in neuronal ceroid lipofuscinoses (NCL): More than a mere co-morbidity.

The neuronal ceroid lipofuscinoses (NCLs) are mostly seen as diseases affecting the central nervous system, but there is accumulating evidence that they have co-morbidities outside the brain. One of these co-morbidities is a decline in cardiac function. This is becoming increasingly recognised in teenagers and adolescents with juvenile CLN3, but it may also occur in individuals with other NCLs. The purpose of this review is to summarise the current knowledge of the structural and functional changes found in the hearts of animal models and people diagnosed with NCL. In addition, we present evidence of structural changes that were observed in a systematic comparison of the cardiomyocytes from CLN3Δex7/8 mice.

T-type calcium channels drive the proliferation of androgen-receptor negative prostate cancer cells.

BACKGROUND: Androgen deprivation therapy (ADT) is the treatment of choice for metastatic prostate cancer (PCa). After an initial response to ADT, PCa cells can generate castration resistant (CRPC) or neuroendocrine (NEPC) malignancies, which are incurable. T-type calcium channels (TTCCs) are emerging as promising therapeutic targets for several cancers, but their role in PCa progression has never been investigated. METHODS: To examine the role of TTCCs in PCa, we analyzed their expression level, copy number variants (CNV) and prognostic significance using clinical datasets (Oncomine and cBioPortal). We then evaluated TTCC expression in a panel of PCa cell lines and measured the effect of their inhibition on cell proliferation and survival using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and caspase assays. RESULTS: TTCCs were upregulated in PCas harboring androgen receptor (AR) mutations; CNV rate was positively associated with PCa progression. Higher expression of one TTCC isoform (CACNA1G) predicted poorer postoperative prognosis in early stage PCa samples. Pharmacological or small interfering RNA (siRNA)-based inhibition of TTCCs caused a decrease in PC-3 cell survival and proliferation. CONCLUSIONS: Our results show that TTCCs are overexpressed in advanced forms of PCa and correlate with a poorer prognosis. TTCC inhibition reduces cell proliferation and survival, suggesting that there may be possible value in the therapeutic targeting of TTCCs in advanced PCa.

Constitutive IP3 signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP3 receptor disruptor BIRD-2.

Anti-apoptotic Bcl-2 proteins are upregulated in different cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), enabling survival by inhibiting pro-apoptotic Bcl-2-family members and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-mediated Ca2+-signaling. A peptide tool (Bcl-2/IP3R Disruptor-2; BIRD-2) was developed to abrogate the interaction of Bcl-2 with IP3Rs by targeting Bcl-2's BH4 domain. BIRD-2 triggers cell death in primary CLL cells and in DLBCL cell lines. Particularly, DLBCL cells with high levels of IP3R2 were sensitive to BIRD-2. Here, we report that BIRD-2-induced cell death in DLBCL cells does not only depend on high IP3R2-expression levels, but also on constitutive IP3 signaling, downstream of the tonically active B-cell receptor. The basal Ca2+ level in SU-DHL-4 DLBCL cells was significantly elevated due to the constitutive IP3 production. This constitutive IP3 signaling fulfilled a pro-survival role, since inhibition of phospholipase C (PLC) using U73122 (2.5 µM) caused cell death in SU-DHL-4 cells. Milder inhibition of IP3 signaling using a lower U73122 concentration (1 µM) or expression of an IP3 sponge suppressed both BIRD-2-induced Ca2+ elevation and apoptosis in SU-DHL-4 cells. Basal PLC/IP3 signaling also fulfilled a pro-survival role in other DLBCL cell lines, including Karpas 422, RI-1 and SU-DHL-6 cells, whereas PLC inhibition protected these cells against BIRD-2-evoked apoptosis. Finally, U73122 treatment also suppressed BIRD-2-induced cell death in primary CLL, both in unsupported systems and in co-cultures with CD40L-expressing fibroblasts. Thus, constitutive IP3 signaling in lymphoma and leukemia cells is not only important for cancer cell survival, but also represents a vulnerability, rendering cancer cells dependent on Bcl-2 to limit IP3R activity. BIRD-2 seems to switch constitutive IP3 signaling from pro-survival into pro-death, presenting a plausible therapeutic strategy.

Examining Cardiomyocyte Dysfunction Using Acute Chemical Induction of an Ageing Phenotype.

