An NH2-terminal multi-basic RKR motif is required for the ATP-dependent regulation of hIK1

We previously demonstrated that the ATP/PKA-dependent activation of the human intermediate conductance, Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal motif. The NH2-terminus of hIK1 contains a multi-basic 13RRRKR17 motif, known to be important in the trafficking and function of ion channels. While individual mutations within this domain have no effect on channel function, the triple mutation (15RKR17/AAA), as well as additional double mutations, result in a near complete loss of functional channels, as assessed by whole-cell patch-clamp. However, cell-surface immunoprecipitation studies confirmed expression of these mutated channels at the plasma membrane. To elucidate the functional consequences of the (15)RKR(17)/AAA mutation we performed inside-out patch clamp recordings where we observed no difference in Ca2+ affinity between the wild-type and mutated channels. However, in contrast to wild-type hIK1, channels expressing the 15RKR17/AAA mutation exhibited rundown, which could not be reversed by the addition of ATP. Wild-type hIK1 channel activity was reduced by alkaline phosphatase both in the presence and absence of ATP, indicative of a phosphorylation event, whereas the 15RKR17/AAA mutation eliminated this effect of alkaline phosphatase. Further, single channel analysis demonstrated that the 15RKR17/AAA mutation resulted in a four-fold lower channel open probability (P(o)), in the presence of saturating Ca2+ and ATP, compared to wild-type hIK1. In conclusion, these results represent the first demonstration for a role of the NH2-terminus in the second messenger-dependent regulation of hIK1 and, in combination with our previous findings, suggest that this regulation is dependent upon a close NH2/C-terminal association.     

Functional and molecular identification of intermediate-conductance Ca(2+)-activated K(+) channels in breast cancer cells: association with cell cycle progression

We have previously reported that the hEAG K⁺ channels are responsible for the potential membrane hyperpolarization that induces human breast cancer cell progression into the G1 phase of the cell cycle. In the present study, we evaluate the role and functional expression of the intermediate-conductance Ca²⁺-activated K⁺ channel, hIK1-like, in controlling cell cycle progression. Our results demonstrate that hIK1 current density increased in cells synchronized at the end of the G1 or S phase compared with those in the early G1 phase. This increased current density paralleled the enhancement in hIK1 mRNA levels and the highly negative membrane potential. Furthermore, in cells synchronized at the end of G1 or S phases, basal cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was also higher than in cells arrested in early G1. Blocking hIK1 channels with a specific blocker, clotrimazole, induced both membrane potential depolarization and a decrease in the [Ca²⁺]_i in cells arrested at the end of G1 and S phases but not in cells arrested early in the G1 phase. Blocking hIK1 with clotrimazole also induced cell proliferation inhibition but to a lesser degree than blocking hEAG with astemizole. The two drugs were essentially additive, inhibiting MCF-7 cell proliferation by 82% and arresting >90% of cells in the G1 phase. Thus, although the progression of MCF-7 cells through the early G1 phase is dependent on the activation of hEAG K⁺ channels, when it comes to G1 and checkpoint G1/S transition, the membrane potential appears to be primarily dependent on the hIK1-activity level. breast cancer; calcium-activated potassium channels; proliferation     

Mechanisms of a Human Skeletal Myotonia Produced by Mutation in the C-Terminus of NaV1.4: Is Ca2+ Regulation Defective?

Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNa_V1.4_F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNa_V1.4_F1705I are incompletely understood. The location of the hNa_V1.4_F1705I in the CT prompted us to examine the role of Ca²⁺ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human Na_V1.4 channel regulation by Ca²⁺ and CaM. hNa_V1.4_F1705I inactivation gating is Ca²⁺-sensitive compared to wild type hNa_V1.4 which is Ca²⁺ insensitive and the mutant channel exhibits a depolarizing shift of the V_1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNa_V1.4 channel background (rNa_V1.4_F1698I) eliminates Ca²⁺ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca²⁺ sensitivity of gating between wild type and mutant human and rat Na_V1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca²⁺/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows I_Na decay in a Ca²⁺-sensitive fashion. The combination of the altered voltage dependence and kinetics of I_Na decay contribute to the myotonic phenotype and may involve the Ca²⁺-sensing apparatus in the CT of Na_V1.4.     

Purinergic (ATP) signaling stimulates JNK1 but not JNK2 MAPK in osteoblast-like cells: contribution of intracellular Ca2+ release, stress activated and L-voltage-dependent calcium influx, PKC and Src kinases

This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y₂ receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP₃ receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca²⁺-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y₂ receptors. These results show that P2Y₂ receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP₃/intracellular Ca²⁺ release and Ca²⁺ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.     

