Showing papers in "Channels in 2012"

Gating the mechanical channel Piezo1: a comparison between whole-cell and patch recording.

Abstract: Piezo1 is a eukaryotic cation-selective mechanosensitive ion channel To understand channel function in vivo, we first need to analyze and compare the response in the whole cell and the patch In patches, Piezo1 inactivates and the current is fit well by a 3-state model with a single pressure-dependent rate However, repeated stimulation led to an irreversible loss of inactivation Remarkably, the loss of inactivation did not occur on a channel-by-channel basis but on all channels at the same time Thus, the channels are in common mechanical domain Divalent ions decreased the unitary conductance from ~68 pS to ~37 pS, irrespective of the cation species Mg and Ca did not affect inactivation rates, but Zn caused a 3-fold slowing CytochalasinD (cytoD) does not alter inactivation rates or the transition to the non-inactivating mode but does reduce the steady-state response Whole-cell currents were similar to patch currents but also had significant differences In contrast to the patch, cytoD inhibited the current suggesting that the activating forces were transmitted through the actin cytoskeleton Hypotonic swelling that prestressed the cytoskeleton and the bilayer greatly increased the sensitivity of both control and cytoD cells so there are two pathways to transmit force to the channels In contrast to patch, removing divalent ions decreased the whole-cell current The difference between whole cell and patch properties provide new insights into our understanding of the Piezo1 gating mechanisms and cautions against generalization to in situ behavior

Voltage-gated sodium channels and metastatic disease.

Abstract: Voltage-gated Na+ channels (VGSCs) are macromolecular protein complexes containing a pore-forming α subunit and smaller non-pore-forming β subunits. VGSCs are expressed in metastatic cells from a number of cancers. In these cells, Na+ current carried by α subunits enhances migration, invasion and metastasis in vivo. In contrast, the β subunits mediate cellular adhesion and process extension. The prevailing hypothesis is that VGSCs are upregulated in cancer, in general favoring an invasive/metastatic phenotype, although the mechanisms are still not fully clear. Expression of the Nav1.5 α subunit associates with poor prognosis in clinical breast cancer specimens, suggesting that VGSCs may have utility as prognostic markers for cancer progression. Furthermore, repurposing existing VGSC-blocking therapeutic drugs may provide a new strategy to improve outcomes in patients suffering from metastatic disease, which is the major cause of cancer-related deaths, and for which there is currently no cure.

Touch sense: functional organization and molecular determinants of mechanosensitive receptors.

Abstract: Cutaneous mechanoreceptors are localized in the various layers of the skin where they detect a wide range of mechanical stimuli, including light brush, stretch, vibration and noxious pressure. This variety of stimuli is matched by a diverse array of specialized mechanoreceptors that respond to cutaneous deformation in a specific way and relay these stimuli to higher brain structures. Studies across mechanoreceptors and genetically tractable sensory nerve endings are beginning to uncover touch sensation mechanisms. Work in this field has provided researchers with a more thorough understanding of the circuit organization underlying the perception of touch. Novel ion channels have emerged as candidates for transduction molecules and properties of mechanically gated currents improved our understanding of the mechanisms of adaptation to tactile stimuli. This review highlights the progress made in characterizing functional properties of mechanoreceptors in hairy and glabrous skin and ion channels that detect mechanical inputs and shape mechanoreceptor adaptation.

Characterization of three novel mechanosensitive channel activities in Escherichia coli

Abstract: Mechanosensitive channels sense elevated membrane tension that arises from rapid water influx occurring when cells move from high to low osmolarity environments (hypoosmotic shock). These non-specific channels in the cytoplasmic membrane release osmotically-active solutes and ions. The two major mechanosensitive channels in Escherichia coli are MscL and MscS. Deletion of both proteins severely compromises survival of hypoosmotic shock. However, like many bacteria, E. coli cells possess other MscS-type genes (kefA, ybdG, ybiO, yjeP and ynaI). Two homologs, MscK (kefA) and YbdG, have been characterized as mechanosensitive channels that play minor roles in maintaining cell integrity. Additional channel openings are occasionally observed in patches derived from mutants lacking MscS, MscK and MscL. Due to their rare occurrence, little is known about these extra pressure-induced currents or their genetic origins. Here we complete the identification of the remaining E. coli mechanosensitive channels YnaI, YbiO a...

