Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca²⁺ is required for both structural and biophysical roles. In addition, changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) orchestrate responses to developmental and environmental signals. In many instances, [Ca²⁺]_cyt is increased by Ca²⁺ influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca²⁺-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca²⁺-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca²⁺-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca²⁺ channels, the genetic counterparts of specific Ca²⁺-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca²⁺ channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca²⁺ channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La³⁺ and nifedipine, and their increased activity as [Ca²⁺]_cyt is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca²⁺-permeable) K⁺ channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.     

Abnormal expression, localization and interaction of canonical transient receptor potential ion channels in human breast cancer cell lines and tissues: a potential target for breast cancer diagnosis and therapy

Background  

Ca²⁺ is known to be involved in a number of metastatic processes including motility and proliferation which can result in store-depletion of Ca²⁺. Up regulation of genes which contribute to store operated channel (SOC) activity may plausibly be necessary for these processes to take place efficiently. TRPC proteins constitute a family of conserved Ca²⁺-permeable channels that have been shown to contribute to SOC activity.     

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.     

Personal recollections on the discovery of the ryanodine receptors of muscle

The intracellular Ca²⁺ release channels are indispensable molecular machinery in practically all eukaryotic cells of multicellular animals. They serve a key role in cell signaling by way of Ca²⁺ as a second messenger. In response to a signaling event, the channels release Ca²⁺ from intracellular stores. The resulting rise in cytoplasmic Ca²⁺ concentration triggers the cell to carry out its specialized role, after which the intracellular Ca²⁺ concentration must be reduced so that the signaling event can again be repeated. There are two types of intracellular Ca²⁺ release channels, i.e., the ryanodine receptors and the inositol triphosphate receptors. My focus in this minireview is to present a personal account, from the vantage point our laboratory, of the discovery, isolation, and characterization of the ryanodine receptors from mammalian muscle. There are three isoforms: ryanodine receptor 1 (RyR1), first isolated from rabbit fast twitch skeletal muscle; ryanodine receptor 2 (RyR2), first isolated from dog heart; and ryanodine receptor 3 (RyR3), first isolated from bovine diaphragm muscle. The ryanodine receptors are the largest channel structures known. The RyR isoforms are very similar albeit with important differences. Natural mutations in humans in these receptors have already been associated with a number of muscle diseases.     

Apamin Boosting of Synaptic Potentials in CaV2.3 R-Type Ca2+ Channel Null Mice

SK2- and K_V4.2-containing K⁺ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca²⁺ source that provides Ca²⁺-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca²⁺ entry through NMDARs, whereas K_V4.2-containing channel availability is increased by Ca²⁺ entry through SNX-482 (SNX) sensitive Ca_V2.3 R-type Ca²⁺ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca²⁺ entry through R-type Ca²⁺ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca²⁺ channels and K_V4.2-containing K⁺ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca²⁺ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from Ca_V2.3 null mice. The results show that in the absence of Ca_V2.3 channels, apamin application still boosted EPSPs. The boosting effect of Ca_V2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from Ca_V2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and K_V4.2-containing channels to Ca_V2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.     

Calcium-dependent inactivation and the dynamics of calcium puffs and sparks

Localized intracellular Ca²⁺ elevations known as puffs and sparks arise from the cooperative activity of inositol 1,4,5-trisphosphate receptor Ca²⁺ channels (IP₃Rs) and ryanodine receptor Ca²⁺ channels (RyRs) clustered at Ca²⁺ release sites on the surface of the endoplasmic reticulum or sarcoplasmic reticulum. When Markov chain models of these intracellular Ca²⁺-regulated Ca²⁺ channels are coupled via a mathematical representation of a Ca²⁺ microdomain, simulated Ca²⁺ release sites may exhibit the phenomenon of “stochastic Ca²⁺ excitability” reminiscent of Ca²⁺ puffs and sparks where channels open and close in a concerted fashion. To clarify the role of Ca²⁺ inactivation of IP₃Rs and RyRs in the dynamics of puffs and sparks, we formulate and analyze Markov chain models of Ca²⁺ release sites composed of 10–40 three-state intracellular Ca²⁺ channels that are inactivated as well as activated by Ca²⁺. We study how the statistics of simulated puffs and sparks depend on the kinetics and dissociation constant of Ca²⁺ inactivation and find that puffs and sparks are often less sensitive to variations in the number of channels at release sites and strength of coupling via local [Ca²⁺] when the average fraction of inactivated channels is significant. Interestingly, we observe that the single channel kinetics of Ca²⁺ inactivation influences the thermodynamic entropy production rate of Markov chain models of puffs and sparks. While excessively fast Ca²⁺ inactivation can preclude puffs and sparks, moderately fast Ca²⁺ inactivation often leads to time-irreversible puffs and sparks whose termination is facilitated by the recruitment of inactivated channels throughout the duration of the puff/spark event. On the other hand, Ca²⁺ inactivation may be an important negative feedback mechanism even when its time constant is much greater than the duration of puffs and sparks. In fact, slow Ca²⁺ inactivation can lead to release sites with a substantial fraction of inactivated channels that exhibit puffs and sparks that are nearly time-reversible and terminate without additional recruitment of inactivated channels.     