Much effort is focussed on understanding the structural and functional changes in the heart that underlie age-dependent deterioration of cardiac performance. Longitudinal studies, using aged animals, have pinpointed changes occurring to the contractile myocytes within the heart. However, whilst longitudinal studies are important, other experimental approaches are being advanced that can recapitulate the phenotypic changes seen during ageing. This study investigated the induction of an ageing cardiomyocyte phenotypic change by incubation of cells with hydroxyurea for several days ex vivo. Hydroxyurea incubation has been demonstrated to phenocopy age- and senescence-induced changes in neurons, but its utility for ageing studies with cardiac cells has not been examined. Incubation of neonatal rat ventricular myocytes with hydroxyurea for up to 7 days replicated specific aspects of cardiac ageing including reduced systolic calcium responses, increased alternans and a lesser ability of the cells to follow electrical pacing. Additional functional and structural changes were observed within the myocytes that pointed to ageing-like remodelling, including lipofuscin granule accumulation, reduced mitochondrial membrane potential, increased production of reactive oxygen species, and altered ultrastructure, such as mitochondria with disrupted cristae and disorganised myofibres. These data highlight the utility of alternative approaches for exploring cellular ageing whilst avoiding the costs and co-morbid factors that can affect longitudinal studies.

Deleterious effects of calcium indicators within cells; an inconvenient truth.

The study of cellular Ca2+ signalling is indebted to Roger Tsien for the invention of fluorescent indicators that can be readily loaded into living cells and provide the means to measure cellular Ca2+ changes over long periods of time with sub-second resolution and microscopic precision. However, a recent study [1] reminds us that as useful as these tools are they need to be employed with caution as there can be off-target effects. This article summarises these recent findings within the wider context of confounding issues that can be encountered when using chemical and genetically-encoded Ca2+ indicators, and briefly discusses some approaches that may mitigate against misleading outcomes.

The regulation of autophagy by calcium signals: Do we have a consensus?

Macroautophagy (hereafter called 'autophagy') is a cellular process for degrading and recycling cellular constituents, and for maintenance of cell function. Autophagy initiates via vesicular engulfment of cellular materials and culminates in their degradation via lysosomal hydrolases, with the whole process often being termed 'autophagic flux'. Autophagy is a multi-step pathway requiring the interplay of numerous scaffolding and signalling molecules. In particular, orthologs of the family of ∼30 autophagy-regulating (Atg) proteins that were first characterised in yeast play essential roles in the initiation and processing of autophagic vesicles in mammalian cells. The serine/threonine kinase mTOR (mechanistic target of rapamycin) is a master regulator of the canonical autophagic response of cells to nutrient starvation. In addition, AMP-activated protein kinase (AMPK), which is a key sensor of cellular energy status, can trigger autophagy by inhibiting mTOR, or by phosphorylating other downstream targets. Calcium (Ca2+) has been implicated in autophagic signalling pathways encompassing both mTOR and AMPK, as well as in autophagy seemingly not involving these kinases. Numerous studies have shown that cytosolic Ca2+ signals can trigger autophagy. Moreover, introduction of an exogenous chelator to prevent cytosolic Ca2+ signals inhibits autophagy in response to many different stimuli, with suggestions that buffering Ca2+ affects not only the triggering of autophagy, but also proximal and distal steps during autophagic flux. Observations such as these indicate that Ca2+ plays an essential role as a pro-autophagic signal. However, cellular Ca2+ signals can exert anti-autophagic actions too. For example, Ca2+ channel blockers induce autophagy due to the loss of autophagy-suppressing Ca2+ signals. In addition, the sequestration of Ca2+ by mitochondria during physiological signalling appears necessary to maintain cellular bio-energetics, thereby suppressing AMPK-dependent autophagy. This article attempts to provide an integrated overview of the evidence for the proposed roles of various Ca2+ signals, Ca2+ channels and Ca2+ sources in controlling autophagic flux.

Tissue Specificity: Store-Operated Ca2+ Entry in Cardiac Myocytes.