Piezo1: Properties of a cation selective mechanical channel

Abstract: Piezo ion channels have been found to be essential for mechanical responses in cells. These channels were first shown to exist in Neuro2A cells, and the gene was identified by siRNAs that diminished the mechanical response. Piezo channels are approximately 2500 amino acids long, have between 24–32 transmembrane regions, and appear to assemble into tetramers and require no other proteins for activity. They have a reversal potential around 0 mV and show voltage dependent inactivation. The channel is constitutively active in liposomes, indicating that no cytoskeletal elements are required. Heterologous expression of the Piezo protein can create mechanical sensitivity in otherwise insensitive cells. Piezo1 currents in outside-out patches were blocked by the extracellular MSC inhibitor peptide GsMTx4. Both enantiomeric forms of GsMTx4 inhibited channel activity in a manner similar to endogenous mechanical channels. Piezo1 can adopt a tonic (non-inactivating) form with repeated stimulation. The transition to th...

Is ZD7288 a selective blocker of hyperpolarization-activated cyclic nucleotide-gated channel currents?

Abstract: ZD7288 has been widely used as a tool in the study of hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels), and to test the relationships between HCN channels and heart and brain function. ZD7288 is widely considered a selective blocker of HCN currents. Here we show that ZD7288 inhibits not only HCN channel currents, but also Na+ currents in DRG neurons and ZD7288 was confirmed to inhibit Na+ current in HEK293 cells transfected with Nav1.4 plasmids. Thus our findings challenge the view that ZD7288 is a selective blocker of HCN channels. Conclusions about the role of NCN channels in neuronal function should be re-evaluated if based exclusively on the effect of ZD7288.

2-aminoethyl diphenyl borinate (2-APB) inhibits TRPM7 channels through an intracellular acidification mechanism.

Abstract: 2-APB is a widely used compound in ion channel research. It affects numerous channels including inositol 1,4,5-trisphosphate receptors, store-operated calcium channels and TRP channels, TRPV3 and TRPM7 among them. A characteristic property of TRPM7 channels is their sensitivity to intracellular Mg ( 2+) and pH. Using patch clamp electrophysiology we find that in Jurkat T lymphocytes, 100-300 µM extracellular 2-APB reversibly inhibits TRPM7 channels when internal HEPES concentration is low (1 mM). Increasing the concentration to 140 mM abolishes the 2-APB effect. Using single-cell fluorescence pH video imaging, we show that at concentrations of 100 µM and higher, 2-APB potently acidifies the cytoplasm. We conclude that TRPM7 sensitivity to 2-APB is not direct but rather, can be explained by cytoplasmic acidification and a resulting channel inhibition.

Functional outcome of pannexin-deficient mice after cerebral ischemia.

Abstract: Pannexin (Px, Panx) channels have been implicated in several physiological and pathological processes. We recently studied the potential contribution of pannexins in ischemic brain damage using Px1-/- Px2-/- mice and provided evidence that (1) the release of IL-1β and hemichannel function in astrocytes are, in contrast to published data, not affected by the absence of Px1 and Px2, (2) channel function in neurons lacking Px1 and Px2 is impaired and (3) Px1-/- Px2-/- mice had a better functional outcome and smaller infarcts than wild-type mice when subjected to ischemic stroke. Here, we further investigate the neurological outcome of wild-type and pannexin double-knockout mice 48 h after permanent occlusion of the distal middle cerebral artery (MCAO). Pannexin double-knockout mice (Px1-/- Px2-/-) were less impaired in parameters such as exploration, anxiety, sensorimotor function and behavioral symmetry.

Potassium channels in articular chondrocytes

Abstract: Chondrocytes are the resident cells of cartilage, which synthesize and maintain the extracellular matrix. The range of known potassium channels expressed by these unique cells is continually increasing. Since chondrocytes are non-excitable, and do not need to be repolarized following action potentials, the function of potassium channels in these cells has, until recently, remained completely unknown. However, recent advances in both traditional physiology and “omic” technologies have enhanced our knowledge and understanding of the chondrocyte channelome. A large number of potassium channels have been identified and a number of putative, but credible, functions have been proposed. Members of each of the potassium channel sub-families (calcium activated, inward rectifier, voltage-gated and tandem pore) have all been identified. Mechanotransduction, cell volume regulation, apoptosis and chondrogenesis all appear to involve potassium channels. Since evidence suggests that potassium channel gene transcription ...

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers.

Abstract: We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave k(on) = 13 x 10 ( 6) M (-1) s (-1) and k(off) = 1609 sec (-1) with K(D) = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a K(D) = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

Abstract: In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40-48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40-48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.

Mechanosensitive ion channels: an evolutionary and scientific tour de force in mechanobiology.

