Dynamic interplay of excitatory and inhibitory coupling modes of neuronal L-type calcium channels

L-type voltage-gated calcium channels (LTCCs) have long been considered as crucial regulators of neuronal excitability. This role is thought to rely largely on coupling of LTCC-mediated Ca(2+) influx to Ca(2+)-dependent conductances, namely Ca(2+)-dependent K(+) (K(Ca)) channels and nonspecific cation (CAN) channels, which mediate afterhyperpolarizations (AHPs) and afterdepolarizations (ADPs), respectively. However, in which manner LTCCs, K(Ca) channels, and CAN channels co-operate remained scarcely known. In this study, we examined how activation of LTCCs affects neuronal depolarizations and analyzed the contribution of Ca(2+)-dependent potassium- and cation-conductances. With the use of hippocampal neurons in primary culture, pulsed current-injections were applied in the presence of tetrodotoxin (TTX) for stepwise depolarization and the availability of LTCCs was modulated by BAY K 8644 and isradipine. By varying pulse length and current strength, we found that weak depolarizing stimuli tend to be enhanced by LTCC activation, whereas in the course of stronger depolarizations LTCCs counteract excitation. Both effect modes appear to involve the same channels that mediate ADP and AHP, respectively. Indeed, ADPs were activated at lower stimulation levels than AHPs. In the absence of TTX, activation of LTCCs prolonged or shortened burst firing, depending on the initial burst duration, and invariably augmented brief unprovoked (such as excitatory postsynaptic potentials) and provoked electrical events. Hence, regulation of membrane excitability by LTCCs involves synchronous activity of both excitatory and inhibitory Ca(2+)-activated ion channels. The overall enhancing or dampening effect of LTCC stimulation on excitability does not only depend on the relative abundance of the respective coupling partner but also on the stimulus intensity.     

Calcium-permeable channels in plant cells

Calcium signal transduction is a central mechanism by which plants sense and respond to endogenous and environmental stimuli. Cytosolic Ca(2+) elevation is achieved via two cellular pathways, Ca(2+) influx through Ca(2+) channels in the plasma membrane and Ca(2+) release from intracellular Ca(2+) stores. Because of the significance of Ca(2+) channels in cellular signaling, interaction with the environment and developmental processes in plants, a great deal of effort has been invested in recent years with regard to these important membrane proteins. Because of limited space, in this review we focus on recent findings giving insight into both the molecular identity and physiological function of channels that have been suggested to be responsible for the elevation in cytosolic Ca(2+) level, including cyclic nucleotide gated channels, glutamate receptor homologs, two-pore channels and mechanosensitive Ca(2+) -permeable channels. We provide an overview of the regulation of these Ca(2+) channels and their physiological roles and discuss remaining questions.     

Molecular cloning of a cDNA encoding a novel Ca(2+)-dependent nuclease of Arabidopsis that is similar to staphylococcal nuclease

We have isolated a cDNA from Arabidopsis thaliana for a protein consisting of 323 amino acids with similarity to an extracellular nuclease from Staphylococcus. Nuclease assay using toluidine blue-DNA plates has demonstrated that the gene product has nuclease activity dependent on Ca(2+) and inhibited by Zn(2+), designated CAN (Ca(2+)-dependent nuclease). Differing from the staphylococcal nuclease, CAN has neither a signal peptide nor any long hydrophobic regions, suggesting that it is not a secreted protein.     

Voltage- and calcium-dependent inactivation of calcium channels in Lymnaea neurons.

Ca(2+) channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca(2+) buffer (5 mM EGTA), the inactivation of these Ca(2+) channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca(2+) influx, or reduced by higher levels of intracellular Ca(2+) buffering. Inactivation measured under these conditions, despite being independent of Ca(2+) influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca(2+) buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca(2+)-dependent inactivation does not increase linearly with Ca(2+) influx, but saturates for relatively small amounts of Ca(2+) influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca(2+)-dependent inactivation suggests that the Ca(2+) binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca(2+) channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca(2+) channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca(2+) channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca(2+)-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca(2+) channels.     