4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer

Calcium (Ca²⁺) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca²⁺ release was associated with the ER Ca²⁺ release channels, inositol 1,4,5?triphosphate receptor (IP₃R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca²⁺ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca²⁺ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca²⁺ concentration ([Ca²⁺]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca²⁺ release mechanism distinct from classical Ca²⁺ release channels.     

Developmental alterations in the biophysical properties of Cav1.3 Ca2+ channels in mouse inner hair cells

Prior to hearing onset, spontaneous action potentials activate voltage-gated Ca_v1.3 Ca²⁺ channels in mouse inner hair cells (IHCs), which triggers exocytosis of glutamate and excitation of afferent neurons. In mature IHCs, Ca_v1.3 channels open in response to evoked receptor potentials, causing graded changes in exocytosis required for accurate sound transmission. Developmental alterations in Ca_v1.3 properties may support distinct roles of Ca_v1.3 in IHCs in immature and mature IHCs, and have been reported in various species. It is not known whether such changes in Ca_v1.3 properties occur in mouse IHCs, but this knowledge is necessary for understanding the roles of Ca_v1.3 in developing and mature IHCs. Here, we describe age-dependent differences in the biophysical properties of Ca_v1.3 channels in mouse IHCs. In mature IHCs, Ca_v1.3 channels activate more rapidly and exhibit greater Ca²⁺-dependent inactivation (CDI) than in immature IHCs. Consistent with the properties of Ca_v1.3 channels in heterologous expression systems, CDI in mature IHCs is not affected by increasing intracellular Ca²⁺ buffering strength. However, CDI in immature IHCs is significantly reduced by strong intracellular Ca²⁺ buffering, which both slows the onset of, and accelerates recovery from, inactivation. These results signify a developmental decline in the sensitivity of CDI to global elevations in Ca²⁺, which restricts negative feedback regulation of Ca_v1.3 channels to incoming Ca²⁺ ions in mature IHCs. Together with faster Ca_v1.3 activation kinetics, increased reliance of Ca_v1.3 CDI on local Ca²⁺ may sharpen presynaptic Ca²⁺ signals and improve temporal aspects of sound coding in mature IHCs.     

Calcium signalling in T-lymphocytes

Calcium signalling is essential for most of the biological T-cell activities, including in Th2 lymphocytes, a T-cell subset that produce interleukin 4, 5 and 13 and which is involved in allergic diseases. T-cell receptor engagement induces the production of inositol trisphosphate that binds to its receptor, releasing intracellular Ca²⁺ stores. STIM in the endo (sarco) plasmic reticulum (ER/SR) is a Ca²⁺ sensor that perceives the depletion of intracellular Ca²⁺ stores, localizes near the cell membrane and allows the activation of ORAI, the main calcium channels at the cell membrane. However, other calcium channels at the membrane of intracellular compartments and at the cell membrane can also contribute to the TCR-driven intracellular Ca²⁺ rise. Among them, voltage-dependent calcium (Ca_v1) channels have been reported in several types of T-lymphocytes, although how they are gated in these non-excitable cells remains unsolved. We have shown that Cav1 channel expression was selectively up regulated in Th2 lymphocytes. In this review, we will discuss about the diversity of the Ca²⁺ channels responsible for Ca²⁺ homeostasis in the different cell subsets and the interactions between these molecules, which can account for the variety of the calcium responses depending upon the functions of effector T-cells.     