Abstract: Mechanosensitive (MS) ion channels are to date the best characterized biological force-sensing systems. They present the best example of coupling protein conformations to the mechanics of the surrounding cell membrane. Studies of MS channels conducted over the last 20 eight years have from their serendipitous discovery1,2 and confusion about their artifactual nature3 to their molecular identification4-6 and structural determination7-10 greatly contributed to our understanding of molecular mechanisms underlying the physiology of mechanosensory transduction.

PKC phosphorylates GluA1-Ser831 to enhance AMPA receptor conductance.

Abstract: AMPA receptors mediate fast excitatory synaptic transmission in the brain, and are dynamically regulated by phosphorylation of multiple residues within the C-terminal domain. CaMKII phosphorylates Ser831 within the AMPA receptor GluA1 subunit to increase single channel conductance, and biochemical studies show that PKC can also phosphorylate this residue. In light of the discovery of additional PKC phosphorylation sites within the GluA1 C-terminus, it remains unclear whether PKC phosphorylation of Ser831 increases GluA1 conductance in intact receptors. Here, we report that the purified, catalytic subunit of PKC significantly increases the conductance of wild-type GluA1 AMPA receptors expressed in the presence of stargazin in HEK293T cells. Furthermore, the mutation GluA1-S831A blocks the functional effect of PKC. These findings suggest that GluA1 AMPA receptor conductance can be increased by activated CaMKII or PKC, and that phosphorylation at this site provides a mechanism for channel modulation via a variety of protein signaling cascades.

KCNE2 and the K (+) channel: the tail wagging the dog.

Abstract: KCNE2, originally designated MinK-related peptide 1 (MiRP1), belongs to a five-strong family of potassium channel ancillary (β) subunits that, despite the diminutive size of the family and its members, has loomed large in the field of ion channel physiology. KCNE2 dictates K (+) channel gating, conductance, α subunit composition, trafficking and pharmacology, and also modifies functional properties of monovalent cation-nonselective HCN channels. The Kcne2 (-/-) mouse exhibits cardiac arrhythmia and hypertrophy, achlorhydria, gastric neoplasia, hypothyroidism, alopecia, stunted growth and choroid plexus epithelial dysfunction, illustrating the breadth and depth of the influence of KCNE2, mutations which are also associated with human cardiac arrhythmias. Here, the modus operandi and physiological roles of this potent regulator of membrane excitability and ion secretion are reviewed with particular emphasis on the ability of KCNE2 to shape the electrophysiological landscape of both excitable and non-excitable cells.

Inorganic polyphosphate--an unusual suspect of the mitochondrial permeability transition mystery.

Abstract: Inorganic polyphosphate (polyP) is a naturally occurring polyanion made of ten to several hundred orthophosphates (Pi) linked together by phosphoanhydride bonds. PolyP is ubiquitously present in all organisms from bacteria to humans. Specific physiological roles of polyP vary dramatically depending on its size, concentration, tissue and subcellular localization. Recently we reported that mitochondria of ventricular myocytes contain significant amounts (280 ± 60 pmol/mg of protein) of polyP with an average length of 25 orthophosphates, and that polyP is involved in Ca2+-dependent activation of the mitochondrial permeability transition pore (mPTP). Here we extend our study to demonstrate the involvement of mitochondrial polyP in cardiac cell death. Furthermore, we show that polyP levels depend on the activity of the respiratory chain and are lower in myocytes from failing hearts. We conclude that polyP is a dynamically regulated macromolecule that plays an important role in mPTP-dependent cell death pathway.

State-independent intracellular access of quaternary ammonium blockers to the pore of TREK-1

Abstract: We previously reported that TREK-1 gating by internal pH and pressure occurs close to or within the selectivity filter. These conclusions were based upon kinetic measurements of high-affinity block by quaternary ammonium (QA) ions that appeared to exhibit state-independent accessibility to their binding site within the pore. Intriguingly, recent crystal structures of two related K2P potassium channels were also both found to be open at the helix bundle crossing. However, this did not exclude the possibility of gating at the bundle crossing and it was suggested that side-fenestrations within these structures might allow state-independent access of QA ions to their binding site. In this addendum to our original study we demonstrate that even hydrophobic QA ions do not access the TREK-1 pore via these fenestrations. Furthermore, by using a chemically reactive QA ion immobilized within the pore via covalent cysteine modification we provide additional evidence that the QA binding site remains accessible to the...

