Histamine Increases [Ca2+] in and Activates Ca-K and Nonselective Cation Currents in Cultured Human Capillary Endothelial Cells

We characterized the effects of histamine on intracellular Ca²⁺ and activation of ionic currents in human capillary endothelial cells. Histamine produced both a transient and sustained increase in intracellular Ca²⁺. The transient response was mediated largely through intracellular Ca²⁺ release and the sustained response was due to extracellular Ca²⁺ entry. The increase in intracellular Ca²⁺ by histamine was not affected by the H2 blocker cimetidine. But was entirely blocked by the H1 antagonist diphenhydramine showing that the histamine response in these cells is mediated through the H1 receptor. A transient ionic current is coactivated with the histamine-induced increase in intracellular Ca²⁺ and this current has several properties of a nonselective, Ca²⁺ permeable, cation channel (NSC). The magnitude of the NSC current does not strictly correlate with intracellular Ca²⁺ levels. A Ca²⁺-activated K⁺ current (BKCA) is activated by the increase in intracellular Ca²⁺ and this current is blocked by the selective BKCA blocker iberiotoxin. Received: 16 June 1999/Revised: 22 September 1999     

TRPC6 inhibited NMDA receptor activities and protected neurons from ischemic excitotoxicity

Excitotoxicity induced by NMDA receptor‐mediated intracellular Ca²⁺ ([Ca²⁺]_i) overload is a major cause of delayed neuronal death in cerebral ischemia. Transient receptor potential canonical (TRPC) 6 protects neurons from ischemic brain damage. However, the mechanisms by which TRPC6 protects neurons are largely unknown. Here, we reported that TRPC6 suppressed the [Ca²⁺]_i elevation induced by NMDA and protected neurons from excitotoxicity. Over‐expressing or down‐regulating TRPC6 suppressed or aggravated Ca²⁺ overload under excitotoxicity, respectively. TRPC6 protected cultured neurons from damage caused by NMDA toxicity or oxygen glucose deprivation (OGD). Moreover, the infarct volume in TRPC6 transgenic (Tg) mice was smaller than that in wild‐type (WT) littermates. The TRPC6 Tg mice had better behavior performance and lower mortality than their WT littermates. Thus, TRPC6 inhibited NMDA receptor‐triggered neurotoxicity and protected neurons from ischemic brain damage. Increase in TRPC6 activity could be a potential strategy for stroke prevention and therapy.     

Brain-derived Neurotrophic Factor (BDNF) Induces Sustained Intracellular Ca2+ Elevation through the Up-regulation of Surface Transient Receptor Potential 3 (TRPC3) Channels in Rodent Microglia

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca²⁺]_i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca²⁺ elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca²⁺ elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.     

The Ca2+ release-activated Ca2+ current (ICRAC) mediates store-operated Ca2+ entry in rat microglia

Ca²⁺ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca²⁺ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca²⁺ permeable channels, and limited electrophysiological analyses of Ca²⁺ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are ‘transient receptor potential’ (TRP) channels and the recently cloned Orai1, which produces a Ca²⁺-release-activated Ca²⁺ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca²⁺ entry (SOCE) pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca²⁺-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca²⁺ current with the same properties, including high selectivity for Ca²⁺ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca²⁺-dependent current potentiation, and block by SKF-96365, DES and 50 μM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.     

Activation of TRPC4β by Gαi subunit increases Ca selectivity and controls neurite morphogenesis in cultured hippocampal neuron

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca²⁺-permeable channels. TRPC channels are activated by stimulation of Gα_q-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gα_i. We studied the essential role of Gα_i subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gα_i2 subunit. Activation of TRPC4 by Gα_i2 increased Ca²⁺ permeability and Ca²⁺ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca²⁺ influx. Moreover, both TRPC4β and the TRPC4β-Gα_i2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gα_i2^Q205L-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gα_i proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.     

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ entry pathway that is activated in response to depletion of ER-Ca²⁺ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled I_CRAC, the highly Ca²⁺-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca²⁺ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust I_CRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca²⁺ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca²⁺ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca²⁺]_i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca²⁺]_i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.     

