Multiple roles of calmodulin and other Ca2+-binding proteins in the functional regulation of TRP channels

Transient receptor potential channels (TRP) have emerged as cellular sensors of various internal and external cues. Generally, the activation of TRP canonical (TRPC) channels is triggered by the stimulation of phospholipase C; however, multiple factors are involved in the regulation of these channels. Among them, Ca²⁺-mediated feedback channel modulations are often mediated by calmodulin (CaM) and other Ca²⁺-binding proteins. In vitro binding studies have revealed multiple CaM-binding sites on TRPC proteins. Among them, a common CaM/inositol 1,4,5-trisphosphate receptor-binding site is found at the carboxyl terminus of every TRPC isoform. Additional non-conserved CaM-binding sites are present at the amino and carboxyl termini of several TRPC proteins. Likewise, multiple CaM-binding sites were found in other TRP proteins. These, together with the presence in close vicinity of the interaction sites for the related neuronal Ca²⁺-binding proteins, such as CaBP1, suggest a multitude of diverse intracellular Ca²⁺-dependent regulations of TRP channels. Functional studies have begun to reveal the unique roles of CaM and CaBP1 binding to several TRP channels. This review will focus on the CaM- and CaBP1-mediated regulations of TRPC channels. Related studies on TRPM and TRPV channels will also be highlighted.     

Differential Ca2+ binding properties in the human cerebellar cortex: distribution of parvalbumin and calbindin D-28k immunoreactivity

Summary The distribution of the Ca²⁺-binding proteins parvalbumin (PV) and calbindin D-28k (CaBP) was investigated in the human cerebellar cortex. Purkinje cells contain both PV and CaBP. PV but not CaBP stains stellate and basket cells in the molecular layer. In the granular layer Golgi neurons can be subdivided into a majority, devoid of both Ca²⁺-binding proteins, and a scanty population which appears to be PV- and CaBP-immunoreactive. Thus GABAergic neurons in the human cerebellar cortex show selective differences in their Ca²⁺-binding properties, and these differences might reflect a heterogeneity in the processing of Ca²-mediated events.Abbreviations CaBP calbindin D-28k - CNS central nervous system - GABA -aminobutyric acid - -IR immunoreactivity - LTD long-term depression - PV parvalbumin     

Neuropeptide Y2-type receptor-mediated activation of large-conductance Ca2+-sensitive K+ channels in a human neuroblastoma cell line

We have proposed recently that a pertussistoxin-insensitive Ca²⁺ influx stimulated by Y₂-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca²⁺ and independent of internal Ca²⁺ stores. We describe here the actions of NPY in these same cells, using the activity of Ca²⁺-activated K⁺ channels as an indicator of [Ca²⁺]_i. The elementary slope conductance of these channels was 110±3 pS (with an asymmetrical K⁺gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca²⁺, increased the channel open probability. ATP applied in the absence of external Ca²⁺ caused rises both in channel open probability and [Ca²⁺]_i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca²⁺ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5-triphosphate (GTP) by (guanosine 5-O-(2-thiodiphosphate) (GDP[S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y₂-type receptors to Ca²⁺ influx in CHP-234 cells.     

Ca2+ entry and vasoconstriction during osmotic swelling of vascular smooth muscle cells

Exposure of aortic strips from guinea-pigs to hypotonic extracellular fluid is followed by marked vasoconstriction, which is inhibited by D-600 (3 M), a blocker of voltage-sensitive Ca²⁺ channels. Conventional electrophysiology, patch-clamp studies, pH determination with 2, 7 bis(2-carboxyethyl)-5, 6-carboxyfluorescein (BCECF) and Ca²⁺ measurements with Fura-2 have been performed on smooth muscle cells cultured either from rat or human aorta to further elucidate the underlying mechanisms. Exposure of the cells to a 25% hypotonic extracellular fluid leads to a rapid and fully reversible depolarization, paralleled by an increase of the selectivity and conductance of the cell membrane to Cl^–, an acidification of the cytoplasm and an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The latter is inhibited by the Ca²⁺ channel blocker D-600 (1–3 M). It is concluded that osmotic cell swelling leads to the activation of an anion channel. The subsequent depolarization of the cell membrane activates voltage-sensitive Ca²⁺ channels which increases [Ca²⁺]_i, thus stimulating the contraction of vascular smooth muscle cells.     