Algal toxin yessotoxin signalling pathways involve immunocyte mussel calcium channels

A fragment of a putative L-type Ca(2+) channel has been identified by molecular biology experiments in immunocytes from the mussel Mytilus galloprovincialis. Using the cell permeable and Ca(2+)-specific fluorochrome FURA 2-AM, we have demonstrated that the algal toxin yessotoxin (YTX) is able to increase intracellular Ca(2+) concentration in M. galloprovincialis immunocytes. The YTX effect on Ca(2+) increase is inhibited by the L-type Ca(2+) channel inhibitor, verapamil, which is cAMP- and cGMP-dependent, but PKA- and nitric oxide-independent. On the basis of these observations, a possible role for YTX as a potential disturber of mussel immune efficiency is suggested.     

STIM1 and the noncapacitative ARC channels

Our understanding of the nature and regulation of receptor-activated Ca(2+) entry in nonexcitable cells has recently undergone a radical change that began with the identification of the stromal interacting molecule proteins (e.g., STIM1) as playing a critical role in the regulation of the capacitative, or store-operated, Ca(2+) entry. As such, current models emphasize the role of STIM1 located in the endoplasmic reticulum membrane, where it senses the status of the intracellular Ca(2+) stores via a luminal N-terminal Ca(2+)-binding EF-hand domain. Dissociation of Ca(2+) from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca(2+) channels (e.g., CRAC channels). Thus, the specific dependence on store-depletion, and the role of the Ca(2+)-binding EF-hand domain in this process, are critical to all current models of the action of STIM1 on Ca(2+) entry. However, until recently, the effects of STIM1 on other modes of receptor-activated Ca(2+) entry have not been examined. Surprisingly, we found that STIM1 exerts similar, although not identical, actions on the arachidonic acid-regulated Ca(2+)-selective (ARC) channels-a widely expressed mode of agonist-activated Ca(2+) entry whose activation is completely independent of Ca(2+) store depletion. Regulation of the ARC channels by STIM1 is not only independent of store depletion, but also of the Ca(2+)-binding function of the EF-hand, and translocation of STIM1 to the plasma membrane. Instead, it is the pool of STIM1 that constitutively resides in the plasma membrane that is critical for the regulation of the ARC channels. Thus, ARC channel activity is selectively inhibited by exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1. Similarly, introducing mutations in STIM1 that prevent the N-linked glycosylation-dependent constitutive expression of the protein in the plasma membrane specifically inhibits the activity of the ARC channels without affecting the CRAC channels. These studies demonstrate that STIM1 is a far more universal regulator of Ca(2+) entry pathways than previously assumed, and has multiple, and entirely distinct, modes of action. Precisely how this same protein can act in such separate and specific ways on these different pathways of agonist-activated Ca(2+)entry remains an intriguing, yet currently unresolved, question.     

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 Ca(2+) permeable ion channel using Ca(2+) indicators like fluo-3. These Single Channel Ca(2+) Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca(2+) sparks and Ca(2+) puffs caused by Ca(2+) release from intracellular stores (due to the opening of ryanodine receptors and IP(3) receptors, respectively). In contrast to intracellular Ca(2+) 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 Ca(2+) handling in the vicinity of a channel with a known Ca(2+) influx, to obtain the Ca(2+) current passing through plasma membrane cation channels in near physiological solutions, to localize Ca(2+) permeable ion channels on the plasma membrane, and to estimate the Ca(2+) currents underlying those elementary events where the Ca(2+) currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca(2+) channels, and stretch-activated channels. For the L-type Ca(2+) channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca(2+) currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.     

