Caffeine activates a Ca(2+)-permeable, nonselective cation channel in smooth muscle cells

The effects of caffeine on cytoplasmic [Ca2+] ([Ca2+]i) and plasma membrane currents were studied in single gastric smooth muscle cells dissociated from the toad, Bufo marinus. Experiments were carried out using Fura-2 for measuring [Ca2+]i and tight-seal voltage-clamp techniques for recording membrane currents. When the membrane potential was held at -80 mV, in 15% of the cells studied caffeine increased [Ca2+]i without having any effect on membrane currents. In these cells ryanodine completely abolished any caffeine induced increase in [Ca2+]i. In the other cells caffeine caused both an increase in [Ca2+]i and activation of an 80-pS nonselective cation channel. In this group of cells ryanodine only partially blocked the increase in [Ca2+]i induced by caffeine; moreover, the change in [Ca2+]i that did occur was tightly coupled to the time course and magnitude of the cation current through these channels. In the presence of ryanodine, blockade of the 80-pS channel by GdCl3 or decreasing the driving force for Ca2+ influx through the plasma membrane by holding the membrane potential at +60 mV almost completely blocked the increase in [Ca2+]i induced by caffeine. Thus, the channel activated by caffeine appears to be permeable to Ca2+. Caffeine activated the cation channel even when [Ca2+]i was clamped to below 10 nM when the patch pipette contained 10 mM BAPTA suggesting that caffeine directly activates the channel and that it is not being activated by the increase in Ca2+ that occurs when caffeine is applied to the cell. Corroborating this suggestion were additional results showing that when the membrane was depolarized to activate voltage-gated Ca2+ channels or when Ca2+ was released from carbachol- sensitive internal Ca2+ stores, the 80-pS channel was not activated. Moreover, caffeine was able to activate the channel in the presence of ryanodine at both positive and negative potentials, both conditions preventing release of Ca2+ from stores and the former preventing its influx. In summary, in gastric smooth muscle cells caffeine transiently releases Ca2+ from a ryanodine-sensitive internal store and also increases Ca2+ influx through the plasma membrane by activating an 80- pS cation channel by a mechanism which does not seem to involve an elevation of [Ca2+]i.     

Allosteric regulation of BK channel gating by Ca(2+) and Mg(2+) through a nonselective, low affinity divalent cation site.

The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K(+) channels is enhanced by cytosolic calcium (Ca(2+)). Activation is sensitive to a range of [Ca(2+)] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 alpha subunits at [Ca(2+)] and [Mg(2+)] up to 100 mM. The half-activation voltage (V(0.5)) is steeply dependent on [Ca(2+)] in the micromolar range, but shows a tendency towards saturation over the range of 60-300 microM Ca(2+). As [Ca(2+)] is increased to millimolar levels, the V(0.5) is strongly shifted again to more negative potentials. When channels are activated by 300 microM Ca(2+), further addition of either mM Ca(2+) or mM Mg(2+) produces similar negative shifts in steady-state activation. Millimolar Mg(2+) also produces shifts of similar magnitude in the complete absence of Ca(2+). The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca(2+)] increase activation rates, addition of 10 mM Mg(2+) to 0 Ca(2+) produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg(2+) does not participate in Ca(2+)-dependent steps that influence current activation rate. We conclude that millimolar Mg(2+) and Ca(2+) concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca(2+)-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca(2+) binding steps, four voltage sensors, and four independent lower affinity Ca(2+)/Mg(2+) binding steps describes well the behavior of G-V curves over a range of Ca(2+) and Mg(2+). The ability of a broad range of [Ca(2+)] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.     

TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization

Calcium-activated nonselective (CAN) cation channels are expressed in various excitable and nonexcitable cells supporting important cellular responses such as neuronal bursting activity, fluid secretion, and cardiac rhythmicity. We have cloned and characterized a second form of TRPM4, TRPM4b, a member of the TRP channel family, as a molecular candidate of a CAN channel. TRPM4b encodes a cation channel of 25 pS unitary conductance that is directly activated by [Ca2+]i with an apparent K(D) of approximately 400 nM. It conducts monovalent cations such as Na+ and K+ without significant permeation of Ca2+. TRPM4b is activated following receptor-mediated Ca2+ mobilization, representing a regulatory mechanism that controls the magnitude of Ca2+ influx by modulating the membrane potential and, with it, the driving force for Ca2+ entry through other Ca2+-permeable pathways.     