Estradiol inhibits the effects of extracellular ATP in human sperm by a non genomic mechanism of action

Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17βE₂) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17βE₂ induced a rapid increase of intracellular calcium (Ca²⁺) concentrations dependent on an influx of Ca²⁺ from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17βE₂ showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca²⁺ in the extracellular medium since it was absent in Ca²⁺ free-medium. When sperm were pre-incubated in the presence of the K⁺ channel inhibitor tetra-ethylammonium, the 17βE₂ induced plasma membrane hyperpolarization was blunted suggesting the involvement of K⁺ channels in the hyperpolarizing effects of 17βE₂. Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm pre-incubation with 17βE₂ inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17βE₂ were specific since its inactive steroisomer 17αE₂ was inactive. Furthermore the effects of 17βE₂ were not inhibited by tamoxifen, an antagonist of the classic 17βE₂ intracellular receptor.     

ATP-dependent activation of the intermediate conductance, Ca2+-activated K+ channel, hIK1, is conferred by a C-terminal domain.

We previously demonstrated that hIK1 is activated directly by ATP in excised, inside-out patches in a protein kinase A inhibitor 5-24 dependent manner, suggesting a role for phosphorylation in the regulation of this Ca(2+)-dependent channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol. Chem. 275, 585-598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca(2+)-dependent K(+) channels, hSK1, rSK2, and rSK3. To define the region of hIK1 responsible for the ATP-dependent regulation, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not activate a chimera containing the N terminus plus S1-S4 from hIK1. In contrast, ATP activated a chimera containing the hIK1 C-terminal amino acids His(299)-Lys(427). Furthermore, truncation of hIK1 at Leu(414) resulted in an ATP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/rSK2 chimeras defined the minimal region of hIK1 required to confer complete ATP sensitivity as including amino acids Arg(355)-Ala(413). An alanine scan of all non-conserved serines and threonines within this region failed to alter the response of hIK1 to ATP, suggesting that hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg(355)-Met(368) of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was approximately 50% of that of hIK1. These results demonstrate that amino acids Arg(355)-Ala(413) within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca(2+).     

ATP-dependent activation of the intermediate conductance, Ca2+-activated K+ channel, hIK1, is conferred by a C-terminal domain

We previously demonstrated that hIK1 is activated directly by ATP in excised, inside-out patches in a protein kinase A inhibitor 5-24 dependent manner, suggesting a role for phosphorylation in the regulation of this Ca(2+)-dependent channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol. Chem. 275, 585-598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca(2+)-dependent K(+) channels, hSK1, rSK2, and rSK3. To define the region of hIK1 responsible for the ATP-dependent regulation, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not activate a chimera containing the N terminus plus S1-S4 from hIK1. In contrast, ATP activated a chimera containing the hIK1 C-terminal amino acids His(299)-Lys(427). Furthermore, truncation of hIK1 at Leu(414) resulted in an ATP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/rSK2 chimeras defined the minimal region of hIK1 required to confer complete ATP sensitivity as including amino acids Arg(355)-Ala(413). An alanine scan of all non-conserved serines and threonines within this region failed to alter the response of hIK1 to ATP, suggesting that hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg(355)-Met(368) of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was approximately 50% of that of hIK1. These results demonstrate that amino acids Arg(355)-Ala(413) within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca(2+).     

Effect of Chloride Channel Inhibitors on Cytosolic Ca2+ Levels and Ca2+-Activated K+ (Gardos) Channel Activity in Human Red Blood Cells

