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.     

Cholesterol depletion-induced inhibition of stretch-activated channels is mediated via actin rearrangement

Cholesterol is a critical regulator of lipid bilayer dynamics and plasma membrane organization in eukaryotes. A variety of ion channels have been shown to be modulated by cellular cholesterol and partition into cholesterol-enriched membrane rafts. However, very little is known about functional role of membrane cholesterol in regulation of mechanically gated channels that are ubiquitously present in living cells. In our previous study, the effect of methyl-beta-cyclodextrin (MbCD), cholesterol-sequestering agent, on Ca²⁺-permeable stretch-activated cation channels (SACs) has been described. Here, cell-attached patch-clamp method was employed to search for the mechanisms of cholesterol-dependent regulation of SACs and to clarify functional contribution of lipid bilayer and submembranous cytoskeleton to channel gating. Cholesterol-depleting treatment with MbCD significantly decreased open probability of SACs whereas alpha-cyclodextrin had no effect. F-actin disassembly fully restored high level of SAC activity in cholesterol-depleted cells. Particularly, treatment with cytochalasin D or latrunculin B abrogated inhibitory effect of MbCD on stretch-activated currents. Single channel analysis and fluorescent imaging methods indicate that inhibition of SACs after cholesterol depletion is mediated via actin remodeling initiated by disruption of lipid rafts. Our data reveal a novel mechanism of channel regulation by membrane cholesterol and lipid rafts.     

Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca(2+) channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca(2+) channel. Perfusion resulted in activation of L-type Ca(2+) channels and an increase in outward current from 664 +/- 57 to 773 +/- 72 pA at +60 mV. Membrane potential hyperpolarized from -42 +/- 4 to -50 +/- 5 mV. In the presence of nifedipine (10 microM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca(2+) channel current in human jejunal circular smooth muscle cells results in increased Ca(2+) entry and cell contraction. Ca(2+) entry activates large-conductance Ca(2+)-activated K(+) channels, resulting in membrane hyperpolarization and relaxation.     

Functional coupling of ion channels in cellular mechanotransduction

The major players in the processes of cellular mechanotransduction are considered to be mechanosensitive (MS) or mechano-gated ion channels. Non-selective Ca²⁺-permeable channels, whose activity is directly controlled by membrane stretch (stretch-activated channels, SACs) are ubiquitously present in mammalian cells of different origin. Ca²⁺ entry mediated by SACs presumably has a significant impact on various Ca²⁺-dependent intracellular and membrane processes. It was proposed that SACs could play a crucial role in the different cellular reactions and pathologies, including oncotransformation, increased metastatic activity and invasion of malignant cells. In the present work, coupling of ion channels in transformed fibroblasts in course of stretch activation was explored with the use of patch-clamp technique. The combination of cell-attached and inside-out single-current experiments showed that Ca²⁺ influx via SACs triggered the activity of Ca²⁺-sensitive K⁺ channels indicating functional compartmentalization of different channel types in plasma membrane. Importantly, the analysis of single channel behavior demonstrated that K⁺ currents could be activated by the rise of intracellular calcium but displayed no direct mechanosensitivity. Taken together, our data imply that local changes in Ca²⁺ concentration due to SAC activity may provide a functional link between various Ca²⁺-dependent molecules in the processes of cellular mechanotransduction.     

TRPA1 is differentially modulated by the amphipathic molecules trinitrophenol and chlorpromazine

TRPA1, a poorly selective Ca(2+)-permeable cation channel, is expressed in peripheral sensory neurons, where it is considered to contribute to a variety of sensory processes such as the detection of painful stimuli. Furthermore, TRPA1 was also identified in hair cells of the inner ear, but its involvement in sensing mechanical forces is still being controversially discussed. Amphipathic molecules such as trinitrophenol and chlorpromazine have been shown to provide useful tools to study mechanosensitive channels. Depending on their charge, they partition in the inner or outer sheets of the lipid bilayer, causing a curvature of the membrane, which has been demonstrated to activate or inhibit mechanosensitive ion channels. In the present study, we investigated the effect of these molecules on TRPA1 gating. TRPA1 was robustly activated by the anionic amphipathic molecule trinitrophenol. The whole-cell and single channel properties resemble those previously described for TRPA1. Moreover, we could show that the toxin GsMTx-4 acts on TRPA1. In addition to its recently described role as an inhibitor of stretch-activated ion channels, it serves as a potent activator of TRPA1 channels. On the other hand, the positively charged drug chlorpromazine modulates activated TRPA1 currents in a voltage-dependent way. The exposure of activated TRPA1 channels to chlorpromazine led to a block at positive potentials and an increased open probability at negative potentials. The variability in the shape of the I-V curve gives a first indication that native mechanically activated TRPA1 currents must not necessarily exhibit the same biophysical properties as ligand-activated TRPA1 currents.     

