Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities

Regulation of nonspecific cation channels often underlies neuronal bursting and other prolonged changes in neuronal activity. In bag cell neurons of Aplysia, it recently has been suggested that an intracellular messenger-induced increase in the activity of a nonspecific cation channel may underlie the onset of a 30-min period of spontaneous action potentials referred to as the “afterdischarge.” In patch clamp studies of the channel, we show that the open probability of the channel can be increased by an average of 10.7-fold by application of ATP to the cytoplasmic side of patches. Duration histograms indicate that the increase is primarily a result of a reduction in the duration and percentage of channel closures described by the slowest time constant. The increase in open probability was not observed using 5′-adenylylimidodiphosphate, a nonhydrolyzable ATP analog, and was blocked in the presence of H7 or the more specific calcium/phospholipid-dependent protein kinase C (PKC) inhibitor peptide(19–36). Because the increase in activity observed in response to ATP occurred without application of protein kinase, our results indicate that a kinase endogenous to excised patches mediates the effect. The effect of ATP could be reversed by exogenously applied protein phosphatase 1 or by a microcystin-sensitive phosphatase also endogenous to excised patches. These results, together with work demonstrating the presence of a protein tyrosine phosphatase in these patches, suggest that the cation channel is part of a regulatory complex including at least three enzymes. This complex may act as a molecular switch to activate the cation channel and, thereby, trigger the afterdischarge.

Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein–protein interactions

Previously we showed that the functional activity of the epithelial chloride channel that is encoded by the cystic fibrosis gene (CFTR) is reciprocally modulated by two components of the vesicle fusion machinery, syntaxin 1A and Munc-18. Here we report that syntaxin 1A inhibits CFTR chloride channels by means of direct and domain-specific protein–protein interactions. Syntaxin 1A stoichiometrically binds to the N-terminal cytoplasmic tail of CFTR, and this binding is blocked by Munc-18. The modulation of CFTR currents by syntaxin 1A is eliminated either by deletion of this tail or by injecting this tail as a blocking peptide into coexpressing Xenopus oocytes. The CFTR binding site on syntaxin 1A maps to the third predicted helical domain (H3) of this membrane protein. Moreover, CFTR Cl− currents are effectively inhibited by a minimal syntaxin 1A construct (i.e., the membrane-anchored H3 domain) that cannot fully substitute for wild-type syntaxin 1A in membrane fusion reactions. We also show that syntaxin 1A binds to and inhibits the activities of disease-associated mutants of CFTR, and that the chloride current activity of recombinant ΔF508 CFTR (i.e., the most common cystic fibrosis mutant) can be potentiated by disrupting its interaction with syntaxin 1A in cultured epithelial cells. Our results provide evidence for a direct physical interaction between CFTR and syntaxin 1A that limits the functional activities of normal and disease-associated forms of this chloride channel.

Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R

The type IIA rat brain sodium channel is composed of three subunits: a large pore-forming α subunit and two smaller auxiliary subunits, β1 and β2. The β subunits are single membrane-spanning glycoproteins with one Ig-like motif in their extracellular domains. The Ig motif of the β2 subunit has close structural similarity to one of the six Ig motifs in the extracellular domain of the cell adhesion molecule contactin (also called F3 or F11), which binds to the extracellular matrix molecules tenascin-C and tenascin-R. We investigated the binding of the purified sodium channel and the extracellular domain of the β2 subunit to tenascin-C and tenascin-R in vitro. Incubation of purified sodium channels on microtiter plates coated with tenascin-C revealed saturable and specific binding with an apparent Kd of ≈15 nM. Glutathione S-transferase-tagged fusion proteins containing various segments of tenascin-C and tenascin-R were purified, digested with thrombin to remove the epitope tag, immobilized on microtiter dishes, and tested for their ability to bind purified sodium channel or the epitope-tagged extracellular domain of β2 subunits. Both purified sodium channels and the extracellular domain of the β2 subunit bound specifically to fibronectin type III repeats 1–2, A, B, and 6–8 of tenascin-C and fibronectin type III repeats 1–2 and 6–8 of tenascin-R but not to the epidermal growth factor-like domain or the fibrinogen-like domain of these molecules. The binding of neuronal sodium channels to extracellular matrix molecules such as tenascin-C and tenascin-R may play a crucial role in localizing sodium channels in high density at axon initial segments and nodes of Ranvier or in regulating the activity of immobilized sodium channels in these locations.