Calcium (Ca2+) is a key regulator of cardiomyocyte contraction. The Ca2+ channels, pumps, and exchangers responsible for the cyclical cytosolic Ca2+ signals that underlie contraction are well known. In addition to those Ca2+ signaling components responsible for contraction, it has been proposed that cardiomyocytes express channels that promote the influx of Ca2+ from the extracellular milieu to the cytosol in response to depletion of intracellular Ca2+ stores. With non-excitable cells, this store-operated Ca2+ entry (SOCE) is usually easily demonstrated and is essential for prolonging cellular Ca2+ signaling and for refilling depleted Ca2+ stores. The role of SOCE in cardiomyocytes, however, is rather more elusive. While there is published evidence for increased Ca2+ influx into cardiomyocytes following Ca2+ store depletion, it has not been universally observed. Moreover, SOCE appears to be prominent in embryonic cardiomyocytes but declines with postnatal development. In contrast, there is overwhelming evidence that the molecular components of SOCE (e.g., STIM, Orai, and TRPC proteins) are expressed in cardiomyocytes from embryo to adult. Moreover, these proteins have been shown to contribute to disease conditions such as pathological hypertrophy, and reducing their expression can attenuate hypertrophic growth. It is plausible that SOCE might underlie Ca2+ influx into cardiomyocytes and may have important signaling functions perhaps by activating local Ca2+-sensitive processes. However, the STIM, Orai, and TRPC proteins appear to cooperate with multiple protein partners in signaling complexes. It is therefore possible that some of their signaling activities are not mediated by Ca2+ influx signals, but by protein-protein interactions.

Investigating interactions between epicardial adipose tissue and cardiac myocytes: what can we learn from different approaches?

Heart disease is a major cause of morbidity and mortality throughout the world. Some cardiovascular conditions can be modulated by lifestyle factors such as increased exercise or a healthier diet, but many require surgical or pharmacological interventions for their management. More targeted and less invasive therapies would be beneficial. Recently, it has become apparent that epicardial adipose tissue plays an important role in normal and pathological cardiac function, and it is now the focus of considerable research. Epicardial adipose tissue can be studied by imaging of various kinds, and these approaches have yielded much useful information. However, at a molecular level, it is more difficult to study as it is relatively scarce in animal models and, for practical and ethical reasons, not always available in sufficient quantities from patients. What is needed is a robust model system in which the interactions between epicardial adipocytes and cardiac myocytes can be studied, and physiologically relevant manipulations performed. There are drawbacks to conventional culture methods, not least the difficulty of culturing both cardiac myocytes and adipocytes, each of which has special requirements. We discuss the benefits of a three-dimensional co-culture model in which in vivo interactions can be replicated. LINKED ARTICLES: This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic ß Cells.

Pancreatic ß cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic ß cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the ß cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in ß cells.

Pulmonary vein sleeve cell excitation-contraction-coupling becomes dysynchronized by spontaneous calcium transients.

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia. Substantial evidence indicates that cardiomyocytes located in the pulmonary veins [pulmonary vein sleeve cells (PVCs)] cause AF by generating ectopic electrical activity. Electrical ablation, isolating PVCs from their left atrial junctions, is a major treatment for AF. In small rodents, the sleeve of PVCs extends deep inside the lungs and is present in lung slices. Here we present data, using the lung slice preparation, characterizing how spontaneous Ca2+ transients in PVCs affect their capability to respond to electrical pacing. Immediately after a spontaneous Ca2+ transient the cell is in a refractory period and it cannot respond to electrical stimulation. Consequently, we observe that the higher the level of spontaneous activity in an individual PVC, the less likely it is that this PVC responds to electrical field stimulation. The spontaneous activity of neighbouring PVCs can be different from each other. Heterogeneity in the Ca2+ signalling of cells and in their responsiveness to electrical stimuli are known pro-arrhythmic events. The tendency of PVCs to show spontaneous Ca2+ transients and spontaneous action potentials (APs) underlies their potential to cause AF.

Calcium-sensing receptor antagonists abrogate airway hyperresponsiveness and inflammation in allergic asthma.

Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyperreactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics.

Reconstituted human TPC1 is a proton-permeable ion channel and is activated by NAADP or Ca2+.

