ER Ca2+ release and store-operated Ca2+ entry – partners in crime or independent actors in oncogenic transformation?

Ca²⁺ is a pleiotropic messenger that controls life and death decisions from fertilisation until death. Cellular Ca²⁺ handling mechanisms show plasticity and are remodelled throughout life to meet the changing needs of the cell. In turn, as the demands on a cell alter, for example through a change in its niche environment or its functional requirements, Ca²⁺ handling systems may be targeted to sustain the remodelled cellular state. Nowhere is this more apparent than in cancer. Oncogenic transformation is a multi-stage process during which normal cells become progressively differentiated towards a cancerous state that is principally associated with enhanced proliferation and avoidance of death. Ca²⁺ signalling is intimately involved in almost all aspects of the life of a transformed cell and alterations in Ca²⁺ handling have been observed in cancer. Moreover, this remodelling of Ca²⁺ signalling pathways is also required in some cases to sustain the transformed phenotype. As such, Ca²⁺ handling is hijacked by oncogenic processes to deliver and maintain the transformed phenotype. Central to generation of intracellular Ca²⁺ signals is the release of Ca²⁺ from the endoplasmic reticulum intracellular (ER) Ca²⁺ store via inositol 1,4,5-trisphosphate receptors (InsP₃Rs). Upon depletion of ER Ca²⁺, store-operated Ca²⁺ entry (SOCE) across the plasma membrane occurs via STIM-gated Orai channels. SOCE serves to both replenish stores but also sustain Ca²⁺ signalling events. Here, we will discuss the role and regulation of these two signalling pathways and their interplay in oncogenic transformation.     

Does the A3333G mutation in the CACNL1A3 gene, detected in malignant hyperthermia, also occur in central core disease?

Malignant hyperthermia (MH) and central core disease (CCD) are two conditions associated with susceptibility to volatile anesthetics and depolarizing muscle relaxants. The gene RYR1, encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum, is responsible for about 50% of the cases of MH and some cases of CCD. However, genetic heterogeneity occurs in MH and a mutation in a second gene (CACLN1A3), encoding the alpha1-subunit of the dihydropyridine (DHP) channel, has recently been found in a large MH French family. The presence of this mutation in patients with CCD has not yet been reported. In this study, we analyzed the A3333G mutation in 5 unrelated patients affected by CCD and 31 MH-susceptible relatives (from 19 MH families) and did not find this mutation in any of them. Nevertheless, the report of data on newly described mutations in different populations is important to estimate the contributions of each gene mutation to the phenotype of MH and CCD.     

Store-operated Ca2+ entry and Ca2+ responses to hypothalamic releasing hormones in anterior pituitary cells from Orai1−/− and heptaTRPC knockout mice

Store operated Ca²⁺ entry (SOCE) is the most important Ca²⁺ entry pathway in non-excitable cells. However, SOCE can also play a pivotal role in excitable cells such as anterior pituitary (AP) cells. The AP gland contains five different cell types that release six major AP hormones controlling most of the entire endocrine system. AP hormone release is modulated by Ca²⁺ signals induced by different hypothalamic releasing hormones (HRHs) acting on specific receptors in AP cells. TRH and LHRH both induce Ca²⁺ release and Ca²⁺ entry in responsive cells while GHRH and CRH only induce Ca²⁺ entry. SOCE has been shown to contribute to Ca²⁺ responses induced by TRH and LHRH but no molecular evidence has been provided. Accordingly, we used AP cells isolated from mice devoid of Orai1 channels (noted as Orai1−/− or Orai1 KO mice) and mice lacking expression of all seven canonical TRP channels (TRPC) from TRPC1 to TRPC7 (noted as heptaTRPC KO mice) to investigate contribution of these putative channel proteins to SOCE and intracellular Ca²⁺ responses induced by HRHs. We found that thapsigargin-evoked SOCE is lost in AP cells from Orai1−/− mice but unaffected in cells from heptaTRPC KO mice. Conversely, while spontaneous intracellular Ca²⁺-oscillations related to electrical activity were not affected in the Orai1−/− mice, these responses were significantly reduced in heptaTRPC KO mice. We also found that Ca²⁺ entry induced by TRH and LHRH is decreased in AP cells isolated from Orai1−/−. In addition, Ca²⁺ responses to several HRHs, particularly TRH and GHRH, are decreased in the heptaTRPC KO mice. These results indicate that expression of Orai1, and not TRPC channel proteins, is necessary for thapsigargin-evoked SOCE and is required to support Ca²⁺ entry induced by TRH and LHRH in mouse AP cells. In contrast, TRPC channel proteins appear to contribute to spontaneous Ca²⁺-oscillations and Ca²⁺ responses induced by TRH and GHRH. We conclude that expression of Orai1 and TRPC channels proteins may play differential and significant roles in AP physiology and endocrine control.     