DIDS, NPPB, tannic acid (TA) and AO1 are widely used inhibitors of Cl^– channels. Some Cl^– channel inhibitors (NPPB, DIDS, niflumic acid) were shown to affect phosphatidylserine (PS) scrambling and, thus, the life span of human red blood cells (hRBCs). Since a number of publications suggest Ca²⁺ dependence of PS scrambling, we explored whether inhibitors of Cl^– channels (DIDS, NPPB) or of Ca²⁺-activated Cl^? channels (DIDS, NPPB, TA, AO1) modified intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and activity of Ca²⁺-activated K⁺ (Gardos) channel in hRBCs. According to Fluo-3 fluorescence in flow cytometry, a short treatment (15 min, +37 °C) with Cl^? channels inhibitors decreased [Ca²⁺]_i in the following order: TA > AO1 > DIDS > NPPB. According to forward scatter, the decrease of [Ca²⁺]_i was accompanied by a slight but significant increase in cell volume following DIDS, NPPB and AO1 treatments. TA treatment resulted in cell shrinkage. According to whole-cell patch-clamp experiments, TA activated and NPPB and AO1 inhibited Gardos channels. The Cl^– channel blockers further modified the alterations of [Ca²⁺]_i following ATP depletion (glucose deprivation, iodoacetic acid, 6-inosine), oxidative stress (1 mM t-BHP) and treatment with Ca²⁺ ionophore ionomycin (1 μM). The ability of the Cl^? channel inhibitors to modulate PS scrambling did not correlate with their influence on [Ca²⁺]_i as TA and AO1 had a particularly strong decreasing effect on [Ca²⁺]_i but at the same time enhanced PS exposure. In conclusion, Cl^– channel inhibitors affect Gardos channels, influence Ca²⁺ homeostasis and induce PS exposure of hRBCs by Ca²⁺-independent mechanisms.     

Roles of the NH2-terminal Domains of Cardiac Ryanodine Receptor in Ca2+ Release Activation and Termination

The NH₂-terminal region (residues 1–543) of the cardiac ryanodine receptor (RyR2) harbors a large number of mutations associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH₂-terminal region is involved in the activation and termination of Ca²⁺ release. The three-dimensional structure of the NH₂-terminal region has recently been solved. It is composed of three domains (A, B, and C). However, the roles of these individual domains in Ca²⁺ release activation and termination are largely unknown. To understand the functional significance of each of these NH₂-terminal domains, we systematically deleted these domains and assessed their impact on caffeine- or Ca²⁺-induced Ca²⁺ release and store overload-induced Ca²⁺ release (SOICR) in HEK293 cells. We found that all deletion mutants were capable of forming caffeine- and ryanodine-sensitive functional channels, indicating that the NH₂-terminal region is not essential for channel gating. Ca²⁺ release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination but had no effect on caffeine or Ca²⁺ activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca²⁺ activation and lowered the threshold for SOICR activation and termination. Conversely, deleting domain C suppressed caffeine activation, abolished Ca²⁺ activation and SOICR, and diminished protein expression. These results suggest that domain A is involved in channel termination, domain B is involved in channel suppression, and domain C is critical for channel activation and expression. Our data shed new insights into the structure-function relationship of the NH₂-terminal domains of RyR2 and the action of NH₂-terminal disease mutations.     

Apamin Boosting of Synaptic Potentials in CaV2.3 R-Type Ca2+ Channel Null Mice

SK2- and K_V4.2-containing K⁺ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca²⁺ source that provides Ca²⁺-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca²⁺ entry through NMDARs, whereas K_V4.2-containing channel availability is increased by Ca²⁺ entry through SNX-482 (SNX) sensitive Ca_V2.3 R-type Ca²⁺ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca²⁺ entry through R-type Ca²⁺ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca²⁺ channels and K_V4.2-containing K⁺ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca²⁺ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from Ca_V2.3 null mice. The results show that in the absence of Ca_V2.3 channels, apamin application still boosted EPSPs. The boosting effect of Ca_V2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from Ca_V2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and K_V4.2-containing channels to Ca_V2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.     

Flow-sensitive K+-coupled ATP Secretion Modulates Activity of the Epithelial Na+ Channel in the Distal Nephron

The epithelial Na⁺ channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under tonic inhibition by a local purinergic signaling system responding to changes in dietary sodium intake. Normal BK_Ca channel function is required for flow-sensitive ATP secretion in the ASDN. We tested here whether ATP secreted through connexin channels in a coupled manner with K⁺ efflux through BK_Ca channels is required for inhibitory purinergic regulation of ENaC in response to increases in sodium intake. Inhibition of connexin channels relieves purinergic inhibition of ENaC. Deletion of the BK-β4 regulatory subunit, which is required for normal BK_Ca channel function and flow-sensitive ATP secretion in the ASDN, suppresses increases in urinary ATP in response to increases in sodium intake. As a consequence, ENaC activity, particularly in the presence of high sodium intake, is inappropriately elevated in BK-β4 null mice. ENaC in BK-β4 null mice, however, responds normally to exogenous ATP, indicating that increases in activity do not result from end-organ resistance but rather from lowered urinary ATP. Consistent with this, disruption of purinergic regulation increases ENaC activity in wild type but not BK-β4 null mice. Consequently, sodium excretion is impaired in BK-β4 null mice. These results demonstrate that the ATP secreted in the ASDN in a BK_Ca channel-dependent manner is physiologically available for purinergic inhibition of ENaC in response to changes in sodium homeostasis. Impaired sodium excretion resulting form loss of normal purinergic regulation of ENaC in BK-β4 null mice likely contributes to their elevated blood pressure.     