STIM1 long and STIM1 gate differently TRPC1 during store-operated calcium entry

During myogenesis, a long splice variant of STIM1, called STIM1L is getting expressed, while the level of STIM1 remains constant. Previous work demonstrated that STIM1L is more efficient in eliciting store-operated Ca²⁺ entry (SOCE), but no current analysis of the channel(s) activated by this new STIM1L isoform was performed until now. In this study, we investigate the ionic channel(s) activated by STIM1L and whether differences exist between the two STIM1 isoforms, using HEK-293 T cells as a model system. Our data show that STIM1 and STIM1L activate Orai1 channel but also the endogenously expressed TRPC1. The channel activation occurs in two steps, with first Orai1 activation followed, in a subset of cells, by TRPC1 opening. Remarkably, STIM1L more frequently activates TRPC1 and preferentially interacts with TRPC1. In low intracellular Ca²⁺ buffering condition, the frequency of TRPC1 opening increases significantly, strongly suggesting a Ca²⁺-dependent channel activation. The ability of STIM1L to open Orai1 appears decreased compared to STIM1, which might be explained by its stronger propensity towards TRPC1. Indeed, increasing the amount of STIM1L results in an enhanced Orai1 current. The role of endogenous TRPC1 in STIM1- and STIM1L-induced SOCE was confirmed by Ca²⁺ imaging experiments. Overall, our findings provide a detailed analysis of the channels activated by both STIM1 isoforms, revealing that STIM1L is more prone to open TRPC1, which might explain the larger SOCE elicited by this isoform.     

Transient receptor potential (TRP) channel function in the reproductive axis

Transient receptor potential (TRP) channels play important functional roles in the signal transduction machinery of hormone-secreting cells and have recently been implicated in reproductive physiology. While expression studies have demonstrated TRP channel expression at all levels of the hypothalamic–pituitary–gonadal (hpg) axis, functional details about TRP channel action at the level of the individual cells controlling reproduction are just beginning to emerge. Canonical TRP (TRPC) channels are prominently expressed in the reproductive center of the neuroendocrine brain, i.e. in kisspeptin and gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin neurons are depolarized by leptin via activation of TRPC channels and kisspeptin depolarizes GnRH neurons through TRPC4 activation. Recent studies have functionally identified TRPC channels also in gonadotrope cells in the anterior pituitary gland, which secrete gonadotropins in response to GnRH and thus regulate gonadal function. TRP channel expression in these cells exhibits remarkable plasticity and depends on the hormonal status of the animal. Subsequent functional analyses have demonstrated that TRPC5 in gonadotropes contributes to depolarization of the plasma membrane upon GnRH stimulation and increases the intracellular Ca²⁺ concentration via its own Ca²⁺ permeability and via the activation of voltage-gated Ca²⁺ channels. However, conditional gene targeting experiments will be needed to unambiguously dissect the physiological role of TRPC channels in the different cell types of the reproductive axis in vivo.     

Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

TRPC5 is a calcium (Ca²⁺)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca²⁺: replacement by Ba²⁺ or Mg²⁺ abolishes it, whereas the addition of 10 mM Ca²⁺ accelerates it. The site of action for Ca²⁺ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca²⁺]. This potentiation is prevented when intracellular Ca²⁺ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca²⁺]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca²⁺] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca²⁺-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.     

On the endothelial cell ISOC

Ca²⁺ store depletion activates both Ca²⁺ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca²⁺ selective current, I_SOC. Indeed, I_SOC is a relatively small inward Ca²⁺ current that exhibits an approximate +40 mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I_SOC, although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I_SOC requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca²⁺ store depletion and channel activation. Thus, at present findings indicate a TRPC4–protein 4.1 physical linkage regulates I_SOC activation following Ca²⁺ store depletion.     

Divergence in Endothelin-1- and Bradykinin-Activated Store-Operated Calcium Entry in Afferent Sensory Neurons