Intracellular citrate induces regenerative calcium release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum was studied in voltage-clamped guinea-pig atrial myocytes. Cells were dialysed with a pipette solution containing the Ca²⁺ indicator 1- [2-amino-5-(6-carboxyindol-2-yl) phenoxy]-2-(2-amino-5-methylphenoxy) ethane-N,N,N,N-tetraacetic acid](Indo-1, 100 M) and as main anion either chloride or the low-affinity Ca²⁺ buffer citrate. Intracellular Ca²⁺ transients (Ca_i transients) were elicited by depolarizations from a holding potential of –50 mV. In chloride-dialysed cells, Ca_i transients showed a bell-shaped dependence on the amplitude of the depolarizing pulse. In citratedialysed cells, membrane depolarizations were associated with a small rise in [Ca²⁺]_i. These small changes in [Ca²⁺]_i were either followed by a large Ca_i. transient or failed to induce large changes in [Ca²⁺]_i. The peak amplitude of the large Ca_i transient did not vary with the amplitude of the depolarizing pulse. These results demonstrate that in the presence of intracellular chloride, Ca²⁺ release in atrial cells is a graded process triggered by Ca²⁺ influx. Using citrate as the main intracellular anoin, Ca²⁺ release triggered by Ca²⁺ entry was no longer graded but occurred in a regenerative manner. The results are discussed in terms of two models in which citrate, affects the spatial distribution of [Ca²⁺]_i or the loading state of the sarcoplasmic reticulum.     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Modulation of calcium current by ATP in guinea-pig adrenal chromaffin cells

The effects of extracellular adenosine 5-triphosphate (ATP) on voltage-dependent Ca²⁺ currents were examined using the whole-cell voltage-clamp technique in guinea-pig isolated adrenal chromaffin cells. ATP (500 M) reversibly suppressed Ca²⁺ currents in the presence of 5 mM Ca²⁺ in the extracellular solution. The inhibitory effect of ATP on Ca²⁺ currents tended to increase with increases in the peak amplitude of ATP-evoked current when the intracellular solution contained 0.1 or 1 mM ethylenebis(oxonitrilo)tetraacetate(EGTA). Using the intracellular solution containing 10 mM EGTA, on the other hand, the inhibitory efftect did not change regardless of the amplitude of current responses to ATP In the presence of 10 mM Ba²⁺, ATP (100 mol/l). reduced Ba²⁺ currents in a manner similar to Ca²⁺ currents. This reduction was decreased by dialysis of cells with the internal solution containing guanosine 5-O-(2-thiodiphosphate) (GDP [-S]; 1 mM) or guanosine 5-O-(3-thiotriphos-phate) (GTP [-S]; 100 mol/l). A depolarizing prepulse channels. In addition, ATP seems to modulate Ca²⁺ channels via the pathway related to G-protein. Adenine nucleotides and adenosine may play a role in controlling secretory activity in guinea-pig adrenal chromaffin cells.     

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in HT29 colonic carcinoma cells

Cl^– secretion in HT₂₉ cells is regulated by agonists such as carbachol, neurotensin and adenosine 5-triphosphate (ATP). These agonists induce Ca²⁺ store release as well as Ca²⁺ influx from the extracellular space. The increase in cytosolic Ca²⁺ enhances the Cl^– and K⁺ conductances of these cells. Removal of extracellular Ca²⁺ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca²⁺ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=6) inhibited ATP (0.1 mmol·l^–1) induced increases in whole-cell conductance (G _m). When Cl^– and K⁺ currents were inhibited by the presence of Cs₂SO₄ in the patch pipette and gluconate in the bath, ATP (0.1 mmol·l^–1) still induced a significant increase in G _m from 1.2±0.3 nS to 4.7±1 nS (n=24). This suggests that ATP induces a cation influx with a conductance of approximately 3–4 nS. This cation influx was inhibited by flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=9). When Ba²⁺ (5 mmol·l^–1) and 4,4-diisothiocyanatostilbene-2-2-disulphonic acid (DIDS, 0.1 mmol·l^–1) were added to the KCl/K-gluconate pipette solution to inhibit K⁺ and Cl^– currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol·l^–1) reduced the membrane current (I _m) significantly from 86±14 pA to 54±11 pA (n=13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5–10 mmol·l^–1 1,2-bis-(2-aminoethoxy)ethane-N,N,N,N-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA). The zero-current membrane voltage (V _m) and I _m (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30–120 s after membrane rupture. V _m depolarised significantly from –33±2 mV to –12±1 mV, and I _m fell significantly from 17±2 pA to 8.9±1.0 pA (n=71). This negative current, representing a cation inward current, was activated when Ca²⁺ stores were emptied and was reduced significantly (I _m) when Ca²⁺ and/or Na⁺ were removed from the bathing solution: removal of Ca²⁺ in the absence of Na⁺ caused a I _m of 5.0±1.2 pA (n=12); removal of Na⁺ in the absence of Ca²⁺ caused a I _m of 12.8±3.5 pA (n=4). The cation inward current was also reduced significantly by La³⁺, Gd³⁺, and flufenamate. We conclude that store depletion induces a Ca²⁺/Na⁺ influx current in these cells. With 145 mmol·l^–1 Na⁺ and 1 mmol·l^–1 Ca²⁺, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.     