The cardiac α1C subunit can support excitation-triggered Ca2+ entry in dysgenic and dyspedic myotubes

Depolarization-induced entry of divalent ions into skeletal muscle has been attributed to a process termed Excitation-Coupled Ca²⁺ Entry (ECCE), which is hypothesized to require the interaction of the ryanodine receptor (RyR1), the L-type Ca²⁺ channel (DHPR) and another unidentified cation channel. Thus, ECCE is absent in myotubes lacking either the DHPR (dysgenic) or RyR1 (dyspedic). Furthermore, ECCE, as measured by Mn²⁺ quench of Fura-2, is reconstituted by expression of a mutant DHPR α_1S subunit (SkEIIIK) thought to be impermeable to divalent cations. Previously, we showed that the bulk of depolarization-induced Ca²⁺ entry could be explained by the skeletal L-type current. Accordingly, one would predict that any Ca²⁺ current similar to the endogenous current would restore such entry and that this entry would not require coupling to either the DHPR or RyR1. Here, we show that expression of the cardiac α_1C subunit in either dysgenic or dyspedic myotubes does result in Ca²⁺ entry similar to that ascribed to ECCE. We also demonstrate that, when potentiated by strong depolarization and Bay K 8644, SkEIIIK supports entry of Mn²⁺. These results strongly support the idea that the L-type channel is the major route of Ca²⁺ entry in response to repetitive or prolonged depolarization of skeletal muscle.     

Structure and function of the CBL–CIPK Ca2+-decoding system in plant calcium signaling

Calcium is a crucial messenger in many growth and developmental processes in plants. The central mechanism governing how plant cells perceive and respond to environmental stimuli is calcium signal transduction, a process through which cellular calcium signals are recognized, decoded, and transmitted to elicit downstream responses. In the initial decoding of calcium signals, Ca²⁺ sensor proteins that bind Ca²⁺ and activate downstream signaling components are implicated, thereby regulating specific physiological and biochemical processes. After calcineurin B-like proteins (CBLs) sense these Ca²⁺ signatures, these proteins interact selectively with CBL-interacting protein kinases (CIPKs), thereby forming CBL/CIPK complexes, which are involved in decoding calcium signals. Therefore, specificity, diversity, and complexity are the main characteristics of the CBL-CIPK signaling system. However, additional CBLs, CIPKs, and CBL/CIPK complexes remain to be identified in plants, and the specific functions of their abiotic and biotic stress signaling will need to be further dissected. Therefore, a much-needed synthesis of recent findings is important to further the study of CBL-CIPK signaling systems. Here, we review the structure of CBLs and CIPKs, discuss the current knowledge of CBL–CIPK pathways that decode calcium signals in Arabidopsis, and link plant responses to a variety of environmental stresses with specific CBL/CIPK complexes. This will provide a foundation for future research on genetically engineered resistant plants with enhanced tolerance to various environmental stresses.     

Ca2+ signaling down-regulation in ageing and Alzheimer's disease: why is Ca2+ so difficult to measure?

Ca2+ signaling is a central process in brain function, but the direction of its change in ageing and in the presymptomatic stages of Alzheimer's disease has been controversial. A great deal of studies have been interpreted as supportive to the current hypothesis that intracellular Ca2+ levels are steadily increased in ageing. We, however, believe that, although current studies have provided valuable knowledge for the mechanisms of the signal transduction process, they have not furnished relevant information regarding the global Ca2+ changes in the ageing brain, because the cognition-related Ca2+ pulses exist only in the intact brain.     

Tamoxifen depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase Cα in rat cerebral cortex nerve terminals

This study was aimed at examining the effect of tamoxifen, a selective estrogen receptor modulator, on the release of endogenous glutamate in rat cerebral cortex nerve terminals (synaptosomes) and exploring the possible mechanism. Tamoxifen inhibited the release of glutamate that was evoked by the K(+) channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration-dependent and insensitive to the estrogen receptor antagonist. The effect of tamoxifen on the evoked glutamate release was prevented by the chelating extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate did not have any effect on the action of tamoxifen. Tamoxifen did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in cytosolic [Ca(2+)]. Furthermore, the inhibitory effect of tamoxifen on the evoked glutamate release was abolished by the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Na(+)/Ca(2+) exchanger blocker CGP37157. In addition, the protein kinase C (PKC) inhibitors GF109203X or Ro318220 prevented tamoxifen from inhibiting glutamate release. Western blotting showed that tamoxifen significantly decreased the 4-AP-induced phosphorylation of PKC and PKCα. Together, these results suggest that tamoxifen inhibits glutamate release from rat cortical synaptosomes, through the suppression of presynaptic voltage-dependent Ca(2+) entry and PKC activity.     

Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain?