TRPC4 and TRPC5: receptor-operated Ca-permeable nonselective cation channels

The seven mammalian channels from the classical (TRPC) subfamily of transient receptor potential (TRP) channels are thought to be receptor-operated cation channels activated in a phospholipase C (PLC)-dependent manner. Based on sequence similarity, TRPC channels can be divided into four subgroups. Group 4 comprises TRPC4 and TRPC5, and is most closely related to group 1 (TRPC1). The functional properties observed following heterologous expression of TRPC4 or TRPC5 in mammalian cells are contradictory and, therefore, controversial. In our hands, and in several independent studies, both channels, probably as homotetramers, form receptor-operated, Ca²⁺-permeable, nonselective cation channels activated independently of inositol 1,4,5-trisphosphate (InsP₃) receptor activation or Ca²⁺ store-depletion. As heteromultimers with TRPC1, TRPC4 and TRPC5 form receptor-operated, Ca²⁺-permeable, nonselective cation channels with biophysical properties distinct from homomeric TRPC4 or TRPC5. In other studies, TRPC4 and TRPC5 have been shown to be store-operated channels, with moderate to high Ca²⁺ permeabilities. At present there is no clear explanation for these major differences in functional properties. To date, little is known as to which native cation channels are formed by TRPC4 and TRPC5. Endothelial cells from TRPC4^−/− mice lack a highly Ca²⁺-permeable, store-dependent current, and data support a role for TRPC4 in endothelium-mediated vasorelaxation. A similar current in adrenal cortical cells is reduced by TRPC4 antisense. From similarities in the properties of the currents and expression of appropriate isoforms in the tissues, it is likely that heteromultimers of TRPC1 and TRPC4 or TRPC5 form receptor-operated nonselective cation channels in central neurones, and that TRPC4 contributes to nonselective cation channels in intestinal smooth muscle.     

P2Y13 Receptor-Mediated Rapid Increase in Intracellular Calcium Induced by ADP in Cultured Dorsal Spinal Cord Microglia

P2Y receptors have been implicated in the calcium mobilization by the response to neuroexcitatory substances in neurons and astrocytes, but little is known about P2Y receptors in microglia cells. In the present study, the effects of ADP on the intracellular calcium concentration ([Ca²⁺]i) in cultured dorsal spinal cord microglia were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescence indicator that could monitor real-time alterations of [Ca²⁺]i. Here we show that ADP (0.01–100 μM) causes a rapid increase in [Ca²⁺]i with a dose-dependent manner in cultured microglia. The action of ADP on [Ca²⁺]i was significantly blocked by MRS2211 (a selective P2Y₁₃ receptor antagonist), but was unaffected by MRS2179 (a selective P2Y₁ receptor antagonist) or MRS2395 (a selective P2Y₁₂ receptor antagonist), which suggest that P2Y₁₃ receptor may be responsible for ADP-evoked Ca²⁺ mobilization in cultured microglia. P2Y₁₃-evoked Ca²⁺ response can be obviously inhibited by BAPTA-AM and U-73122, respectively. Moreover, removal of extracellular Ca²⁺ (by EGTA) also can obvious suppress the Ca²⁺ mobilization. These results means both intracellular calcium and extracellular calcium are potentially important mechanisms in P2Y₁₃ receptor-evoked Ca²⁺ mobilization. However, P2Y₁₃ receptor-evoked Ca²⁺ response was not impaired after CdCl₂ and verapamil administration, which suggest that voltage-operated Ca²⁺ channels may be not related with P2Y₁₃-evoked Ca²⁺ response. In addition, Ca²⁺ mobilization induced by ADP was abolished by different store-operated Ca²⁺ channels (SOCs) blocker, 2-APB (50 μM) and SKF-96365 (1 mM), respectively. These observations suggest that the activation of P2Y₁₃ receptor might be involved in the effect of ADP on [Ca²⁺]i in cultured dorsal spinal cord microglia. Furthermore, our results raise a possibility that P2Y₁₃ receptor activation causes Ca²⁺ release from Ca²⁺ store, which leads to the opening of SOCs.     