L-type calcium channels are preferentially coupled to endocytosis in bovine chromaffin cells

Exocytosis and endocytosis are Ca(2+)-dependent processes. The contribution of high-voltage activated Ca(2+) channels subtypes to exocytosis has been thoroughly studied in chromaffin cells. However, similar reports concerning endocytosis are unavailable. Thus, we studied here the effects of blockers of L (nifedipine), N (omega-conotoxin GVIA) and P/Q (omega-agatoxin IVA) Ca(2+) channel on Ca(2+) currents (I(Ca)), Ca(2+) entry (Q(Ca)), as well as on the changes in membrane capacitance (C(m)) in perforated-patch voltage-clamped bovine adrenal chromaffin cells. Using 500-ms pulses to 0 or +10 mV, given from a holding potential of -80 mV and 2 mM Ca(2+) we found that omega-conotoxin GVIA affected little the exo-endocytotic responses while omega-agatoxin IVA markedly blocked those responses. However, nifedipine blocked little exocytosis but almost completely inhibited endocytosis. We conclude that L-type Ca(2+) channels seem to be selectively coupled to endocytosis.     

Elementary mechanisms producing facilitation of Cav2.1 (P/Q-type) channels

The regulation of Ca(V)2.1 (P/Q-type) channels by calmodulin (CaM) showcases the powerful Ca(2+) decoding capabilities of CaM in complex with the family of Ca(V)1-2 Ca(2+) channels. Throughout this family, CaM does not simply exert a binary on/off regulatory effect; rather, Ca(2+) binding to either the C- or N-terminal lobe of CaM alone can selectively trigger a distinct form of channel modulation. Additionally, Ca(2+) binding to the C-terminal lobe triggers regulation that appears preferentially responsive to local Ca(2+) influx through the channel to which CaM is attached (local Ca(2+) preference), whereas Ca(2+) binding to the N-terminal lobe triggers modulation that favors activation via Ca(2+) entry through channels at a distance (global Ca(2+) preference). Ca(V)2.1 channels fully exemplify these features; Ca(2+) binding to the C-terminal lobe induces Ca(2+)-dependent facilitation of opening (CDF), whereas the N-terminal lobe yields Ca(2+)-dependent inactivation of opening (CDI). In mitigation of these interesting indications, support for this local/global Ca(2+) selectivity has been based upon indirect inferences from macroscopic recordings of numerous channels. Nagging uncertainty has also remained as to whether CDF represents a relief of basal inhibition of channel open probability (P(o)) in the presence of external Ca(2+), or an actual enhancement of P(o) over a normal baseline seen with Ba(2+) as the charge carrier. To address these issues, we undertake the first extensive single-channel analysis of Ca(V)2.1 channels with Ca(2+) as charge carrier. A key outcome is that CDF persists at this level, while CDI is entirely lacking. This result directly upholds the local/global Ca(2+) preference of the lobes of CaM, because only a local (but not global) Ca(2+) signal is here present. Furthermore, direct single-channel determinations of P(o) and kinetic simulations demonstrate that CDF represents a genuine enhancement of open probability, without appreciable change of activation kinetics. This enhanced-opening mechanism suggests that the CDF evoked during action-potential trains would produce not only larger, but longer-lasting Ca(2+) responses, an outcome with potential ramifications for short-term synaptic plasticity.     

Lack of evidence for presenilins as endoplasmic reticulum Ca2+ leak channels

Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca(2+) ([Ca(2+)](i)) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca(2+)](i) signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca(2+) leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca(2+) release channel opening. To investigate this hypothesis, we employed multiple Ca(2+) imaging protocols and indicators to directly measure ER Ca(2+) dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca(2+) leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca(2+)](i) versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca(2+) indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca(2+) leak channels and highlight the need to use ER-targeted Ca(2+) indicators when studying ER Ca(2+) dynamics.     