C2-domains are widespread protein modules with diverse Ca2+-regulatory functions. Although multiple Ca2+ ions are known to bind at the tip of several C2-domains, the exact number of Ca2+-binding sites and their functional relevance are unknown. The first C2-domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca2+-dependent interactions with syntaxin and phospholipids. We have studied the Ca2+-binding mode of this C2-domain as a prototypical C2-domain using NMR spectroscopy and site-directed mutagenesis. The C2-domain is an elliptical module composed of a beta-sandwich with a long axis of 50 A. Our results reveal that the C2-domain binds three Ca2+ ions in a tight cluster spanning only 6 A at the tip of the module. The Ca2+-binding region is formed by two loops whose conformation is stabilized by Ca2+ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C2-domains. All three Ca2+ ions are required for the interactions of the C2-domain with syntaxin and phospholipids. These results support an electrostatic switch model for C2-domain function whereby the beta-sheets of the domain provide a fixed scaffold for the Ca2+-binding loops, and whereby interactions with target molecules are triggered by a Ca2+-induced switch in electrostatic potential.     

β₃-Adrenergic activation of sequential Ca(2+) release from mitochondria and the endoplasmic reticulum and the subsequent Ca(2+) entry in rodent brown adipocytes

We studied how mitochondrial uncoupling by β(3)-adrenergic stimulation elicits Ca(2+) signals in rodent brown adipocytes by fluorometry of Ca(2+) concentrations ([Ca(2+)](i), [Ca(2+)](m) and [Ca(2+)](ER)) in the cytoplasm, mitochondria and the endoplasmic reticulum (ER), respectively, and mitochondrial membrane potential, using fura-2, rhod-5N, cameleon and rhodamine 123. Immunoblotting demonstrated α(1A)- and β(3)-adrenergic receptor and UCP1 in adipocytes, while RT-PCR revealed the mRNA of type 3, 7 and 9 adenylate cyclase, UCP1, UCP2, UCP3 and type 1 and 2 inositoltrisphosphate receptors. Isoproterenol and BRL37344, β-agonist, caused triphasic rises in [Ca(2+)](i) (β-responses) with mitochondrial depolarization in adipocytes. BRL37344 transiently decreased [Ca(2+)](m). β-Responses were blocked by propranolol, β-antagonist, H-89, protein kinase A blocker, and knockout of UCP1 gene. The late phase of β-responses was depressed by a Ca(2+) free, EGTA solution, U73122, a phospholipase C blocker, and thapsigargin, ER-Ca(2+) pump blocker, and by transfecting siRNA for type 2 IP(3)R. Intracellular loading of BAPTA/AM depressed the late phase more strongly than the initial phase. β-Agonists, phenylephrine, α-agonist, and cyclopiazonic acid, ER-Ca(2+) pump blocker, decreased [Ca(2+)](ER). Thus, the mitochondrial uncoupling by β(3)-adrenergic activation causes Ca(2+) release from mitochondria and subsequently from the ER and further evokes plasmalemmal Ca(2+) entries, including the store-operated Ca(2+) entry.     

Cloning,Expression, and Characterization of the Squid Na+–Ca2+ Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na⁺–Ca²⁺ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na⁺–Ca²⁺ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca²⁺ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na⁺-dependent ⁴⁵Ca²⁺ uptake; the uptake was inhibited by injection of Ca²⁺ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na⁺-dependent inactivation, secondary activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F⁻ (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na⁺ and Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca²⁺ extrusion were much larger. For NCX-SQ1, charge movements of Ca²⁺ transport could be defined in voltage jump experiments with a low cytoplasmic Ca²⁺ (2 μM) in the presence of high extracellular Ca²⁺ (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s⁻¹ at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.     

SEK-1 MAPKK mediates Ca2+ signaling to determine neuronal asymmetric development in Caenorhabditis?elegans

The mitogen-activated protein kinase (MAPK) pathway is a highly conserved signaling cascade that converts extracellular signals into various outputs. In Caenorhabditis elegans, asymmetric expression of the candidate odorant receptor STR-2 in either the left or the right of two bilaterally symmetrical olfactory AWC neurons is regulated by axon contact and Ca²⁺ signaling. We show that the MAPK kinase (MAPKK) SEK-1 is required for asymmetric expression in AWC neurons. Genetic and biochemical analyses reveal that SEK-1 functions in a pathway downstream of UNC-43 and NSY-1, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and MAPK kinase kinase (MAPKKK), respectively. Thus, the NSY-1–SEK-1–MAPK cascade is activated by Ca²⁺ signaling through CaMKII and establishes asymmetric cell fate decision during neuronal development.     

Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse,a model of Huntington’s disease

Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor–based Ca²⁺ signaling, which is crucial for skeletal muscle excitation–contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca²⁺ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca²⁺ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca²⁺ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca²⁺ removal from the myoplasm and Ca²⁺ release flux from the sarcoplasmic reticulum were characterized using a Ca²⁺ binding and transport model, which indicated a significant reduction in slow Ca²⁺ removal activity and Ca²⁺ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca²⁺ channel conductance. These results indicate profound changes of Ca²⁺ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD.     

Can Ca2+ Fluxes to the Root Xylem Be Sustained by Ca2+-ATPases in Exodermal and Endodermal Plasma Membranes?

The pathway of Ca2+ movement from the soil solution into the root stele has been a subject of controversy. If transport through the endodermis is assumed to be through the cytoplasm, the limiting factor is believed to be the active pumping of Ca2+ from the cytoplasm into the stele apoplast through the plasma membrane lying on the stele side of the Casparian band. By analogy, for similar transport through the exodermis, the limiting step would be the active pumping into the apoplast on the central cortical side of the layer. Such effluxes are mediated by Ca2+-ATPases. To assess whether or not known Ca2+ fluxes to the stele in onion (Allium cepa) roots could be supported by Ca2+-ATPases, the percentages of total membrane protein particles required to effect the transport were calculated using measured values of membrane surface areas, an animal literature value for Ca2+-ATPase V(max), plant literature values for Ca2+-ATPase K(m), and protein densities of relevant membranes. Effects of a putative symplastic movement of Ca2+ from the exo- or endodermis into the next cell layer, which would increase the surface areas available for pumping, were also considered. Depending on the assumptions applied, densities of Ca2+ pumps, calculated as a percentage of total membrane protein particles, varied tremendously between three and 1,600 for the endodermis, and between 0.94 and 1,900 for the exodermis. On the basis of the data, the possibility of Ca2+ transport through the cytoplasm and membranes of the exodermis and endodermis cannot be discounted. Thus, it is premature to assign an entirely apoplastic pathway for Ca2+ movement from the soil solution to the tracheary elements of the xylem. To verify any conclusion with certainty, more detailed data are required for the characteristics of exo- and endodermal Ca2+-ATPases.     

Myofilament sensitivity to Ca2+ in ventricular myocytes from the Goto–Kakizaki diabetic rat

The chronic effects of type 2 diabetes mellitus on myofilament sensitivity to Ca(2+) in ventricular myocytes from the Goto-Kakizaki (GK) rat have been investigated. Experiments were performed in ventricular myocytes isolated from 17-month GK rats and age-matched Wistar controls. Myocytes were loaded with fura-2 (an indicator for intracellular Ca(2+) concentration) and the fura-2 ratio (340/380 nm), and shortening were measured simultaneously in electrically stimulated myocytes. Myofilament sensitivity to Ca(2+) was assessed from phase-plane diagrams of fura-2 versus cell length by measuring the gradient of the fura-2-cell length trajectory during late relaxation of the twitch contraction. Non-fasting and fasting blood glucose were elevated in GK rats compared to controls. Fasting blood glucose was 151.5 +/- 15.3 mg/dl (n = 8) in GK rats compared to 72.1 +/- 3.6 mg/dl (n = 9) in controls. At 120 min after intraperitoneal injection of glucose (2 g/kg body weight), blood glucose was 570.8 +/- 36.8 mg/dl in GK rats compared to 148 +/- 8.6 mg/dl in controls. Amplitude of shortening was significantly increased in myocytes from GK rats (6.56 +/- 0.54%, n = 31) compared to controls (5.05 +/- 0.43%, n = 36), and the amplitude of the Ca(2+) transient was decreased in myocytes from GK rats (0.23 +/- 0.02 RU, n = 31) compared to controls (0.30 +/- 0.02 RU, n = 36). The fura-2-cell length trajectory during the late stages of relaxation of the twitch contraction was steeper in myocytes from GK rats (89.2 +/- 16.6 microm/RU, n = 27) compared to controls (31.9 +/- 5.9 microm/RU, n = 35). Increased amplitude of shortening, accompanied by a decrease in amplitude of the Ca(2+) transient, might be explained by an increased myofilament sensitivity to Ca(2+).     

Ca2+–Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1

Most chemical neurotransmission occurs through Ca²⁺-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca²⁺ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca²⁺ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca²⁺ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H⁺-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca²⁺–Calmodulin (CaM)-dependent manner. Ca²⁺–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca²⁺-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca²⁺–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.     

Distinct properties of Ca2+–calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel

Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM (Ca²⁺–CaM) in complex with the TRPV1-CT segment, determined to 1.95-Å resolution. The two lobes of Ca²⁺–CaM wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of Ca²⁺–CaM, respectively. This structure is similar to canonical Ca²⁺–CaM-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity Ca²⁺–CaM-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the Ca²⁺–CaM-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the Ca²⁺–CaM-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.