VOCCs and TREK-1 ion channel expression in human tenocytes

Mechanosensitive and voltage-gated ion channels are known to perform important roles in mechanotransduction in a number of connective tissues, including bone and muscle. It is hypothesized that voltage-gated and mechanosensitive ion channels also may play a key role in some or all initial responses of human tenocytes to mechanical stimulation. However, to date there has been no direct investigation of ion channel expression by human tenocytes. Human tenocytes were cultured from patellar tendon samples harvested from five patients undergoing routine total knee replacement surgery (mean age: 66 yr; range: 63-73 yr). RT-PCR, Western blotting, and whole cell electrophysiological studies were performed to investigate the expression of different classes of ion channels within tenocytes. Human tenocytes expressed mRNA and protein encoding voltage-operated calcium channel (VOCC) subunits (Ca alpha(1A), Ca alpha(1C), Ca alpha(1D), Ca alpha(2)delta(1)) and the mechanosensitive tandem pore domain potassium channel (2PK(+)) TREK-1. They exhibit whole cell currents consistent with the functional expression of these channels. In addition, other ionic currents were detected within tenocytes consistent with the expression of a diverse array of other ion channels. VOCCs and TREK channels have been implicated in mechanotransduction signaling pathways in numerous connective tissue cell types. These mechanisms may be present in human tenocytes. In addition, human tenocytes may express other channel currents. Ion channels may represent potential targets for the pharmacological management of chronic tendinopathies.     

GDNF acutely potentiates Ca2+ channels and excitatory synaptic transmission in midbrain dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) is best known for its long-term survival effect on dopaminergic neurons in the ventral midbrain. A recent study showed that acute application of GDNF to these neurons suppresses A-type potassium channels and potentiates neuronal excitability. Here we have characterized the acute effects of GDNF on Ca(2+) channels and synaptic transmission. GDNF rapidly and reversibly potentiated the high voltage-activated (HVA) Ca(2+) channel currents in cultured dopaminergic neurons. Analyses of channel kinetics indicate that GDNF decreased the activation time constant, increased the inactivation and deactivation time constants of HVA Ca(2+) channel currents. Ca(2+) imaging experiments demonstrate that GDNF facilitated Ca(2+) influx induced by membrane depolarization. To investigate the physiological consequences of the Ca(2+) channel modulation, we examined the acute effects of GDNF on excitatory synaptic transmission at synapses made by these dopaminergic neurons, which co-release the transmitter glutamate. Within 3 min of application, GDNF increased the amplitude of spontaneous and evoked excitatory autaptic- or multiple-postsynaptic currents. The frequency as well as the amplitude of miniature excitatory postsynaptic currents was also increased. These results reveal, for the first time, an acute effect of GDNF on synaptic transmission and its potential mechanisms, and suggest that an important function of GDNF for midbrain dopaminergic neurons is the acute modulation of transmission and ion channels.     