Disruption of NMDAR–CRMP-2 signaling protects against focal cerebral ischemic damage in the rat middle cerebral artery occlusion model

Abstract: Collapsin response mediator protein 2 (CRMP-2), traditionally viewed as an axon/dendrite specification and axonal growth protein, has emerged as nidus in regulation of both pre- and post-synaptic Ca ( 2+) channels. Building on our discovery of the interaction and regulation of Ca ( 2+) channels by CRMP-2, we recently identified a short sequence in CRMP-2 which, when appended to the transduction domain of HIV TAT protein, suppressed acute, inflammatory and neuropathic pain in vivo by functionally uncoupling CRMP-2 from the Ca ( 2+) channel. Remarkably, we also found that this region attenuated Ca ( 2+) influx via N-methylD-Aspartate receptors (NMDARs) and reduced neuronal death in a moderate controlled cortical impact model of traumatic brain injury (TBI). Here, we sought to extend these findings by examining additional neuroprotective effects of this peptide (TAT-CBD3) and exploring the biochemical mechanisms by which TAT-CBD3 targets NMDARs. We observed that an intraperitoneal injection of TAT-CBD3 peptide significantly reduced infarct volume in an animal model of focal cerebral ischemia. Neuroprotection was observed when TAT-CBD3 peptide was given either prior to or after occlusion but just prior to reperfusion. Surprisingly, a direct biochemical complex was not resolvable between the NMDAR subunit NR2B and CRMP-2. Intracellular application of TAT-CBD3 failed to inhibit NMDAR current. NR2B interactions with the post synaptic density protein 95 (PSD-95) remained intact and were not disrupted by TAT-CBD3. Peptide tiling of intracellular regions of NR2B revealed two 15-mer sequences, in the carboxyl-terminus of NR2B, that may confer binding between NR2B and CRMP-2 which supports CRMP-2's role in excitotoxicity and neuroprotection.

Two-pore domain potassium channels: variation on a structural theme.

Abstract: The ability of cells to reliably fire action potentials is critically dependent upon the maintenance of a hyperpolarized resting potential, which allows voltage-gated Na(+) and Ca(2+) channels to recover from inactivation and open in response to a subsequent stimulus. Hodgkin and Huxley first recognized the functional importance a small, steady outward leak of K(+) ions to the resting potential, action potential generation and cellular excitability, and we now appreciate the contribution of inward rectifier-type K(+) channels (Kir or KCNJ channels) to this process. More recently, however, it has become evident that two-pore domain K(+) (K2P) channels also contribute to the steady outward leak of K(+) ions, and thus, maintenance of the resting potential. Molecular cloning efforts have demonstrated that K2P channel exist in yeast to humans, and represent a major branch in the K(+) channel superfamily. Humans express 15 types of K2P channels, which are grouped into six subfamilies, based on similarities in amino acid sequence and functional properties. Although K2P channels are not voltage-gated, due to the absence of a canonical voltage sensor domain, their activity can be regulated by a variety of stimuli, including mechanical force, polyunsaturated fatty acids (PUFAs) (e.g., arachidonic acid), volatile anesthetics, acidity/pH, pharmacologic agents, heat and signaling events, such as phosphorylation and protein-protein interactions. K2P channels thus represent important regulators of cellular excitability by virtue of their impact on the resting potential, and as such, have garnered considerable attention in recent years.

Rem-GTPase regulates cardiac myocyte L-type calcium current

Abstract: Rationale: The L-type calcium channels (LTCC) are critical for maintaining Ca2+-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK–LTCC interactions is untested. Objective: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca2+ current (ICa,L) via LTCC in murine cardiomyocytes. Methods and Results: Rem knockout mice (Rem−/−) were engineered, and ICa,L and Ca2+-handling properties were assessed. Rem−/− ventricular cardiomyocytes displayed increased ICa,L density. ICa,L activation was shifted positive on the voltage axis, and β-adrenergic stimulation normalized this shift compared with wild-type ICa,L. Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem−/−. Cell shortening was not significantly different. Increased ICa,L density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite...

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations: From tension sensing to channel opening

Abstract: One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.

The roles of G proteins in the activation of TRPC4 and TRPC5 transient receptor potential channels

Abstract: TRPC4 and TRPC5 channels are important regulators of electrical excitability in both gastrointestinal myocytes and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Gαq protein coupled receptor or epidermal growth factor in particular. However, our understanding of the roles of Gαi/o proteins on TRPC4/5 channels is still rudimentary. We discuss potential roles for Gαi/o proteins in channel activation in addition to their known role in cellular signaling.

A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels, an arachidonate-regulated store-independent Orai channel

Abstract: The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.

PtdIns(4,5)P2 interacts with CaM binding domains on TRPM3 N-terminus.

