Ca2+-activated Cl− channel in plasmalemma ofNitellopsis obtusa

Summary The mechanisms of Cl^–-channel activation in the plasmalemma ofNitellopsis obtusa was studied by measuring both the transient inward current under voltage clamp and Cl^– efflux during the action potential. 9-anthracenecarboxylic acid (A-9-C) at 1.0mm inhibited both the transient inward current and the Cl^– efflux, but did not uncouple the sudden cessation of the cytoplasmic streaming. Since this excitation-cessation coupling is caused by a transient increase in the cytoplasmic Ca²⁺ concentration, these results suggest that A-9-C inhibited not the Ca²⁺ channel but specifically the Cl^– channel. The following results were found between the Ca²⁺-channel activation and the Cl^–-channel activation: (1) The Ca²⁺-channel blocker La³⁺ uncoupled the excitation-cessation coupling and inhibited both the transient inward current and the Cl^– efflux, although the Cl^–-channel blocker A-9-C did not affect the excitation-cessation coupling. (2) The Cl^– efflux was greatly reduced by depletion of Ca²⁺ from the external solution and restored by an increase in the external Ca²⁺ concentration. (3) An increase in the external ionic, strength which increases Ca²⁺ entry (T. Shiina & M. Tazawa,J. Membrane Biol. 96:263–276, 1987) enhanced the Cl^– efflux. (4) Mg²⁺, which cannot pass through the Ca²⁺ channel, reduced both the transient inward current and the Cl^– efflux. (5) Although Sr²⁺ can pass through the plasmalemma Ca²⁺ channel, Cl^–-channel activation by Sr²⁺ was only partial. These findings support the hypothesis that voltage-dependent Ca²⁺-channel activation, which increases the free Ca²⁺ concentration in the cytoplasm, is necessary for the subsequent Cl^–-channel activation.     

Role of Ca2+-activated Cl- channels in the mechanism of apoptosis induced by cyclosporin A in a human hepatoma cell line

The mechanism of apoptosis induced by cyclosporin A (CsA) in a human hepatoma cell line was investigated. CsA induced apoptosis in a dose- and time-dependent manner in HepG2 human hepatoma cells. CsA induced Cl- efflux, which was significantly blocked by niflumic acid (NA), a specific inhibitor, and flufenamic acid (FA), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), and 4,4'-diisothiocyanoto-stibene-2,2'-disulfonic acid (DIDS), non-specific inhibitors of Ca2+-activated Cl- channels (CaCCs), not by calyculin A, an inhibitor of K+,Cl- -cotransport. In addition, CsA did not alter intracellular K+ concentration. Moreover, CsA increased intracellular Ca2+ concentration, and treatment with BAPTA/AM, an intracellular Ca2+ chelator, significantly inhibited the CsA-induced Cl- efflux, indicating that CsA induced Cl- efflux through the activation of CaCCs. Treatment with these CaCC inhibitors (NA, FA, NPPB, and DIDS) markedly prevented the CsA-induced apoptosis. Taken together, these results suggest that CaCCs may mediate apoptosis induced by CsA in HepG2 cells. Furthermore, these results provide a new insight into the novel function of CaCCs in the regulation of cancer cell apoptosis associated with perturbation of intracellular Ca2+ signal.     

The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes

We examined the effects of the mitochondrial Ca(2+)-activated K(+) (mitoBK(Ca)) channel activator NS 1619 on L-type Ca(2+) channels in rat ventricular myocytes. NS 1619 inhibited the Ca(2+) current in a dose-dependent manner. NS 1619 shifted the activation curve to more positive potentials, but did not have a significant effect on the inactivation curve. Pretreatment with inhibitors of membrane BK(Ca) channel, mitoBK(Ca) channel, protein kinase C, protein kinase A, and protein kinase G had little effect on the Ca(2+) current and did not alter the inhibitory effect of NS 1619 significantly. The application of additional NS 1619 in the presence of isoproterenol, a selective beta-adrenoreceptor agonist, reduced the Ca(2+) current to approximately the same level as a single application of NS 1619. In conclusion, our results suggest that NS 1619 inhibits the Ca(2+) current independent of the mitoBK(Ca) channel and protein kinases. Since NS 1619 is widely used to study mitoBK(Ca) channel function, it is essential to verify these unexpected effects of NS 1619 before experimental data can be interpreted accurately.     