A type of voltage-dependent Ca2+ channel on Vicia faba guard cell plasma membrane outwardly permeates K+

The fine regulation of stomatal aperture is important for both plant photosynthesis and transpiration, while stomatal closing is an essential plant response to biotic and abiotic stresses such as drought, salinity, wounding, and pathogens. Quick stomatal closing is primarily due to rapid solute loss. Cytosolic free calcium ([Ca(2+)](cyt)) is a ubiquitous second messenger, and its elevation or oscillation plays important roles in stomatal movements, which can be triggered by the opening of Ca(2+)-permeable channels on the plasma membrane. For Ca(2+)-permeable channel recordings, Ba(2+) is preferred as a charge-carrying ion because it has higher permeability to Ca(2+) channels and blocks K(+) channel activities to facilitate current recordings; however, it prevents visualization of Ca(2+) channels' K(+) permeability. Here, we employed Ca(2+) instead of Ba(2+) in recording Ca(2+)-permeable channels on Vicia faba guard cell plasma membrane to mimic physiological solute conditions inside guard cells more accurately. Inward Ca(2+) currents could be recorded at the single-channel level, and these currents could be inhibited by micromolar Gd(3+), but their reversal potential is far away from the theoretical equilibrium potential for Ca(2+). Further experiments showed that the discrepancy of the reversal potential of the recorded Ca(2+) currents is influenced by cytosolic K(+). This suggests that voltage-dependent Ca(2+) channels also mediate K(+) efflux at depolarization voltages. In addition, a new kind of high-conductance channels with fivefold to normal Ca(2+) channel and 18-fold to normal outward K(+) conductance was found. Our data presented here suggest that plants have their own saving strategies in their rapid response to stress stimuli, and multiple kinds of hyperpolarization-activated Ca(2+)-permeable channels coexist on plasma membranes.     

Plasma membrane ion channels in suicidal cell death

The machinery leading to apoptosis includes altered activity of ion channels. The channels contribute to apoptotic cell shrinkage and modify intracellular ion composition. Cl(-) channels allow the exit of Cl(-), osmolytes and HCO(3)(-) leading to cell shrinkage and cytosolic acidification. K(+) exit through K(+) channels contributes to cell shrinkage and decreases intracellular K(+) concentration, which in turn favours apoptotic cell death. K(+) channel activity further determines the cell membrane potential, a driving force for Ca(2+) entry through Ca(2+) channels. Ca(2+) may enter through unselective cation channels. An increase of cytosolic Ca(2+) may stimulate several enzymes executing apoptosis. Specific ion channel blockers may either promote or counteract suicidal cell death. The present brief review addresses the role of ion channels in the regulation of suicidal cell death with special emphasis on the role of channels in CD95 induced apoptosis of lymphocytes and suicidal death of erythrocytes or eryptosis.     

Slow vacuolar channels from barley mesophyll cells are regulated by 14-3-3 proteins

The conductance of the vacuolar membrane at elevated cytosolic Ca(2+) levels is dominated by the slow activating cation selective (SV) channel. At physiological, submicromolar Ca(2+) concentrations the SV currents are very small. Only recently has the role of 14-3-3 proteins in the regulation of voltage-gated and Ca(2+)-activated plasma membrane ion channels been investigated in Drosophila, Xenopus and plants. Here we report the first evidence that plant 14-3-3 proteins are involved in the down-regulation of ion channels in the vacuolar membrane as well. Using the patch-clamp technique we have demonstrated that 14-3-3 protein drastically reduces the current carried by SV channels. The current decline amounted to 80% and half-maximal reduction was reached within 5 s after 14-3-3-addition to the bath. The voltage sensitivity of the channel was not affected by 14-3-3. A coordinating role for 14-3-3 proteins in the regulation of plasma membrane and tonoplast ion transporters is discussed.     

The ATP binding cassette transporter AtMRP5 modulates anion and calcium channel activities in Arabidopsis guard cells

Stomatal guard cells control CO(2) uptake and water loss between plants and the atmosphere. Stomatal closure in response to the drought stress hormone, abscisic acid (ABA), results from anion and K(+) release from guard cells. Previous studies have shown that cytosolic Ca(2+) elevation and ABA activate S-type anion channels in the plasma membrane of guard cells, leading to stomatal closure. However, membrane-bound regulators of abscisic acid signaling and guard cell anion channels remain unknown. Here we show that the ATP binding cassette (ABC) protein AtMRP5 is localized to the plasma membrane. Mutation in the AtMRP5 ABC protein impairs abscisic acid and cytosolic Ca(2+) activation of slow (S-type) anion channels in the plasma membrane of guard cells. Interestingly, atmrp5 insertion mutant guard cells also show impairment in abscisic acid activation of Ca(2+)-permeable channel currents in the plasma membrane of guard cells. These data provide evidence that the AtMRP5 ABC transporter is a central regulator of guard cell ion channel during abscisic acid and Ca(2+) signal transduction in guard cells.     