Expression of scavenger receptor class B type 1 (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstituted system

In the “selective” cholesteryl ester (CE) uptake process, surface-associated lipoproteins [high density lipoprotein (HDL) and low density lipoprotein] are trapped in the space formed between closely apposed surface microvilli (microvillar channels) in hormone-stimulated steroidogenic cells. This is the same location where an HDL receptor (SR-BI) is found. In the current study, we sought to understand the relationship between SR-BI and selective CE uptake in a heterologous insect cell system. Sf9 (Spodoptera frugiperda) cells overexpressing recombinant SR-BI were examined for (i) SR-BI protein by Western blot analysis and light or electron immunomicroscopy, and (ii) selective lipoprotein CE uptake by the use of radiolabeled or fluorescent (BODIPY-CE)-labeled HDL. Noninfected or infected control Sf9 cells do not express SR-BI, show microvillar channels, or internalize CEs. An unexpected finding was the induction of a complex channel system in Sf9 cells expressing SR-BI. SR-BI-expressing cells showed many cell surface double-membraned channels, immunogold SR-BI, apolipoprotein (HDL) labeling of the channels, and high levels of selective HDL-CE uptake. Thus, double-membraned channels can be induced by expression of recombinant SR-BI in a heterologous system, and these specialized structures facilitate both the binding of HDL and selective HDL-CE uptake.

FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle

FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor β (TGF-β) type I receptor. We now demonstrate that cells from FKBP12-deficient (FKBP12−/−) mice manifest cell cycle arrest in G1 phase and that these cells can be rescued by FKBP12 transfection. This arrest is mediated by marked augmentation of p21(WAF1/CIP1) levels, which cannot be further augmented by TGF-β1. The p21 up-regulation and cell cycle arrest derive from the overactivity of TGF-β receptor signaling, which is normally inhibited by FKBP12. Cell cycle arrest is prevented by transfection with a dominant-negative TGF-β receptor construct. TGF-β receptor signaling to gene expression can be mediated by SMAD, p38, and ERK/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathways. SMAD signaling is down-regulated in FKBP12−/− cells. Inhibition of ERK/MAP kinase fails to affect p21 up-regulation. By contrast, activated phosphorylated p38 is markedly augmented in FKBP12−/− cells and the p21 up-regulation is prevented by an inhibitor of p38. Thus, FKBP12 is a physiologic regulator of cell cycle acting by normally down-regulating TGF-β receptor signaling.

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200–500 μM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 μs) gating events and insensitivity to Ca2+. Addition of ryanodine restores Ca2+-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy

The ATP-sensitive potassium channel (KATP) regulates insulin secretion in pancreatic β cells. Loss of functional KATP channels because of mutations in either the SUR1 or Kir6.2 channel subunit causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We investigated the molecular mechanism by which a single phenylalanine deletion in SUR1 (ΔF1388) causes PHHI. Previous studies have shown that coexpression of ΔF1388 SUR1 with Kir6.2 results in no channel activity. We demonstrate here that the lack of functional expression is due to failure of the mutant channel to traffic to the cell surface. Trafficking of KATP channels requires that the endoplasmic reticulum-retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during channel assembly. To ask whether ΔF1388 SUR1 forms functional channels with Kir6.2, we inactivated the RKR signal in ΔF1388 SUR1 by mutation to AAA (ΔF1388 SUR1AAA). Inactivation of similar endoplasmic reticulum-retention signals in the cystic fibrosis transmembrane conductance regulator has been shown to partially overcome the trafficking defect of a cystic fibrosis transmembrane conductance regulator mutation, ΔF508. We found that coexpression of ΔF1388 SUR1AAA with Kir6.2 led to partial surface expression of the mutant channel. Moreover, mutant channels were active. Compared with wild-type channels, the mutant channels have reduced ATP sensitivity and do not respond to stimulation by MgADP or diazoxide. The RKR → AAA mutation alone has no effect on channel properties. Our results establish defective trafficking of KATP channels as a molecular basis of PHHI and show that F1388 in SUR1 is critical for normal trafficking and function of KATP channels.

Single point mutations affect fatty acid block of human myocardial sodium channel α subunit Na+ channels

Suppression of cardiac voltage-gated Na+ currents is probably one of the important factors for the cardioprotective effects of the n-3 polyunsaturated fatty acids (PUFAs) against lethal arrhythmias. The α subunit of the human cardiac Na+ channel (hH1α) and its mutants were expressed in human embryonic kidney (HEK293t) cells. The effects of single amino acid point mutations on fatty acid-induced inhibition of the hH1α Na+ current (INa) were assessed. Eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced INa in HEK293t cells expressing the wild type, Y1767K, and F1760K of hH1α Na+ channels. The inhibition was voltage and concentration-dependent with a significant hyperpolarizing shift of the steady state of INa. In contrast, the mutant N406K was significantly less sensitive to the inhibitory effect of EPA. The values of the shift at 1, 5, and 10 μM EPA were significantly smaller for N406K than for the wild type. Coexpression of the β1 subunit and N406K further decreased the inhibitory effects of EPA on INa in HEK293t cells. In addition, EPA produced a smaller hyperpolarizing shift of the V1/2 of the steady-state inactivation in HEK293t cells coexpressing the β1 subunit and N406K. These results demonstrate that substitution of asparagine with lysine at the site of 406 in the domain-1-segment-6 region (D1-S6) significantly decreased the inhibitory effect of PUFAs on INa, and coexpression with β1 decreased this effect even more. Therefore, asparagine at the 406 site in hH1α may be important for the inhibition by the PUFAs of cardiac voltage-gated Na+ currents, which play a significant role in the antiarrhythmic actions of PUFAs.