Voltage and Ca2+ activation of single large-conductance Ca2+-activated K+ channels described by a two-tiered allosteric gating mechanism

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.     

Aminoglycoside blockade of Ca2+-activated K+ channel from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Ca²⁺-activated K⁺ channels from rat brain synaptosomal membranes were incorporated into planar lipid bilayers, and the effects of aminoglycoside antibiotics on the single channel conductance (258±13 pS at 100mm K⁺) were investigated. Aminoglycosides reduced the single channel conductance from the cis (cytoplasmic) side in a dose- and voltage-dependent manner. Voltage dependence of the blockade indicated an interaction between positively charged amino residues of aminoglycoside antibiotics and a binding site located within the electric field of the ion-conducting pathway. The order of blocking potency was consistent with that of the number of amino residues of aminoglycosides (neomycin (6)>dibekacin (5)>ribostamycin (4)=kanamycin (4)), while the electrical distance (z=0.46–0.49) of the binding site kept almost constant for each drug. Thesezs were almost the same with those (0.46–0.51) of alkyldiamine blockers with two amino residues (total net charge of +2) and approximately twice of those (0.25–0.26) of alkylmonoamine blockers (total net charge of +1). Assuming that amino residues of aminoglycosides and alkylamines shared the same binding site located at 25% voltage drop from the cytoplasmic surface of the channel, the site would have to be at least large enough to accommodate one diamino sugar residue of the aminoglycoside in order to simultaneously interact with two positively charged amino groups. Dose- and voltage-dependent blockade of the channel by gallamine, an extremely bulky trivalent organic cation, supported the picture that the channel has a wide mouth on the cytoplasmic side and its pore region, where voltage drop occurs, may also be quite wide and nonselective, suddenly tapering to a constriction where most charged cations block the channel by occluding the K⁺-conducting pathway.     

The beta subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states

Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the beta subunit increased open probability (Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the beta subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.     

Intracellular localization and physiological function of a rice Ca2+-permeable channel OsTPC1

Two-pore channels (TPCs) are cation channels with a voltage-sensor domain conserved in plants and animals. Rice OsTPC1 is predominantly localized to the plasma membrane (PM), and assumed to play an important role as a Ca²⁺-permeable cation channel in the regulation of cytosolic Ca²⁺ rise and innate immune responses including hypersensitive cell death and phytoalexin biosynthesis in cultured rice cells triggered by a fungal elicitor, xylanase from Trichoderma viride. In contrast, Arabidopsis AtTPC1 is localized to the vacuolar membrane (VM). To gain further insights into the intracellular localization of OsTPC1, we stably expressed OsTPC1-GFP in tobacco BY-2 cells. Confocal imaging and membrane fractionation revealed that, unlike in rice cells, the majority of OsTPC1-GFP fusion protein was targeted to the VM in tobacco BY-2 cells. Intracellular localization and functions of the plant TPC family is discussed.     

Ca2+-activated K+ permeability in human erythrocytes: Modulation of single-channel events

Elevated levels of intracellular Ca²⁺ activate a K⁺-selective permeability in the membrane of human erythrocytes. Currents through single channels were analysed in excised inside-out membrane patches. The effects of several ions that are known to inhibit K⁺ fluxes are described with respect to the single-channel events. The results suggest that the blocking ions can partly move into the channels (but cannot penetrate) and interact with other ions inside the pore. The reduction of single-channel conductance by Cs⁺, tetraethylammonium and Ba²⁺ and of single-channel activity by quinine and Ba²⁺ is referred to different rates of access to the channel. The concentration- and voltage-dependent inhibition by ions with measurable permeability (Na⁺ and Rb⁺) can be explained by their lower permeability, with single-file movement and ionic interactions inside the pore.     