Magnesium permeation through mechanosensitive channels： single-current measurements

Compelling evidence shows that intracellular free magnesium [Mg^2＋]i may be a critical regulator of cell activity in eukaryotes. However, membrane transport mechanisms mediating Mg^2＋ influx in mammalian cells are poorly understood. Here, we show that mechanosensitive （MS） cationic channels activated by stretch are permeable for Mg^2＋ ions at different extracellular concentrations including physiological ones. Single-channel currents were recorded from cell-attached and inside-out patches on K562 leukaemia cells at various concentrations of MgCl2 when Mg^2＋ was the only available carrier of inward currents. At 2 mM Mg^2＋, inward mechanogated currents representing Mg^2＋ influx through MS channels corresponded to the unitary conductance of about 5 pS. At higher Mg^2＋ levels, only slight increase of single-channel currents and conductance occurred, implying that Mg^2＋ permeation through MS channels is characterized by strong saturation. At 20 and 90 mM Mg^2＋, mean conductance values for inward currents carried by Mg^2＋ were rather similar, being equal to 6.8 ± 0.5 and 6.4 ± 0.5 pS, respectively. The estimation of the channel-selective permeability according to constant field equation is obviously limited due to saturation effects. We conclude that the detection of single currents is the main evidence for Mg^2＋ permeation through membrane channels activated by stretch. Our single-current measurements document Mg^2＋ influx through MS channels in the plasma membrane of leukaemia cells.     

A mechanosensitive ion channel in Schizosaccharomyces pombe.

Protoplast protuberances (blebs) of Schizosaccharomyces pombe were examined using the patch-clamp technique. In addition to several voltage-gated ion channels, we encountered the activities of a mechanosensitive ion channel with a conductance of 180 pS. Microscopic currents of one or two units were observed in some excised patches and ensemble currents of several tens of units were observed in all blebs examined in whole-bleb configuration. This channel opens at pressures of cm Hg applied to whole blebs and it passes cations, including Ca2+. It is inactivated by membrane depolarizations and blocked by Gd3+. We discuss the possible functions of such a channel, including its activation upon cell cycle dependent cytoskeletal reorganizations.     

Interaction of bestrophin-1 and Ca2+ channel β-subunits: identification of new binding domains on the bestrophin-1 C-terminus

Bestrophin-1 modulates currents through voltage-dependent L-type Ca(2+) channels by physically interacting with the β-subunits of Ca(2+) channels. The main function of β-subunits is to regulate the number of pore-forming Ca(V)-subunits in the cell membrane and modulate Ca(2+) channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca(2+) channel subunits, together with modulation of Ca(V)1.3 Ca(2+) channels currents. In heterologeous expression, bestrophin-1 showed co-immunoprecipitation with either, β3-, or β4-subunits. We identified a new highly conserved cluster of proline-rich motifs on the bestrophin-1 C-terminus between amino acid position 468 and 486, which enables possible binding to SH3-domains of β-subunits. A bestrophin-1 that lacks these proline-rich motifs (ΔCT-PxxP bestrophin-1) showed reduced efficiency to co-immunoprecipitate with β3 and β4-subunits. In the presence of ΔCT-PxxP bestrophin-1, β4-subunits and Ca(V)1.3 subunits partly lost membrane localization. Currents from Ca(V)1.3 subunits were modified in the presence of β4-subunit and wild-type bestrophin-1: accelerated time-dependent activation and reduced current density. With ΔCTPxxP bestrophin-1, currents showed the same time-dependent activation as with wild-type bestrophin-1, but the current density was further reduced due to decreased number of Ca(2+) channels proteins in the cell membrane. In summary, we described new proline-rich motifs on bestrophin-1 C-terminus, which help to maintain the ability of β-subunits to regulate surface expression of pore-forming Ca(V) Ca(2+)-channel subunits.     