Abstract: TRPM3 has been reported to play an important role in Ca2+ homeostasis, but its gating mechanisms and regulation via Ca2+ are unknown. Ca2+ binding proteins such as calmodulin (CaM) could be probable modulators of this ion channel. We have shown that this protein binds to two independent domains, A35-K124 and H291-G382 on the TRPM3 N-terminus, which contain conserved hydrophobic as well as positively charged residues in specific positions, and that these residues have a crucial impact on its binding. We also showed that another Ca2+ binding protein, S100A1, is able to bind to these regions and that CaM and S100A1 compete for these binding sites on the TRPM3 N-terminus. Moreover, our results suggest that another very important TRP channel activity modulator, PtdIns(4,5)P2, interacts with the CaM/S100A1 binding sites on the TRPM3 N-terminus with high affinity.

Mechanosensitive Ca 2+ permeant cation channels in human prostate tumor cells

Abstract: 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 Ca(2+)-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd(3+) or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca(2+)](i). Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca(2+) 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 Ca(2+) influx to a small Ca(2+)-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 Ca(2+) signaling that may play a role in tumor progression.

Macrophage function in atherosclerosis: Potential roles of TRP channels

Abstract: Cation channels of the Transient Receptor Potential Canonical (TRPC) group, which belong to the larger TRP superfamily of channel proteins, are critical players in cardiovascular disease. Recent studies underscored a role of TRPC3 in macrophage survival and efferocytosis, two critical events in atherosclerosis lesion development. Also, other members of the TRP channel superfamily are found expressed in monocytes/macrophages, where they participate in processes that might be of significance to atherogenesis. These observations set a framework for future studies aimed at defining the ultimate functions not only of TRPC3, but probably other TRP channels, in macrophage biology. The purpose of this manuscript is to provide a timely revision of existing evidence on the role of members of the TRP channel superfamily, in particular TRPCs, in macrophages and discuss it in the context of the macrophage’s function in atherogenesis.

Mechanical properties of lipid bilayers and regulation of mechanosensitive function: From biological to biomimetic channels

Abstract: Material properties of lipid bilayers, including thickness, intrinsic curvature and compressibility regulate the function of mechanosensitive (MS) channels. This regulation is dependent on phospholipid composition, lateral packing and organization within the membrane. Therefore, a more complete framework to understand the functioning of MS channels requires insights into bilayer structure, thermodynamics and phospholipid structure, as well as lipid-protein interactions. Phospholipids and MS channels interact with each other mainly through electrostatic forces and hydrophobic matching, which are also crucial for antimicrobial peptides. They are excellent models for studying the formation and stabilization of membrane pores. Importantly, they perform equivalent responses as MS channels: (1) tilting in response to tension and (2) dissipation of osmotic gradients. Lessons learned from pore forming peptides could enrich our knowledge of mechanisms of action and evolution of these channels. Here, the current state of the art is presented and general principles of membrane regulation of mechanosensitive function are discussed.

Structural determinants of CaV1.3 L-type calcium channel gating

Abstract: A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Cav1.3 L-type Ca2+ channels, in particular the voltage dependence of activation (V0.5) and Ca2+ dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Cav1.3 (Cav1.3L), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCav1.3scg). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCav1.3scg with a rat cDNA isolated from rat pancreas (rCav1.3L). When expressed in tsA-201 cells under identical experimental conditions rCav1.3L exhibited Ca2+ current properties indistinguishable from human and mouse Cav1.3L, compatible with the presence of a functional CTM. In contrast, rCav1.3scg showed gating properties similar to human short splice variants lacking a CTM. rCav1.3scg differs from rCav1.3L at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-te...

Regulation of CREB signaling through L-type Ca2+ channels by Nipsnap-2.

Abstract: A recent study identified Nipsnap1 as an auxiliary protein inhibiting TRPV6 ion channel activity. Based upon this finding, we investigated the role of Nipsnap1, and the closely related Nipsnap2, in Ca2+ channel regulation. Here, we find that overexpression of Nipsnap2 caused a 45% increase in currents though L-type Ca2+ channels in a neuronal cell line, while siRNA knockdown of Nipsnap2 greatly reduced L-type currents. The increased influx through L-type Ca2+ channels due to Nipsnap2 overexpression led to increased phosphorylation of the transcription factor cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) along with enhanced expression of several transcription factors and CREB target genes. These experiments highlight a novel role of Nipsnap2 in transcriptional regulation via L-type Ca2+ channels.

The intracellular Ca2+ channels of membrane traffic

Abstract: Regulation of organellar fusion and fission by Ca2+ has emerged as a central paradigm in intracellular membrane traffic Originally formulated for Ca2+-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant The recent identification of Ca2+ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca2+ channel that drive intracellular membrane traffic The present review summarizes the recent developments and the current questions relevant to this topic

Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides.

Abstract: TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P(2)-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P(2) regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.