Cell-cycle-dependent expression of the large Ca2+-activated K+ channels in breast cancer cells

In a previous work, we have reported that the ionic nature of the outward current recorded in MCF-7 cells was that of a K+ current. In this study, we have identified a Ca2+-activated K+ channel not yet described in MCF-7 human breast cancer cells. In cells arrested in the early G1 (depolarized cells), increasing [Ca2+]i induced both a shift in the I-V curve toward more negative potentials and an increase in current amplitude at negative and more at positive potential. Currents were inhibited by r-iberiotoxin (r-IbTX, 50 nM) and charybdotoxin (ChTX, 50 nM). These data indicate that human breast cancer cells express large-conductance Ca2+-activated K+ (BK) channels. BK current-density increased in cells synchronized at the end of G1, as compared with those in the early G1 phase. This increased current-density paralleled the enhancement in BK mRNA levels. Blocking BK channels with r-IbTX, ChTX or both induced a slight depolarization in cells arrested in the early G1, late G1, and S phases and accumulated cells in the S phase, but failed to induce cell proliferation. Thus, the expression of the BK channels was cell-cycle-dependent and seems to contribute more to the S phase than to the G1 phase. However, these K+ channels did not regulate the cell proliferation because of their minor role in the membrane potential.     

Calmodulin regulation of TMEM16A and 16B Ca2+-activated chloride channels

Ca²⁺-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca²⁺ sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca²⁺-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca²⁺-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Slo1 tail domains,but not the Ca2+ bowl,are required for the beta 1 subunit to increase the apparent Ca2+ sensitivity of BK channels

Functional large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels can be assembled from four alpha subunits (Slo1) alone, or together with four auxiliary beta1 subunits to greatly increase the apparent Ca(2+) sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the alpha subunit, which includes the RCK2 (regulator of K(+) conductance) domain and Ca(2+) bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca(2+) bowl and high affinity Ca(2+) sensitivity. In the second, the Ca(2+) bowl was disrupted by mutations that greatly reduce the apparent Ca(2+) sensitivity. We found that the beta1 subunit increased the apparent Ca(2+) sensitivity of Slo1 channels, independently of whether the alpha subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca(2+) bowl was mutated. In contrast, beta1 subunits no longer increased Ca(2+) sensitivity when Slo1 tails were replaced by Slo3 tails. The beta1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 beta-estradiol activated these channels in the presence of beta1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca(2+) sensitivity induced by the beta1 subunit does not require either the Ca(2+) bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The beta1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the beta1 subunit-induced increase in apparent Ca(2+) sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the beta1 subunit may be different.     

Essential role of the N-terminus of murine Orai1 in store-operated Ca2+ entry

Store-operated Ca(2+) entry (SOCE) is a physiologically important process that is triggered by intracellular Ca(2+) depletion. Recently, human Orai1 (the channel-forming subunit) and STIM1 (the calcium sensor) were identified as essential molecules for SOCE. Here, we report the cloning and functional analysis of three murine orthologs of Orai1, termed Orai1, 2, and 3. Among the genes identified, Orai1 contains a distinctive proline- and arginine-rich N-terminal cytoplasmic sequence. Co-expression of STIM1 with Orai1 produced a marked effect on SOCE, while co-expression with Orai2 or Orai3 had little effect. Expression of Orai1 without its N-terminal tail had a marginal effect on SOCE, while chimeric Orai2 containing the Orai1 N-terminus produced a marked increase in SOCE. In addition, a truncated version of Orai1 containing the N-terminus without the pore-forming transmembrane domain had a dominant negative effect on SOCE. These results reveal the essential role of Orai1 and its N-terminal sequence in SOCE.     

Anoctamin 1 in secretory epithelia

Fluid and electrolyte releasing from secretory epithelia are elaborately regulated by orchestrated activity of ion channels. The activity of chloride channel at the apical membrane decides on the direction and the rate of secretory fluid and electrolyte. Chloride-dependent secretion is conventionally associated with intracellular increases in two second messengers, cAMP and Ca²⁺, responding to luminal purinergic and basolateral adrenergic or cholinergic stimulation. While it is broadly regarded that cAMP-dependent Cl⁻ secretion is regulated by cystic fibrosis transmembrane conductance regulator (CFTR), Ca²⁺-activated Cl⁻ channel (CaCC) had been veiled for quite some time. Now, Anoctamin 1 (ANO1 or TMEM16A) confers Ca²⁺-activated Cl⁻ currents. Ano 1 and its paralogs have been actively investigated for multiple functions underlying Ca²⁺-activated Cl⁻ efflux and fluid secretion in a variety of secretory epithelial cells. In this review, we will discuss recent advances in the secretory function and signaling of ANO1 in the secretory epithelia, such as airways, intestines, and salivary glands.