Rapid ligand-regulated gating kinetics of single inositol 1,4,5-trisphosphate receptor Ca2+ release channels

The ubiquitous inositol 1,4,5-trisphosphate receptor (InsP(3)R) intracellular Ca(2+) release channel is engaged by thousands of plasma membrane receptors to generate Ca(2+) signals in all cells. Understanding how complex Ca(2+) signals are generated has been hindered by a lack of information on the kinetic responses of the channel to its primary ligands, InsP(3) and Ca(2+), which activate and inhibit channel gating. Here, we describe the kinetic responses of single InsP(3)R channels in native endoplasmic reticulum membrane to rapid ligand concentration changes with millisecond resolution, using a new patch-clamp configuration. The kinetics of channel activation and deactivation showed novel Ca(2+) regulation and unexpected ligand cooperativity. The kinetics of Ca(2+)-mediated channel inhibition showed the single-channel bases for fundamental Ca(2+) release events and Ca(2+) release refractory periods. These results provide new insights into the channel regulatory mechanisms that contribute to complex spatial and temporal features of intracellular Ca(2+) signals.     

Functional consequences of activating store-operated CRAC channels

Store-operated CRAC channels, which are activated by the emptying of the endoplasmic reticulum Ca(2+) stores, are an important and widespread route for triggering rises in cytoplasmic Ca(2+). The cellular responses that are activated in response to Ca(2+) entry through CRAC channels are being dissected out, and recent evidence has established that CRAC channels can induce both short-term (safeguarding the Ca(2+) content of the endoplasmic reticulum, maintenance of cytoplasmic Ca(2+) oscillations, enzyme activation, secretion) and long-term (gene expression) changes in cells. CRAC channel activation is therefore capable of evoking a range of temporally distinct responses, highlighting the versatility of this ubiquitous Ca(2+) entry pathway.     

Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells

Recently, we described a novel 3-pS Ca(2+)-conducting channel that is activated by BAPTA and thapsigargin-induced passive depletion of intracellular Ca(2+) stores and likely to be a native store-operated channel in vascular smooth muscle cells (SMC). Neither Ca(2+) nor inositol 1,4,5-trisphosphate or other second messengers tested activated this channel in membrane patches excised from resting SMC. Here we report that these 3-pS channels are activated in inside-out membrane patches from SMC immediately upon application of Ca(2+) influx factor (CIF) extracted from mutant yeast, which has been previously shown to activate Ca(2+) influx in Xenopus oocytes and Ca(2+) release-activated Ca(2+) current in Jurkat cells. In bioassay experiments depletion of Ca(2+) stores in permeabilized human platelets resulted in the release of endogenous factor, which activated 3-pS channels in isolated inside-out membrane patches excised from SMC and exposed to permeabilized platelets. The same 3-pS channels in excised membrane patches were also activated by acid extracts of CIF derived from human platelets with depleted Ca(2+) stores, which also stimulated Ca(2+) influx upon injection into Xenopus oocytes. Specific high pressure liquid chromatography fractions of platelet extracts were found to have CIF activity when injected into oocytes and activate 3-pS channels in excised membrane patches. These data show for the first time that CIF produced by mammalian cells and yeast with depleted Ca(2+) stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca(2+) store depletion.     

Molecular genetics of ryanodine receptors Ca2+-release channels

The family of ryanodine receptor (RyR) genes encodes three highly related Ca(2+)-release channels: RyR1, RyR2 and RyR3. RyRs are known as the Ca(2+)-release channels that participate to the mechanism of excitation-contraction coupling in striated muscles, but they are also expressed in many other cell types. Actually, in several cells two or three RyR isoforms can be co-expressed and interactive feedbacks among them may be important for generation of intracellular Ca(2+) signals and regulation of specific cellular functions. Important developments have been obtained in understanding the biochemical complexity underlying the process of Ca(2+) release through RyRs. The 3-D structure of these large molecules has been obtained and some regulatory regions have been mapped within these 3-D reconstructions. Recent studies have clarified the role of protein kinases and phosphatases that, by physically interacting with RyRs, appear to play a role in the regulation of these Ca(2+)-release channels. These and other recent advancements in understanding RyR biology will be the object of this review.     