NAADP potently triggers Ca2+ release from acidic lysosomal and endolysosomal Ca2+ stores. Human two-pore channels (TPC1 and TPC2), which are located on these stores, are involved in this process, but there is controversy over whether TPC1 and TPC2 constitute the Ca2+ release channels. We therefore examined the single-channel properties of human TPC1 after reconstitution into bilayers of controlled composition. We found that TPC1 was permeable not only to Ca2+ but also to monovalent cations and that permeability to protons was the highest (relative permeability sequence: H+ >> K+ > Na(+) ≥ Ca2+). NAADP or Ca2+ activated TPC1, and the presence of one of these ligands was required for channel activation. The endolysosome-located lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] had no effect on TPC1 open probability but significantly increased the relative permeability of Na+ to Ca2+ and of H+ to Ca2+. Furthermore, our data showed that, although both TPC1 and TPC2 are stimulated by NAADP, these channels differ in ion selectivity and modulation by Ca2+ and pH. We propose that NAADP triggers H+ release from lysosomes and endolysomes through activation of TPC1, but that the Ca2+ -releasing ability of TPC1 will depend on the ionic composition of the acidic stores and may be influenced by other regulators that affect TPC1 ion permeation.

Spontaneous, pro-arrhythmic calcium signals disrupt electrical pacing in mouse pulmonary vein sleeve cells.

The pulmonary vein, which returns oxygenated blood to the left atrium, is ensheathed by a population of unique, myocyte-like cells called pulmonary vein sleeve cells (PVCs). These cells autonomously generate action potentials that propagate into the left atrial chamber and cause arrhythmias resulting in atrial fibrillation; the most common, often sustained, form of cardiac arrhythmia. In mice, PVCs extend along the pulmonary vein into the lungs, and are accessible in a lung slice preparation. We exploited this model to study how aberrant Ca(2+) signaling alters the ability of PVC networks to follow electrical pacing. Cellular responses were investigated using real-time 2-photon imaging of lung slices loaded with a Ca(2+)-sensitive fluorescent indicator (Ca(2+) measurements) and phase contrast microscopy (contraction measurements). PVCs displayed global Ca(2+) signals and coordinated contraction in response to electrical field stimulation (EFS). The effects of EFS relied on both Ca(2+) influx and Ca(2+) release, and could be inhibited by nifedipine, ryanodine or caffeine. Moreover, PVCs had a high propensity to show spontaneous Ca(2+) signals that arose via stochastic activation of ryanodine receptors (RyRs). The ability of electrical pacing to entrain Ca(2+) signals and contractile responses was dramatically influenced by inherent spontaneous Ca(2+) activity. In PVCs with relatively low spontaneous Ca(2+) activity (<1 Hz), entrainment with electrical pacing was good. However, in PVCs with higher frequencies of spontaneous Ca(2+) activity (>1.5 Hz), electrical pacing was less effective; PVCs became unpaced, only partially-paced or displayed alternans. Because spontaneous Ca(2+) activity varied between cells, neighboring PVCs often had different responses to electrical pacing. Our data indicate that the ability of PVCs to respond to electrical stimulation depends on their intrinsic Ca(2+) cycling properties. Heterogeneous spontaneous Ca(2+) activity arising from stochastic RyR opening can disengage them from sinus rhythm and lead to autonomous, pro-arrhythmic activity.

Ca2+-sensitive fluorescent dyes and intracellular Ca2+ imaging.

Imaging Ca(2+)-sensitive fluorescent indicators provides a common approach for studying Ca(2+) signals in many contexts. Fluorescent indicators are particularly useful for measuring acute Ca(2+) changes in a relatively noninvasive manner. The availability of indicators that can be targeted to specific cellular domains, coupled with variations in affinity, brightness or spectral characteristics, provides tools for exploring spatially and temporally diverse Ca(2+) signals, and moreover, multiplexing the readout of Ca(2+) with other cellular functions. This article aims to give the novice experimentalist some insight into the considerations and potential pitfalls that impinge on the use of fluorescent Ca(2+) indicators.

Loading fluorescent Ca2+ indicators into living cells.