The transient receptor potential family of ion channels

The transient receptor potential (TRP) multigene superfamily encodes integral membrane proteins that function as ion channels. Members of this family are conserved in yeast, invertebrates and vertebrates. The TRP family is subdivided into seven subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin) and TRPN (NOMPC-like); the latter is found only in invertebrates and fish. TRP ion channels are widely expressed in many different tissues and cell types, where they are involved in diverse physiological processes, such as sensation of different stimuli or ion homeostasis. Most TRPs are non-selective cation channels, only few are highly Ca²⁺ selective, some are even permeable for highly hydrated Mg²⁺ ions. This channel family shows a variety of gating mechanisms, with modes of activation ranging from ligand binding, voltage and changes in temperature to covalent modifications of nucleophilic residues. Activated TRP channels cause depolarization of the cellular membrane, which in turn activates voltage-dependent ion channels, resulting in a change of intracellular Ca²⁺ concentration; they serve as gatekeeper for transcellular transport of several cations (such as Ca²⁺ and Mg²⁺), and are required for the function of intracellular organelles (such as endosomes and lysosomes). Because of their function as intracellular Ca²⁺ release channels, they have an important regulatory role in cellular organelles. Mutations in several TRP genes have been implicated in diverse pathological states, including neurodegenerative disorders, skeletal dysplasia, kidney disorders and pain, and ongoing research may help find new therapies for treatments of related diseases.     

Molecular characterization and functional expression of the Apis mellifera voltage-dependent Ca2+ channels

Voltage-gated Ca²⁺ channels allow the influx of Ca²⁺ ions from the extracellular space upon membrane depolarization and thus serve as a transducer between membrane potential and cellular events initiated by Ca²⁺ transients. Most insects are predicted to possess three genes encoding Cavα, the main subunit of Ca²⁺ channels, and several genes encoding the two auxiliary subunits, Cavβ and Cavα2δ; however very few of these genes have been cloned so far. Here, we cloned three full-length cDNAs encoding the three Cavα subunits (AmelCav1a, AmelCav2a and AmelCav3a), a cDNA encoding a novel variant of the Cavβ subunit (AmelCavβc), and three full-length cDNAs encoding three Cavα2δ subunits (AmelCavα2δ1 to 3) of the honeybee Apis mellifera. We identified several alternative or mutually exclusive exons in the sequence of the AmelCav2 and AmelCav3 genes. Moreover, we detected a stretch of glutamine residues in the C-terminus of the AmelCav1 subunit that is reminiscent of the motif found in the human Cav2.1 subunit of patients with Spinocerebellar Ataxia type 6. All these subunits contain structural domains that have been identified as functionally important in their mammalian homologues. For the first time, we could express three insect Cavα subunits in Xenopus oocytes and we show that AmelCav1a, 2a and 3a form Ca²⁺ channels with distinctive properties. Notably, the co-expression of AmelCav1a or AmelCav2a with AmelCavβc and AmCavα2δ1 produces High Voltage-Activated Ca²⁺ channels. On the other hand, expression of AmelCav3a alone leads to Low Voltage-Activated Ca²⁺ channels.     

Physiological Epidermal Growth Factor Concentrations Activate High Affinity Receptors to Elicit Calcium Oscillations

Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca²⁺ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca²⁺ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca²⁺ signal quantitatively similar to the Ca²⁺ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca²⁺ channels that are not activated by internal Ca²⁺ store depletion, while the nanomolar EGF response involved internal Ca²⁺ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K⁺ channels, the nanomolar response was insensitive to the blockade of these ion channels.     

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca²⁺ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca²⁺ imaging, we found that the depletion of ER/SR Ca²⁺ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca²⁺ ([Ca²⁺]_i), followed by sustained increase depending on extracellular Ca²⁺. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na⁺/Ca²⁺ exchanger inhibitors, inhibited [Ca²⁺]_i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl₃) or by an increased extracellular Ca²⁺([Ca²⁺]_o) increased the concentration of intracelluar Ca²⁺, whereas, the sustained elevation of [Ca²⁺]_i was reduced in the presence of SKF96365. Similarly, the duration of [Ca²⁺]_i increase was also shortened in the absence of extracellular Ca²⁺. Western blot analysis showed that GdCl₃ increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl₃. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca²⁺-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.     