Alternative Splicing Generates a Novel Truncated Cav1.2 Channel in Neonatal Rat Heart

L-type Ca_v1.2 Ca²⁺ channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Ca_v1.2 Ca²⁺ channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Ca_v1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Ca_v1.2_33L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Ca_v1.2 channels, Ca_v1.2_33L channels reduced the current density and altered the electrophysiological properties of the wild type Ca_v1.2 channels. Interestingly, the truncated 3.5-domain Ca_v1.2_33L channels also yielded a dominant negative effect on Ca_v1.3 channels, but not on Ca_v3.2 channels, suggesting that Ca_vβ subunits is required for Ca_v1.2_33L regulation. A biochemical study provided evidence that Ca_v1.2_33L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Ca_v1.2_33L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Ca_v1.2 channels. Moreover, the human Ca_v1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Ca_v1.2 channel. An electrophysiological study showed that human Ca_v1.2_33L channel is a functional channel but conducts Ca²⁺ ions at a much lower level.     

The gating of the sheep skeletal sarcoplasmic reticulum Ca2+-release channel is regulated by luminal Ca2+

The effects of changes in luminal [Ca²⁺] have been investigated in sheep skeletal sarcoplasmic reticulum (SR) Ca²⁺-release channels after activation of the channels by different ligands from the cytosolic side of the channel. Native heavy SR membrane vesicles were incorporated into planar phospholipid bilayers under voltage-clamp conditions. Experiments were carried out in symmetrical 250 mm Cs⁺. Lifetime analysis indicates that channels activated solely by cytosolic Ca²⁺ exhibit at least two open and five closed states. The open events are very brief and are close to the minimum resolvable duration. When channels are activated solely by cytosolic Ca²⁺, luminal Ca²⁺ does not appear to exert any regulatory effect. The P ₀ and duration of the open and closed lifetimes are unchanged. However, if channels are activated by ATP alone or by ATP plus cytosolic Ca²⁺, increases in luminal [Ca²⁺] produce marked increases in P ₀ and in the duration of the open lifetimes. Our results demonstrate that maximum activation of the skeletal SR Ca²⁺-release channel by ATP cannot be obtained in the absence of millimolar luminal [Ca²⁺].We are grateful to the British Heart Foundation for financial support.     

Photoreceptor Degeneration in Two Mouse Models for Congenital Stationary Night Blindness Type 2

Light-dependent conductance changes of voltage-gated Ca_v1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the α1F subunit of Ca_v1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Ca_v1.4 channels, but some mutations alter the channels’ gating properties and, presumably, disturb Ca²⁺ influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (ΔEx14–17) in a longitudinal study up to eight months of age. In ΔEx14–17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca²⁺ responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca²⁺ response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ΔEx14–17 mutants.     

Nicotinamide‐rich diet improves physical endurance by up‐regulating SUR2A in the heart

SUR2A is an ATP‐binding protein that serves as a regulatory subunit of cardioprotective ATP‐sensitive K⁺ (K_ATP) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled K_ATP channels, we have suggested that nicotinamide‐rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide‐rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild‐type mice on nicotinamide‐rich diet. The experiments focused on action membrane potential and intracellular Ca²⁺ concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal K_ATP channels and steady Ca²⁺ levels in cardiomyocytes in response to β‐adrenergic stimulation. In contrast, the same challenge in the wild‐type was characterized by a lack of the channel activation and rise in intracellular Ca²⁺. Nicotinamide‐rich diet was ineffective to increase physical endurance in mice lacking K_ATP channels. This study has shown that nicotinamide‐rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide‐rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable.     