Ca(2+) -permeable channels in the hepatocyte plasma membrane and their roles in hepatocyte physiology

Hepatocytes are highly differentiated and spatially polarised cells which conduct a wide range of functions, including intermediary metabolism, protein synthesis and secretion, and the synthesis, transport and secretion of bile acids. Changes in the concentrations of Ca(2+) in the cytoplasmic space, endoplasmic reticulum (ER), mitochondria, and other intracellular organelles make an essential contribution to the regulation of these hepatocyte functions. While not yet fully understood, the spatial and temporal parameters of the cytoplasmic Ca(2+) signals and the entry of Ca(2+) through Ca(2+)-permeable channels in the plasma membrane are critical to the regulation by Ca(2+) of hepatocyte function. Ca(2+) entry across the hepatocyte plasma membrane has been studied in hepatocytes in situ, in isolated hepatocytes and in liver cell lines. The types of Ca(2+)-permeable channels identified are store-operated, ligand-gated, receptor-activated and stretch-activated channels, and these may vary depending on the animal species studied. Rat liver cell store-operated Ca(2+) channels (SOCs) have a high selectivity for Ca(2+) and characteristics similar to those of the Ca(2+) release activated Ca(2+) channels in lymphocytes and mast cells. Liver cell SOCs are activated by a decrease in Ca(2+) in a sub-region of the ER enriched in type1 IP(3) receptors. Activation requires stromal interaction molecule type 1 (STIM1), and G(i2alpha,) F-actin and PLCgamma1 as facilitatory proteins. P(2x) purinergic channels are the only ligand-gated Ca(2+)-permeable channels in the liver cell membrane identified so far. Several types of receptor-activated Ca(2+) channels have been identified, and some partially characterised. It is likely that TRP (transient receptor potential) polypeptides, which can form Ca(2+)- and Na(+)-permeable channels, comprise many hepatocyte receptor-activated Ca(2+)-permeable channels. A number of TRP proteins have been detected in hepatocytes and in liver cell lines. Further experiments are required to characterise the receptor-activated Ca(2+) permeable channels more fully, and to determine the molecular nature, mechanisms of activation, and precise physiological functions of each of the different hepatocyte plasma membrane Ca(2+) permeable channels.     

Inactivation of L-type calcium channels in cardiomyocytes. Experimental and theoretical approaches

The L-type calcium current (ICa) plays an important role in excitation-contraction coupling of heart cells. It is critical for forming the major trigger for Ca(2+)-induced Ca(2+) release from the sarcoplasmic reticulum and hence its feedback regulation is of fundamental biological significance. The channel inactivation sharpens the kinetics and temporal precision of the Ca(2+) signals so that it prevents longer-term increases in free intracellular Ca(2+) concentration. Cardiac L-type Ca(2+) channels are known to inactivate through voltage- and Ca(2+)-dependent mechanisms. Pure voltage-dependent inactivation has a much slower time course of development than Ca(2+)-dependent inactivation and plays minor role in inhibition of Ca(2+) influx into the cell. The major determinant of the inactivation kinetics of Ca(2+) current during depolarization is Ca(2+)-dependent mechanisms. Furthermore, it is possible to distinguish two phases in Ca(2+)-dependent inactivation of calcium current: a slow phase that depends on Ca(2+) flow through the channels (Ca(2+) current-dependent inactivation) and a fast one that depends on Ca(2+) released from the sarcoplasmic reticulum (Ca(2+) release-dependent inactivation). Although both Ca(2+) released from the SR and Ca(2+) permeating channels play a role, SR-released Ca(2+) is the most effective inactivation mechanism in inhibition of Ca(2+) entry through the channel.     