PAC1hop receptor activation facilitates catecholamine secretion selectively through 2-APB-sensitive Ca2+ channels in PC12 cells

PACAP is a critical regulator of long-term catecholamine secretion from the adrenal medulla in vivo, however the receptor or pathways for Ca²⁺ entry triggering acute and sustained secretion have not been adequately characterized. We have previously cloned the bovine adrenal chromaffin cell PAC1 receptor that contains the molecular determinants required for PACAP-induced Ca²⁺ elevation and is responsible for imparting extracellular Ca²⁺ influx-dependent secretory competence in PC12 cells. Here, we use this cell model to gain mechanistic insights into PAC1hop-dependent Ca²⁺ pathways responsible for catecholamine secretion. PACAP-modulated extracellular Ca²⁺ entry in PC12 cells could be partially blocked with nimodipine, an inhibitor of L-type VGCCs and partially blocked by 2-APB, an inhibitor and modulator of various transient receptor potential (TRP) channels. Despite the co-existence of these two modes of Ca²⁺ entry, sustained catecholamine secretion in PC12 cells was exclusively modulated by 2-APB-sensitive Ca²⁺ channels. While IP3 generation occurred after PACAP exposure, most PACAP-induced Ca²⁺ mobilization involved release from ryanodine-gated cytosolic stores. 2-APB-sensitive Ca²⁺ influx, and subsequent catecholamine secretion was however not functionally related to intracellular Ca²⁺ mobilization and store depletion. The reconstituted PAC1hop-expessing PC12 cell model therefore recapitulates both PACAP-induced Ca²⁺ release from ER stores and extracellular Ca²⁺ entry that restores PACAP-induced secretory competence in neuroendocrine cells. We demonstrate here that although bPAC1hop receptor occupancy induces Ca²⁺ entry through two independent sources, VGCCs and 2-APB-sensitive channels, only the latter contributes importantly to sustained vesicular catecholamine release that is a fundamental characteristic of this neuropeptide system. These results emphasize the importance of establishing functional linkages between Ca²⁺ signaling pathways initiated by pleotrophic signaling molecules such as PACAP, and physiologically important downstream events, such as secretion, triggered by them.     

Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs—fundamental calcium events

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca²⁺ permeable ion channel using Ca²⁺ indicators like fluo-3. These Single Channel Ca²⁺ Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca²⁺ sparks and Ca²⁺ puffs caused by Ca²⁺ release from intracellular stores (due to the opening of ryanodine receptors and IP₃ receptors, respectively). In contrast to intracellular Ca²⁺ release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca²⁺ handling in the vicinity of a channel with a known Ca²⁺ influx, to obtain the Ca²⁺ current passing through plasma membrane cation channels in near physiological solutions, to localize Ca²⁺ permeable ion channels on the plasma membrane, and to estimate the Ca²⁺ currents underlying those elementary events where the Ca²⁺ currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca²⁺ channels, and stretch-activated channels. For the L-type Ca²⁺ channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca²⁺ currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Chemico-genetic identification of drebrin as a regulator of calcium responses

Store-operated calcium channels are plasma membrane Ca²⁺ channels that are activated by depletion of intracellular Ca²⁺ stores, resulting in an increase in intracellular Ca²⁺ concentration, which is maintained for prolonged periods in some cell types. Increases in intracellular Ca²⁺ concentration serve as signals that activate a number of cellular processes, however, little is known about the regulation of these channels. We have characterized the immuno-suppressant compound BTP, which blocks store-operated channel mediated calcium influx into cells. Using an affinity purification scheme to identify potential targets of BTP, we identified the actin reorganizing protein, drebrin, and demonstrated that loss of drebrin protein expression prevents store-operated channel mediated Ca²⁺ entry, similar to BTP treatment. BTP also blocks actin rearrangements induced by drebrin. While actin cytoskeletal reorganization has been implicated in store-operated calcium channel regulation, little is known about actin-binding proteins that are involved in this process, or how actin regulates channel function. The identification of drebrin as a mediator of this process should provide new insight into the interaction between actin rearrangement and store-operated channel mediated calcium influx.