A store-operated nonselective cation channel in lymphocytes is activated directly by Ca(2+) influx factor and diacylglycerol

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.     

Characterization of a 25-pS nonselective cation channel in a cultured secretory epithelial cell line

Summary We have studied a 25-pS nonselective cation channel from the apical membranes of cell line ST₈₈₅, derived from neonatal mouse mandibular glands. Its Cl^– permeability was not significantly different from zero. The permeabilities (relative to Na⁺) for inorganic cations were NH ₄ ⁺ (1.87)>K⁺(1.12)>Li⁺ (1.02)>Na⁺(1)>Rb⁺(0.81)>Mg²⁺(0.07)>Ca²⁺(0.002), and for organic cations, guanidinium (1.61)4-aminopyridine (0.66)>diethylamine (0.54)>piperazine (0.25)>Tris (0.18)>N-methylglucamine (0.12). The Tris and N-methylglucamine permeabilities differed significantly from zero. Fitting the Renkin equation indicated that the channel had an equivalent pore radius of 0.49 nm. The channel was activated by Ca²⁺ on the cytosolic surface (>0.1 mmol/liter) with a Hill coefficient of 1.2; it was also activated by depolarization. Open- and closed-time histograms indicated that it had at least two open and two closed states. The channel was blocked by cytosolic AMP or ATP (0.1 mmol/liter). It was also blocked by the Cl^– channel blocker, diphenylamine-2-carboxylate (DPC; 0.1 mmol/liter), applied to the extracellular but not the cytosolic surface. 4-Aminopyridine, which permeated the channel when applied to the extracellular surface, blocked it when applied in low concentrations (5 mmol/liter) to the cytosolic surface. Quinine (0.1 mmol/liter) blocked from both the extracellular and cytosolic surfaces, blockade from either side being enhanced by depolarization. The channel was held open by application of SITS (0.1 mmol/liter) to the cytosolic surface. The channel shows striking similarities to the nicotinic acetylcholine receptor channel,viz., both channel types are abnormally permeable to 4-aminopyridine applied externally, and their selectivity sequences for inorganic ions are similar and for organic cations are identical.     

Ca(2+) sensors of L-type Ca(2+) channel

Ca(2+)-induced inactivation of L-type Ca(2+) is differentially mediated by two C-terminal motifs of the alpha(1C) subunit, L (1572-1587) and K (1599-1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca(2+) sensor and an adjacent Ca(2+)-independent tethering site for calmodulin. The Ca(2+) sensor contributes to higher Ca(2+) sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca(2+)-dependent current decay, the two-site modulation by Ca(2+) and calmodulin may underlie Ca(2+)-induced inactivation of the channel.     

Ryanodine sensitizes the Ca(2+) release channel (ryanodine receptor) to Ca(2+) activation

Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca(2+) signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca(2+) release channel (ryanodine receptor), ryanodine causes two major changes in the channel: a reduction in single-channel conductance and a marked increase in open probability. The molecular mechanisms underlying these alterations are not well understood. In the present study, we investigated the gating behavior and Ca(2+) dependence of the wild type (wt) and a mutant cardiac ryanodine receptor (RyR2) after being modified by ryanodine. Single-channel studies revealed that the ryanodine-modified wt RyR2 channel was sensitive to inhibition by Mg(2+) and to activation by caffeine and ATP. In the presence of Mg(2+), the ryanodine-modified single wt RyR2 channel displayed a sigmoidal Ca(2+) dependence with an EC(50) value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC(50) of 120 microm for Ca(2+) activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca(2+) activation by approximately 1,000-fold. Furthermore, ryanodine is able to restore Ca(2+) activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit approximately 1,000-fold reduction in Ca(2+) sensitivity to activation. The E3987A mutation, however, affects neither [(3)H]ryanodine binding to, nor the stimulatory and inhibitory effects of ryanodine on, the RyR2 channel. These results demonstrate that ryanodine does not "lock" the RyR channel into an open state as generally believed; rather, it sensitizes dramatically the channel to activation by Ca(2+).     