Small-molecule fluorescent Ca(2+) reporters are the most widely used tools in the field of Ca(2+) signaling. The excellent spatial and temporal resolution afforded by fluorescent reporters has driven the understanding of Ca(2+) as a messenger in many different cell types. In many situations, the cellular loading and monitoring of fluorescent Ca(2+) indicators is quite trivial. However, there are numerous pitfalls that require consideration to ensure that optimal data are recorded. Fluorescent Ca(2+) indicators have carboxylic acid groups for binding of Ca(2+). Because these "free-acid" forms of the indicators are hydrophilic they cannot readily cross cell membranes and need to be introduced into cells using techniques such as microinjection, pinocytosis, or diffusion from a patch pipette. However, the most convenient and widely used method for loading indicators into cells is as hydrophobic compounds in which the carboxylic acid groups are esterified (commonly as acetoxymethyl [AM] or acetate esters). The ester versions of the indicators permeate the plasma membrane. The Ca(2+)-sensitive, free-acid form of the indicator is liberated following hydrolysis of the ester groups by intracellular esterases.

Converting fluorescence data into Ca2+ concentration.

In many situations, fluorescent Ca(2+) reporters are used to simply indicate that a change of Ca(2+) concentration has occurred. Monitoring the emission from a Ca(2+)-sensitive indicator can be sufficient to tell whether a signal has arisen, and what its kinetic/spatial parameters were. The emission from an indicator does not have a linear relationship to the Ca(2+) concentration within a cell; rather, the relationship between fluorescence emission and Ca(2+) concentration is described by a logistic function. Simply recording fluorescence emission, therefore, provides a relative indication of the magnitude of a Ca(2+) signal that should not be used for generating mean amplitude data. However, with a little consideration and effort, the fluorescence output can be calibrated to yield actual Ca(2+) concentration.

Loss of activity mutations in phospholipase C zeta (PLCζ) abolishes calcium oscillatory ability of human recombinant protein in mouse oocytes.

BACKGROUND: Mammalian oocyte activation occurs via a series of intracellular calcium (Ca(2+)) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLCζ). There is now strong evidence to indicate that certain types of human male infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Molecular analysis of the PLCζ gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLCζ residue 398 (PLCζ(H398P)), leading to abnormal Ca(2+) release profiles and reduced oocyte activation efficiency. METHODS AND RESULTS: In the present study, we used HEK293T cells to produce recombinant human wild-type PLCζ (PLCζ(WT)) protein which, upon microinjection into mouse oocytes, induced Ca(2+) oscillations characteristic of oocyte activation. Injection of recombinant PLCζ(H398P) was unable to elicit Ca(2+) oscillations in mouse oocytes. Loss of activity mutations, such as PLCζ(H398P) and an artificially induced frameshift mutation (PLCζ(ΔYC2)) did not affect Ca(2+) release when over-expressed in HEK293T cells, whereas PLCζ(WT) inhibited adenosine triphosphate-activated Ca(2+) release. Confocal imaging of fluorescently tagged PLCζ isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLCζ(WT) > PLCζ(H398P) > PLCζ(ΔYC2), indicating that loss of activity mutations may lead to protein instability. This was further indicated by the low proportion of sperm and the lower levels of total PLCζ immunofluorescence from the patient exhibiting PLCζ(H398P) compared with fertile controls. CONCLUSIONS: We demonstrate, for the first time, the production of active recombinant human PLCζ protein which retained the ability to elicit characteristic Ca(2+) oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLCζ, and provide a critical step forward in obtaining purified PLCζ protein as a potential therapeutic agent for oocyte activation deficiency.

Two-pore channels form homo- and heterodimers.

Two-pore channels (TPCs) have been recently identified as NAADP-regulated Ca(2+) release channels, which are localized on the endolysosomal system. TPCs have a 12-transmembrane domain (TMD) structure and are evolutionary intermediates between the 24-TMD α-subunits of Na(+) or Ca(2+) channels and the transient receptor potential channel superfamily, which have six TMDs in a single subunit and form tetramers with 24 TMDs as active channels. Based on this relationship, it is predicted that TPCs dimerize to form functional channels, but the dimerization of human TPCs has so far not been studied. Using co-immunoprecipitation studies and a mass spectroscopic analysis of the immunocomplex, we show the presence of homo- and heteromeric complexes for human TPC1 and TPC2. Despite their largely distinct localization, we identified a discrete number of endosomes that coexpressed TPC1 and TPC2. Homo- and heteromerization were confirmed by a FRET study, showing that both proteins interacted in a rotational (N- to C-terminal/head-to-tail) symmetry. This is the first report describing the presence of homomultimeric TPC1 channels and the first study showing that TPCs are capable of forming heteromers.