Endothelin-1 (ET-1) and bradykinin (BK) are endogenous peptides that signal through Gαq/11-protein coupled receptors (GPCRs) to produce nociceptor sensitization and pain. Both peptides activate phospholipase C to stimulate Ca²⁺ accumulation, diacylglycerol production, and protein kinase C activation and are rapidly desensitized via a G-protein receptor kinase 2-dependent mechanism. However, ET-1 produces a greater response and longer lasting nocifensive behavior than BK in multiple models, indicating a potentially divergent signaling mechanism in primary afferent sensory neurons. Using cultured sensory neurons, we demonstrate significant differences in both Ca²⁺ influx and Ca²⁺ release from intracellular stores following ET-1 and BK treatments. As intracellular store depletion may contribute to the regulation of other signaling cascades downstream of GPCRs, we concentrated our investigation on store-operated Ca²⁺ channels. Using pharmacological approaches, we identified transient receptor potential canonical channel 3 (TRPC3) as a dominant contributor to Ca²⁺ influx subsequent to ET-1 treatment. On the other hand, BK treatment stimulated Orai1 activation, with only minor input from TRPC3. Taken together, data presented here suggest that ET-1 signaling targets TRPC3, generating a prolonged Ca²⁺ signal that perpetuates nocifensive responses. In contrast, Orai1 dominates as the downstream target of BK receptor activation and results in transient intracellular Ca²⁺ increases and abridged nocifensive responses.     

Involvement of ryanodine receptors in muscarinic receptor-mediated membrane current oscillation in urinary bladder smooth muscle

The urinary bladder pressure during micturition consists of two components: an initial, phasic component and a subsequent, sustained component. To investigate the excitation mechanisms underlying the sustained pressure, we recorded from membranes of isolated detrusor cells from the pig, which can be used as a model for human micturition. Parasympathomimetic agents promptly evoke a large transient inward current, and subsequently during its continuous presence, oscillating inward currents of relatively small amplitudes are observed. The two types of inward current are considered to cause the phasic and sustained pressure rises, respectively. Ionic substitution and applications of channel blockers revealed that Ca²⁺-activated Cl^– channels were responsible for the large transient and oscillating inward currents. Furthermore, the inclusion of guanosine 5'-O-(2-thiodiphosphate) in the patch pipette indicates that both inward currents involve G proteins. However, applications of heparin in the patch pipette and of xestospongin C in the bathing solution suggest a signaling pathway other than inositol 1,4,5-trisphosphate (IP₃) operating in the inward current oscillations, unlike the initial transient inward current. This IP₃-independent inward current oscillation system required both sustained Ca²⁺ influx from the extracellular space and Ca²⁺ release from the intracellular stores. These two requirements are presumably SKF-96365-sensitive cation channels and ryanodine receptors, respectively. Experiments with various Ca²⁺ concentrations suggested that Ca²⁺ influx from the extracellular space plays a major role in pacing the oscillatory rhythm. The fact that distinct mechanisms underlie the two types of inward current may help in development of clinical treatments of, for example, urinary incontinence and residual urine volume control. G proteins; micturition; oscillation; carbachol; SKF-96365     

Involvement of TRPC3 channels in calcium oscillations mediated by OX1 orexin receptors

Oscillations of intracellular Ca²⁺ provide a novel mechanism for sustained activation of cellular processes. Receptor-activated oscillations are mainly thought to occur through rhythmic IP₃-dependent store discharge. However, as shown here in HEK293 cells 1 nM orexin-A (Ox-A) acting at OX₁ receptors (OX₁R) triggered oscillatory Ca²⁺ responses, requiring external Ca²⁺. These responses were attenuated by interference with TRPC3 channel (but not TRPC1/4) function using dominant negative constructs, elevated Mg²⁺ (a blocker of many TRP channels) or inhibition of phospholipase A₂. These treatments did not affect Ca²⁺ oscillations elicited by high concentrations of Ox-A (100 nM) in the absence of external Ca²⁺. OX₁R are thus able to activate TRPC(3)-channel-dependent oscillatory responses independently of store discharge.     

Trafficking mechanisms and regulation of TRPC channels

TRPC channels are Ca²⁺-permeable cation channels which are regulated downstream from receptor-coupled PIP₂ hydrolysis. These channels contribute to a wide variety of cellular functions. Loss or gain of channel function has been associated with dysfunction and aberrant physiology. TRPC channel functions are influenced by their physical and functional interactions with numerous proteins that determine their regulation, scaffolding, trafficking, as well as their effects on the downstream cellular processes. Such interactions also compartmentalize the Ca²⁺ signals arising from TRPC channels. A large number of studies demonstrate that trafficking is a critical mode by which plasma membrane localization and surface expression of TRPC channels are regulated. This review will provide an overview of intracellular trafficking pathways as well as discuss the current state of knowledge regarding the mechanisms and components involved in trafficking of the seven members of the TRPC family (TRPC1–TRPC7).     