Permeation of monovalent cations through the non-capacitative arachidonate-regulated Ca2+ channels in HEK293 cells. Comparison with endogenous store-operated channels

In a manner similar to voltage-gated Ca(2+) channels and Ca(2+) release-activated Ca(2+) (CRAC) channels, the recently identified arachidonate-regulated Ca(2+) (ARC) channels display a large monovalent conductance upon removal of external divalent cations. Using whole-cell patch-clamp recording, we have characterized the properties of these monovalent currents in HEK293 cells stably transfected with the m3 muscarinic receptor and compared them with the corresponding currents through the endogenous store-operated Ca(2+) (SOC) channels in the same cells. Although the monovalent currents seen through these two channels displayed certain similarities, several marked differences were also apparent, including the magnitude of the monovalent current/Ca(2+) current ratio, the rate and nature of the spontaneous decline in the currents, and the effects of external monovalent cation substitutions and removal of internal Mg(2+). Moreover, monovalent ARC currents could be activated after the complete spontaneous inactivation of the corresponding SOC current in the same cell. We conclude that the non-capacitative ARC channels share, with voltage-gated Ca(2+) channels and store-operated Ca(2+) channels (e.g. SOC and CRAC the general property of monovalent ion permeation in the nominal absence of extracellular divalent ions. However, the clear differences between the properties of these currents through ARC and SOC channels in the same cell confirm that these represent distinct conductances.     

Tuning the mechanosensitivity of a BK channel by changing the linker length

Some large-conductance Ca(2+) and voltage-activated K(+)(BK) channels are activated by membrane stretch. However, the mechanism of mechano-gating of the BK channels is still not well understood. Previous studies have led to the proposal that the linker-gating ring complex functions as a passive spring, transducing the force generated by intracellular Ca(2+) to the gate to open the channel. This raises the question as to whether membrane stretch is also transmitted to the gate of mechanosensitive (MS) BK channels via the linker-gating complex. To study this, we changed the linker length in the stretch-activated BK channel (SAKCaC), and examined the effect of membrane stretch on the gating of the resultant mutant channels. Shortening the linker increased, whereas extending the linker reduced, the channel mechanosensitivity both in the presence and in the absence of intracellular Ca(2+). However, the voltage and Ca(2+) sensitivities were not significantly altered by membrane stretch. Furthermore, the SAKCaC became less sensitive to membrane stretch at relatively high intracellular Ca(2+) concentrations or membrane depolarization. These observations suggest that once the channel is in the open-state conformation, tension on the spring is partially released and membrane stretch is less effective. Our results are consistent with the idea that membrane stretch is transferred to the gate via the linker-gating ring complex of the MS BK channels.     

A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.     

Hyperpolarization, but not depolarization, increases intracellular Ca(2+) level in cultured chick myoblasts.

Ca(2+) influx appears to be important for triggering myoblast fusion. It remains, however, unclear how Ca(2+) influx rises prior to myoblast fusion. The present study examines a possible involvement of the voltage-dependent Ca(2+) influx pathways. Treatment with the L-type Ca(2+) channel blockers, diltiazem, and nifedipine did not alter cytosolic Ca(2+) levels. Depolarization with high K(+) solution and activation of Ca(2+) channel with Bay K 8644, and agonist of voltage dependent Ca(2+) channels, failed to elicit increases intracellular Ca(2+) level, indicating the absence of depolarization-operated mechanisms. In contrast, phloretin, an agonist of Ca(2+)-activated potassium (K(Ca)) channels, was able to hyperpolarize membrane potential and promoted Ca(2+) influx. These effects were completely abolished by treatment of charybdotoxin, a specific inhibitor of K(Ca) channels. In addition, gadolinium, a potent stretch-activated channel (SAC) blocker, prevented the phloretin-mediated Ca(2+) increase, indicating the involvement of SACs in Ca(2+) influx. Furthermore, phloretin stimulated precocious myoblast fusion and this effect was blocked with gadolinium or charybdotoxin. Taken together, these results suggest that induced hyperpolarization, but not depolarization increases Ca(2+) influx through stretch-activated channels, and in turn triggers myoblast fusion.