Inhibition of Ca2+-dependent Cl− channels from secretory epithelial cells by low internal pH

The actions of intracellular pH (pH _i ) on Ca²⁺dependent Cl^? channels were studied in secretory epithelial cells derived from human colon carcinoma (T84) and in isolated rat parotid acinar cells. Channel currents were measured with the whole cell voltage clamp technique with pipette solutions of different pH. Ca²⁺dependent Cl^? channels were activated by superfusing ionomycin to increase the intracellular calcium concentration ([Ca²⁺] _i ) or by using pipette solutions with buffered Ca²⁺ levels. Large currents were activated in T84 and parotid cells by both methods with pH _i levels of 7.3 or 8.3. Little or no Cl^? channel current was activated with pH _i at 6.4. We used on-cell patch clamp methods to investigate the actions of low pH _i on single Cl^? channel current amplitude in T84 cells. Lowering the pH _i had little or no effect on the current amplitude of a 8 pS Cl^? channel, but did reduce channel activity. These results suggest that cytosolic acidification may be able to modulate stimulus-secretion coupling in fluid-secreting epithelia by inhibiting the activation of Ca²⁺-activated Cl^? channels.     

ATP-sensitive voltage- and calcium-dependent chloride channels in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

Chloride channels in the sarcoplasmic reticulum (SR) are thought to play an essential role in excitation-contraction (E-C) coupling by balancing charge movement during calcium release and uptake. In this study the nucleotide-sensitivity of Cl⁻ channels in the SR from rabbit skeletal muscle was investigated using the lipid bilayer technique. Two distinct ATP-sensitive Cl⁻ channels that differ in their conductance and kinetic properties and in the mechanism of ATP-induced channel inhibition were observed. The first, a nonfrequent 150 pS channel was inhibited by trans (luminal) ATP, and the second, a common 75 pS small chloride (SCl) channel was inhibited by cis (cytoplasmic) ATP. In the case of the SCl channel the ATP-induced reversible decline in the values of current (maximal current amplitude, I _max and integral current, I′) and kinetic parameters (frequency of opening F _O , probability of the channel being open P _O , mean open T _O and closed T _c times) show a nonspecific block of the voltage- and Ca²⁺-dependent SCl channel. ATP was a more potent blocker from the cytoplasmic side than from the luminal side of the channel. The SCl channel block was not due to Ca²⁺ chelation by ATP, nor to phosphorylation of the channel protein. The inhibitory action of ATP was mimicked by the nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP) in the absence of Mg²⁺. The inhibitory potency of the adenine nucleotides was charge dependent in the following order ATP⁴⁻ > ADP³⁻ > > > AMP²⁻. The data suggest that ATP-induced effects are mediated via an open channel block mechanism. Modulation of the SCl channel by [ATP] _cis and [Ca²⁺] _cis indicates that (i) this channel senses the bioenergetic state of the muscle fiber and (ii) it is linked to the ATP-dependent cycling of the Ca²⁺ between the SR and the sarcoplasm. Received: 4 September 1996/Revised: 6 December 1996     

cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca²⁺ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac ₁ and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the ₁ subunit, resulted in Ca²⁺ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the ₁ subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca²⁺ channel functions suggesting that cardiac Ca²⁺ channels expressed in these cells behave, in terms of lack of PKA control, like Ca²⁺ channels of smooth muscle cells.     

The effects of extracellular nucleotides on [Ca2+]i signalling in a human‐derived renal proximal tubular cell line (HKC‐8)

HKC‐8 cells are a human‐derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine [Ca²⁺]_i signalling mediated by P2 receptor in HKC‐8. Fura‐2 and a ratio imaging method were employed to measure [Ca²⁺]_i in HKC‐8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca²⁺]_i that was dependent on an intracellular source of Ca²⁺, while prolonged activation of P2Y receptors induced a rise in [Ca²⁺]_i that was dependent on intra‐ and extracellular sources of Ca²⁺. Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca²⁺ entry in coupling to activation of P2Y in HKC‐8 cells. U73221, an inhibitor of PI‐PLC, did not inhibit the initial ATP‐induced response; whereas D609, an inhibitor of PC‐PLC, caused a significant decrease in the initial ATP‐induced response, suggesting that P2Y receptors are coupled to PC‐PLC. Although P2X were present in HKC‐8, The P2X agonist, α,β me‐ATP, failed to cause a rise in [Ca²⁺]_i. However, PPADS at a concentration of 100 µM inhibits the ATP‐induced rise in [Ca²⁺]_i. Our results indicate the presence of functional P2Y receptors in HKC‐8 cells. ATP‐induced [Ca²⁺]_i elevation via P2Y is tightly associated with PC‐PLC and TRP channel. J. Cell. Biochem. 109: 132–139, 2010. © 2009 Wiley‐Liss, Inc.     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.