Voltage-independent barium-permeable channel activated inLymnaea neurons by internal perfusion or patch excision

Summary Isolated nerve cells fromLymnaea stagnalis were studied using the internal-perfusion and patch-clamp techniques. Patch excision frequently activated a voltage-independent Ba²⁺-permeable channel with a slope conductance of 27 pS at negative potentials (50mm Ba²⁺). This channel is not seen in patches on healthy cells and, unlike the voltage-dependent Ca channel, is not labile in isolated patches. The activity of the channel in inside-out patches is unaffected by intracellular ATP, Ca²⁺ below 1mm or the catalytic subunit of cAMP-dependent protein kinase but is reversibly blocked by millimolar intracellular Ca²⁺ or Ba²⁺. The channel can be activated in on-cell patches by either internal perfusion with high Ca²⁺ or the long-term internal perfusion of low Ca²⁺ solutions not containing ATP. These channels may carry the inward Ca²⁺ current which causes a regenerative increase in intracellular Ca⁺ when snail neurons are perfused with high Ca²⁺ solutions. High internal Ca²⁺, or long periods of internal perfusion with ATP-free solutions, induces an increase in a resting (–50 mV) whole-cell Ba²⁺ conductance. This conductance can be turned off by returning the intracellular perfusate to a low Ca²⁺ solution containing ATP and Mg²⁺. The activity of this channel appears to have an opposite dependence on intracellular conditions to that of the voltage-dependent Ca channel.     

Chronic exposure to stress hormones alters the subtype of store‐operated channels expressed in H19‐7 hippocampal neuronal cells

Differentiating H19‐7 hippocampal precursor cells up‐regulate (～4.3‐fold) store‐operated channel (SOC) activity; relatively linear current‐voltage curves indicate an I_SOC subtype of SOC. In differentiated H19‐7 neurons, the majority of agonist (arginine vasopressin, AVP)‐stimulated Ca²⁺ entry occurs via SOCs, based on 2‐aminoethyldiphenylborinate (2‐APB) inhibition data and the observation that transient receptor potential C1 (TRPC1) channel knock down cells show a dramatic reduction of thapsigargin‐stimulated store‐operated Ca²⁺ entry (SOCE) and inhibition of AVP‐stimulated Ca²⁺ entry. Treatment of H19‐7 cells with the rat stress hormone corticosterone during differentiation induces a significant increase in AVP‐stimulated Ca²⁺ entry, which is virtually eliminated by 2‐APB, suggesting a corticosterone‐induced increase of SOCE. Corticosterone also enhances AVP‐stimulated Mn²⁺ entry, confirming an elevated Ca²⁺ entry pathway, rather than a decreased Ca²⁺ extrusion. When corticosterone addition is delayed until after H19‐7 cells have fully differentiated, it still elevates SOCE. In corticosterone‐treated H19‐7 cells, the knock down of TRPC1 no longer blocks thapsigargin‐stimulated Ca²⁺ entry suggesting that the subtype of SOCs expressed in H19‐7 cells is altered by corticosterone treatment. Electrophysiological studies demonstrate that store‐operated currents in corticosterone‐treated H19‐7 cells exhibit a highly inward rectifying current‐voltage curve consistent with an I_CRAC subtype of SOCs. Consistent with this finding is the observation that corticosterone treatment of H19‐7 cells increases the expression of the I_CRAC channel subunit Orai1. Thus, the subtype of SOCs expressed in H19‐7 hippocampal neurons can be altered from I_SOC to I_CRAC by chronic treatment with stress hormones. J. Cell. Physiol. 228: 1332–1343, 2013. © 2012 Wiley Periodicals, Inc.     

An endogenous elevation of cGMP increases the excitability of identified insect neurosecretory cells

In the moth, Manduca sexta, the neuropeptide, eclosion hormone, triggers a dramatic rise in the levels of intracellular cGMP within a group of 50 neurons. The cells within this group include the segmentally repeated neurosecretory cell, Cell 27. In this study the effect of cGMP on the excitability of Cell 27 was investigated using intracellular recordings. Prior to its normal elevation in cGMP, Cell 27 exhibited a high spike threshold, but this was lowered dramatically when intracellular cGMP levels increased. The latter was also associated with spontaneous action potentials. This change in excitability did not correspond with changes in either resting potential, input resistance, or action potential amplitude. A similar lowering of threshold was induced by perfusion of 8-bromo-cGMP, whereas 8-bromo-cAMP caused the threshold to increase. Intracellular recordings using various ion substitution paradigms and channel blockers provided evidence which suggests indirectly that Ca²⁺ is mostly responsible for the depolarizing phase of the action potential while a Ca²⁺-activated K⁺ current contributes to the hyperpolarization. The results of these manipulations are consistent with the hypothesis that cGMP may partially increase excitability in Cell 27 by enhancing an inward Ca²⁺ current. Accepted: 31 October 1996     