Nicotinic cholinergic signaling in hippocampal astrocytes involves calcium-induced calcium release from intracellular stores

In this report we provide evidence that neuronal nicotinic acetylcholine receptors (nAChRs) are present on hippocampal astrocytes and their activation produces rapid currents and calcium transients. Our data indicate that these responses obtained from astrocytes are primarily mediated by an AChR subtype that is functionally blocked by α-bungarotoxin (αBgt) and contains the α7 subunit (αBgt-AChRs). Furthermore, their action is unusual in that they effectively increase intracellular free calcium concentrations by activating calcium-induced calcium release from intracellular stores, triggered by influx through the receptor channels. These results reveal a mechanism by which αBgt-AChRs on astrocytes can efficiently modulate calcium signaling in the central nervous system in a manner distinct from that observed with these receptors on neurons.

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels

The cystic fibrosis transmembrane conductance regulator (CFTR) protein has the ability to function as both a chloride channel and a channel regulator. The loss of these functions explains many of the manifestations of the cystic fibrosis disease (CF), including lung and pancreatic failure, meconium ileus, and male infertility. CFTR has previously been implicated in the cell regulatory volume decrease (RVD) response after hypotonic shocks in murine small intestine crypts, an effect associated to the dysfunction of an unknown swelling-activated potassium conductance. In the present study, we investigated the RVD response in human tracheal CF epithelium and the nature of the volume-sensitive potassium channel affected. Neither the human tracheal cell line CFT1, expressing the mutant CFTR-ΔF508 gene, nor the isogenic vector control line CFT1-LC3, engineered to express the βgal gene, showed RVD. On the other hand, the cell line CFT1-LCFSN, engineered to express the wild-type CFTR gene, presented a full RVD. Patch-clamp studies of swelling-activated potassium currents in the three cell lines revealed that all of them possess a potassium current with the biophysical and pharmacological fingerprints of the intermediate conductance Ca2+-dependent potassium channel (IK, also known as KCNN4). However, only CFT1-LCFSN cells showed an increase in IK currents in response to hypotonic challenges. Although the identification of the molecular mechanism relating CFTR to the hIK channel remains to be solved, these data offer new evidence on the complex integration of CFTR in the cells where it is expressed.

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga1

The divalent cation Sr2+ induced repetitive transient spikes of the cytosolic Ca2+ activity [Ca2+]cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca2+]cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr2+-induced [Ca2+]cy oscillations. Sr2+ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca2+]cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca2+-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr2+-induced repetitive [Ca2+]cy spikes, whereas the compartmentalization of Sr2+ was not influenced. A repetitive Ca2+ release and Ca2+ re-uptake by the ER probably generated repetitive [Ca2+]cy spikes in E. viridis in the presence of Sr2+. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr2+-induced Ca2+ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca2+ channel. The blockage of Sr2+-induced repetitive [Ca2+]cy spikes by La3+ or Gd3+ indicated the necessity of a certain influx of divalent cations for sustained [Ca2+]cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca2+]cy oscillations in E. viridis.

Anion Channels and the Stimulation of Anthocyanin Accumulation by Blue Light in Arabidopsis Seedlings1

Activation of anion channels by blue light begins within seconds of irradiation in seedlings and is related to the ensuing growth inhibition. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a potent, selective, and reversible blocker of these anion channels in Arabidopsis thaliana. Here we show that 20 μm NPPB blocked 72% of the blue-light-induced accumulation of anthocyanin pigments in seedlings. Feeding biosynthetic intermediates to wild-type and tt5 seedlings provided evidence that NPPB prevented blue light from up-regulating one or more steps between and including phenylalanine ammonia lyase and chalcone isomerase. NPPB was found to have no significant effect on the blue-light-induced increase in transcript levels of PAL1, CHS, CHI, or DFR, which are genes that encode anthocyanin-biosynthetic enzymes. Immunoblots revealed that NPPB also did not inhibit the accumulation of the chalcone synthase, chalcone isomerase, or flavanone-3-hydroxylase proteins. This is in contrast to the reduced anthocyanin accumulation displayed by a mutant lacking the HY4 blue-light receptor, as hy4 displayed reduced expression of the above enzymes. Taken together, the data indicate that blue light acting through HY4 leads to an increase in the amount of biosynthetic enzymes, but blue light must also act through a separate, anion-channel-dependent system to create a fully functional biosynthetic pathway.

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures1

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca2+-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca2+]cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca2+]cyt and inhibition of growth that was similar to the effect of the Ca2+ channel blocker La3+ and the Ca2+ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca2+ channel blockers verapamil and nifedipine did not induce a decrease in [Ca2+]cyt in these cells and also failed to inhibit growth. Al and La3+, but not verapamil or nifedipine, reduced the rate of Mn2+ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca2+- and Mn2+-permeable channels. These results suggest that Al may act to block Ca2+ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.

Diurnal variation of the adenylyl cyclase type 1 in the rat pineal gland.

Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:00-3:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calcium-calmodulin complex. These findings suggest (i) the involvement of AC1 acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of AC1 in the rat pineal gland.

Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.