Mitochondrial regulation of store-operated CRAC channels

In eukaryotic cells, one major route for Ca(2+) influx is through store-operated CRAC channels, which are activated following a fall in Ca(2+) content within the endoplasmic reticulum. Mitochondria are key regulators of this ubiquitous Ca(2+) influx pathway. Respiring mitochondria rapidly take up some of the Ca(2+) released from the stores, resulting in more extensive store depletion and thus robust activation of CRAC channels. As CRAC channels open, the ensuing rise in cytoplasmic Ca(2+) feeds back to inactivate the channels. By buffering some of the incoming Ca(2+) mitochondria reduce Ca(2+)-dependent inactivation of the CRAC channels, resulting in more prolonged Ca(2+) influx. However, mitochondria can release Ca(2+) close to the endoplasmic reticulum, accelerating store refilling and thus promoting deactivation of the CRAC channels. Mitochondria thus regulate all major transitions in CRAC channel gating, revealing remarkable versatility in how this organelle impacts upon Ca(2+) influx. Recent evidence suggests that mitochondria also control CRAC channels through mechanisms that are independent of their Ca(2+)-buffering actions and ability to generate ATP. Furthermore, pyruvic acid, a key intermediary metabolite and precursor substrate for the Krebs cycle, reduces the extent of Ca(2+)-dependent inactivation of CRAC channels. Hence mitochondrial metabolism impacts upon Ca(2+) influx through CRAC channels and thus on a range of key downstream cellular responses.     

Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia

The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca(2+)](cyt)) is essential for pollen-tube growth. Local Ca(2+) influx mediated by Ca(2+)-permeable channels plays a key role in maintaining this [Ca(2+)](cyt) gradient. Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba(2+)) approximately Ca(2+) > magnesium (Mg(2+)) > strontium (Sr(2+)) > manganese (Mn(2+)). This channel conductance was selective for Ca(2+) over chlorine (Cl(-)) (relative permeability P(Ca)/P(Cl) = 14 in 10 mm extracellular Ca(2+)). We also showed that the channel was inhibited by the Ca(2+) channel blockers lanthanum (La(3+)) and gadolinium (Gd(3+)). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca(2+)-permeable channel is likely to play a role in mediating Ca(2+) influx into the growing pollen tubes to maintain the [Ca(2+)](cyt) gradient.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

Ion channels and transporters in cancer. 6. Vascularizing the tumor: TRP channels as molecular targets

Tumor vascularization is a critical process that determines tumor growth and metastasis. In the last decade new experimental evidence obtained from in vitro and in vivo studies have challenged the classical angiogenesis model forcing us to consider new scenarios for tumor neovascularization. In particular, the genetic stability of tumor-derived endothelial cells (TECs) has been recently questioned in several studies, which show that TECs, as well as pericytes, differ significantly from their normal counterparts at genetic and functional levels. In addition to such an epigenetic action of tumor microenvironment on endothelial cells (ECs) commitment, the distinct characteristics of TECs could be due to differences in their origin compared with preexisting differentiated ECs. Intracellular Ca(2+) signals are involved at different critical phases in the regulation of the complex process of angiogenesis and tumor progression. These signals are generated by a wide variety of intrinsic and extrinsic factors. Several key components of Ca(2+) signaling including Ca(2+) channels in the plasma membrane, endoplasmic reticulum, calcium pumps, and mitochondria contribute to the generation, amplitude, and frequency of these Ca(2+) change. In particular, several members of the transient receptor potential (TRP) family of calcium-permeable channels have profound effects on the function of ECs. Because of its multifaceted role in the control of cell function, proliferation, and motility, TRP channels have been suggested as a potential molecular target for control of tumor neovascularization. Since plasma membrane Ca(2+) channels are easily and directly accessible via the bloodstream, they are potential targets for a number of pharmacological and antibody-targeted therapeutic strategies, with specificity being the main limitation. In this review we discuss recent advances in understanding the role of Ca(2+) channels, with specific reference to TRP channels, in tumor vascularization process.