TRPM5 is a voltage-modulated and Ca(2+)-activated monovalent selective cation channel

The TRPM subfamily of mammalian TRP channels displays unusually diverse activation mechanisms and selectivities. One member of this subfamily, TRPM5, functions in taste receptor cells and has been reported to be activated through G protein-coupled receptors linked to phospholipase C. However, the specific mechanisms regulating TRPM5 have not been described. Here, we demonstrate that TRPM5 is a monovalent-specific cation channel with a 23 pS unitary conductance. TRPM5 does not display constitutive activity. Rather, it is activated by stimulation of a receptor pathway coupled to phospholipase C and by IP(3)-mediated Ca(2+) release. Gating of TRPM5 was dependent on a rise in Ca(2+) because it was fully activated by Ca(2+). Unlike any previously described mammalian TRP channel, TRPM5 displayed voltage modulation and rapid activation and deactivation kinetics upon receptor stimulation. The most closely related protein, the Ca(2+)-activated monovalent-selective cation channel TRPM4b, also showed voltage modulation, although with slower relaxation kinetics than TRPM5. Taken together, the data demonstrate that TRPM5 and TRPM4b represent the first examples of voltage-modulated, Ca(2+)-activated, monovalent cation channels (VCAMs). The voltage modulation and rapid kinetics provide TRPM5 with an excellent set of properties for participating in signaling in taste receptors and other excitable cells.     

Cloning of a stretch-inhibitable nonselective cation channel

A homologue of the capsaicin receptor-nonselective cation channel was cloned from the rat kidney to investigate a mechanosensitive channel. We found this channel to be inactivated by membrane stretch and have designated it stretch-inactivated channel (SIC). SIC encodes a 563-amino acid protein with putative six transmembrane segments. The cDNA was expressed in mammalian cells, and electophysiological studies were performed. SIC-induced large cation currents were found to be regulated by cell volume, with currents being stimulated by cell shrinkage and inhibited by cell swelling. Single channel analysis showed a conductance of 250 pS with cation permeability (PCl/PNa < 0.1), and the channel possessed some of the characteristics of a stretch-inactivated channel in that it was permeable to calcium, sensitive to membrane stretch, and blocked by Gd3+. Therefore, we cloned one of the mechanosensitive cation channels of mammals, which is considered to regulate Ca2+ influx in response to mechanical stress on the cell membrane.     

Regulation of the Ca2+ sensitivity of the nonselective cation channel TRPM4

TRPM4, a Ca(2+)-activated cation channel of the transient receptor potential superfamily, undergoes a fast desensitization to Ca(2+). The mechanisms underlying the alterations in Ca(2+) sensitivity are unknown. Here we show that cytoplasmic ATP reversed Ca(2+) sensitivity after desensitization, whereas mutations to putative ATP binding sites resulted in faster and more complete desensitization. Phorbol ester-induced activation of protein kinase C (PKC) increased the Ca(2+) sensitivity of wild-type TRPM4 but not of two mutants mutated at putative PKC phosphorylation sites. Overexpression of a calmodulin mutant unable to bind Ca(2+) dramatically reduced TRPM4 activation. We identified five Ca(2+)-calmodulin binding sites in TRPM4 and showed that deletion of any of the three C-terminal sites strongly impaired current activation by reducing Ca(2+) sensitivity and shifting the voltage dependence of activation to very positive potentials. Thus, the Ca(2+) sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation, and calmodulin binding at the C terminus.     

A helix-breaking mutation in the epithelial Ca(2+) channel TRPV5 leads to reduced Ca(2+)-dependent inactivation

TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca(2+) and is tightly regulated by intracellular Ca(2+) concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca(2+) concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca(2+)-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (τ) and half maximal inhibition constant (IC(50)) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca(2+) permeability. Out of different point mutations created to characterize the importance of M490 in Ca(2+)-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca(2+)-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca(2+)-dependent inactivation.     

Induction of mouse Ca(2+)-sensitive chloride channel 2 gene during involution of mammary gland.

To understand molecular mechanisms that regulate mammary gland involution, we identified involution-induced cDNA clones by suppression subtractive hybridization methods. Nucleotide sequencing of a clone revealed that it was 97% identical to Ca(2+)-sensitive chloride channel 1 (mCLCA1) gene that has been identified in lung tissue. We concluded that our clone was derived from different gene with mCLCA1 and named it mCLCA2. We confirmed that expression of mCLCA2 gene was predominant in mammary gland while mCLCA1 mRNA was mainly detected in lung tissues by RT-PCR. Northern analysis showed that the mCLCA2 gene was induced at involution phase compared to pregnant and lactating phases of mammary gland. Under serum starvation, HC11 mammary epithelial cells showed DNA fragmentation and induction of mCLCA2 expression.     