Calcium entry via TRPC6 mediates albumin overload-induced endoplasmic reticulum stress and apoptosis in podocytes

Albumin, which is the most abundant component of urine proteins, exerts injurious effects on renal cells in chronic kidney diseases. However, the toxicity of albumin to podocytes is not well elucidated. Here, we show that a high concentration of albumin triggers intracellular calcium ([Ca²⁺]_i) increase through mechanisms involving the intracellular calcium store release and extracellular calcium influx in conditionally immortalized podocytes. The canonical transient receptor potential-6 (TRPC6) channel, which is associated with a subset of familial forms of focal segmental glomerulosclerosis (FSGS) and several acquired proteinuric kidney diseases, was shown to be one of the important Ca²⁺ permeable ion channels in podocytes. Therefore we explored the role of TRPC6 on albumin-induced functional and structural changes in podocytes. It was found that albumin-induced increase in [Ca²⁺]_i was blocked by TRPC6 siRNA or SKF-96365, a blocker of TRP cation channels. Long-term albumin exposure caused an up-regulation of TRPC6 expression in podocytes, which was inhibited by TRPC6 siRNA. Additionally, the inhibition of TRPC6 prevented the F-actin cytoskeleton disruption that is induced by albumin overload. Moreover, albumin overload induced expression of the endoplasmic reticulum (ER) stress protein GRP78, led to caspase-12 activation and ultimately podocyte apoptosis, all of which were abolished by the knockdown of TRPC6 using TRPC6 siRNA. These results support the view that albumin overload may induce ER stress and the subsequent apoptosis in podocytes via TRPC6-mediated Ca²⁺ entry.     

Mechanosensitive Ca2+ permeant cation channels in human prostate tumor cells

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca²⁺-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd³⁺ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca²⁺]_i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca²⁺ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca²⁺ influx to a small Ca²⁺-activated K⁺ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca²⁺ signaling that may play a role in tumor progression.     

TRPC5 Is a Ca2+-activated Channel Functionally Coupled to Ca2+-selective Ion Channels

TRPC5 forms non-selective cation channels. Here we studied the role of internal Ca²⁺ in the activation of murine TRPC5 heterologously expressed in human embryonic kidney cells. Cell dialysis with various Ca²⁺ concentrations (Ca²⁺_i) revealed a dose-dependent activation of TRPC5 channels by internal Ca²⁺ with EC₅₀ of 635.1 and 358.2 nm at negative and positive membrane potentials, respectively. Stepwise increases of Ca²⁺_i induced by photolysis of caged Ca²⁺ showed that the Ca²⁺ activation of TRPC5 channels follows a rapid exponential time course with a time constant of 8.6 ± 0.2 ms at Ca²⁺_i below 10 μm, suggesting that the action of internal Ca²⁺ is a primary mechanism in the activation of TRPC5 channels. A second slow activation phase with a time to peak of 1.4 ± 0.1 s was also observed at Ca²⁺_i above 10 μm. In support of a Ca²⁺-activation mechanism, the thapsigargin-induced release of Ca²⁺ from internal stores activated TRPC5 channels transiently, and the subsequent Ca²⁺ entry produced a sustained TRPC5 activation, which in turn supported a long-lasting membrane depolarization. By co-expressing STIM1 plus ORAI1 or the α₁C and β₂ subunits of L-type Ca²⁺ channels, we found that Ca²⁺ entry through either calcium-release-activated-calcium or voltage-dependent Ca²⁺ channels is sufficient for TRPC5 channel activation. The Ca²⁺ entry activated TRPC5 channels under buffering of internal Ca²⁺ with EGTA but not with BAPTA. Our data support the hypothesis that TRPC5 forms Ca²⁺-activated cation channels that are functionally coupled to Ca²⁺-selective ion channels through local Ca²⁺ increases beneath the plasma membrane.