Characterization of a Ca2+-activated nonselective cation channel during dedifferentiation of cultured rat ventricular cardiomyocytes

Cardiac hypertrophy is associated with electrical activity modifications, including sustained depolarization, that lead to a propensity for arrhythmias. The ionic currents underlying the sustained depolarization are not well defined. Similar modifications were reported on adult rat cardiomyocytes in primary culture undergoing dedifferentiation. Using the single-channel measurements on these cells, we identified the appearance of a Ca2+-activated nonselective cation channel (NSCCa) during the dedifferentiation process. In excised inside-out patches the channel presented a linear I/V relationship with a conductance of 26.5 pS. It was equally selective for Na+ and K+ and impermeable to Cl- and Ca2+ ions. The open probability increased with depolarization and with rise in intracellular calcium concentration. The channel activity was reduced by intracellular ATP and suppressed by flufenamic acid. Channel detection increased after incubation with a purinergic receptor agonist (ATPgS) or a PKC activator (PMA). Furthermore, occurrence of the channel developed during the culture. Absent at one day in vitro (d.i.v.), channel activity was present in 5, 46, 27 and 19% of patches after 4, 7, 14 and 21 d.i.v., respectively. We suggest that the channel may be associated with pro-arrhythmic signaling, in particular during the release of transmitters from autonomic nerve endings in the hypertrophied hearts.     

Permeation and gating properties of the novel epithelial Ca(2+) channel

The recently cloned epithelial Ca(2+) channel (ECaC) constitutes the Ca(2+) influx pathway in 1,25-dihydroxyvitamin D(3)-responsive epithelia. We have combined patch-clamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca(2+) concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca(2+) gradient, demonstrating the distinctive Ca(2+) permeability and constitutive activation of ECaC. Cells dialyzed with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid displayed large inward currents through ECaC in response to voltage ramps. The corresponding current-voltage relationship showed pronounced inward rectification. Currents evoked by voltage steps to potentials below -40 mV partially inactivated with a biexponential time course. This inactivation was less pronounced if Ba(2+) or Sr(2+) replaced Ca(2+) and was absent in Ca(2+)-free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratio P(Ca):P(Na) calculated from the reversal potential at 30 mM [Ca(2+)](o) was larger than 100. The divalent cation selectivity profile is Ca(2+) > Mn(2+) > Ba(2+) approximately Sr(2+). Repetitive stimulation of ECaC-expressing cells induced a decay of the current response, which was greatly reduced if Ca(2+) was replaced by Ba(2+) and was virtually abolished if [Ca(2+)](o) was lowered to 1 nM. In conclusion, ECaC is a Ca(2+) selective channel, exhibiting Ca(2+)-dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.     

Epithelial Ca(2+) channel expression and Ca(2+) uptake in developing zebrafish

The purpose of the present work was to study the possible role of the epithelial Ca(2+) channel (ECaC) in the Ca(2+) uptake mechanism in developing zebrafish (Danio rerio). With rapid amplification of cDNA ends, full-length cDNA encoding the ECaC of zebrafish (zECaC) was cloned and sequenced. The cloned zECaC was 2,578 bp in length and encoded a protein of 709 amino acids that showed up to 73% identity with previously described vertebrate ECaCs. The zECaC was found to be expressed in all tissues examined and began to be expressed in the skin covering the yolk sac of embryos at 24 h postfertilization (hpf). zECaC-expressing cells expanded to cover the skin of the entire yolk sac after embryonic development and began to occur in the gill filaments at 96 hpf, and thereafter zECaC-expressing cells rapidly increased in both gills and yolk sac skin. Corresponding to ECaC expression profile, the Ca(2+) influx and content began to increase at 36-72 hpf. Incubating zebrafish embryos in low-Ca(2+) (0.02 mM) freshwater caused upregulation of the whole body Ca(2+) influx and zECaC expression in both gills and skin. Colocalization of zECaC mRNA and the Na(+)-K(+)-ATPase alpha-subunit (a marker for mitochondria-rich cells) indicated that only a portion of the mitochondria-rich cells expressed zECaC mRNA. These results suggest that the zECaC plays a key role in Ca(2+) absorption in developing zebrafish.