Interaction between capacitative Ca2+ influx and Ca2+-dependent Cl− currents inXenopus oocytes

The relationship between capacitative Ca²⁺ influx and activation of Ca²⁺-dependent Cl^– channels was monitored in intactXenopus oocytes following stimulation of 5-hydroxytryptamine (5-HT) receptors, through the activity of Ca²⁺-dependent Cl^– channels using the double-electrode voltage-clamp technique. Under voltage-clamp conditions, 5-HT evoked a rapid transient inward current followed by a slowly developing secondary inward current. The secondary current reflected depletion-activated Ca²⁺ entry. Hyperpolarising pulses evoked sustained Ca²⁺-dependent Cl^– currents when applied during the transient inward current, but evoked hump-like currents which inactivated rapidly when applied during the secondary inward current. Hump currents arose from Ca²⁺ entering through the depletion-activated pathway. The hump currents inactivated with hyperpolarising pulses at <5-s intervals, and recovered monoexponentially with a time constant of around 8 s. Currents in response to hyperpolarising pulses during the transient current did not inactivate, suggesting that inactivation was associated with Ca²⁺ entry. When Ca²⁺ release evoked by inositol 1,4,5-triphosphate [Ins(1,4,5)P ₃] was prevented by heparin injection, hyperpolarising pulses during Ca²⁺ ionophore application also generated hump currents that were dependent on external Ca²⁺, inactivated and recovered from inactivation with a similar time course as the humps following 5-HT treatment. Pretreatment with the Ca²⁺ adenosine 5-triphosphatase (Ca²⁺ATPase) inhibitor thapsigargin reduced the rate of rise of the hump current, increased the time-to-peak of the current and slowed the rate of decay. Pharmacological interventions to disrupt the cytoskeleton reduced the amplitude of the hump current. It is suggested that, following hyperpolarisation in the presence of Ca²⁺ entry, the ensuing Ca²⁺ influx interacts with Cl^– channels in a way that might reflect both Ca²⁺ inhibition of Ca²⁺ entry and clustering of Cl^– channels in the plasma membrane.     

Nonselective cation channels are essential for maintaining intracellular Ca2+ levels and spontaneous firing activity in the midbrain dopamine neurons

Intracellular Ca²⁺ and Ca²⁺-permeable ion channels are important in regulating the firing activity and pattern of midbrain dopamine neurons, but the role of Ca²⁺-permeable nonselective cation channels (NSCCs) on spontaneous firing activity is unclear. Therefore, we investigated how Ca²⁺-permeable NSCCs modulate spontaneous firing activity and cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in acutely isolated midbrain dopamine neurons of the rat. Applications of voltage-dependent Ca²⁺ channels antagonists failed to abolish spontaneous firing activity completely, but they decreased firing rate and [Ca²⁺]_c. However, a blockade of NSCCs by 2-APB or SKF96365 more potently suppressed spontaneous firings with a depolarization of membrane potential and strong decreases in basal [Ca²⁺]_c levels. The depolarization of membrane potentials was attenuated by intracellular dialysis with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). NSCCs blockers inhibited oscillatory potentials and decreased basal [Ca²⁺]_c in the presence of tetrodotoxin. Apamin, a small-conductance Ca²⁺-activated K⁺ channel inhibitor, depolarized membrane potentials and enhanced firing rates. From these data, we conclude that NSCCs not only make up the tonic Ca²⁺ entry pathways to uphold basal [Ca²⁺]_c levels but also contribute to generation of spontaneous firings, thereby regulating spontaneous firing activities of the midbrain dopamine neurons.     

Intracellular neutral carrier-based Ca2+ microelectrode with subnanomolar detection limit

In intracellular electrolyte solutions a Ca²⁺-selective microelectrode based on the synthetic electrically neutral carrier N,N,N,N-tetracyclohexyl-3-oxapentanediamide (ETH 129) shows an improved detection limit when compared with the so far widely used Ca²⁺ microelectrodes based on the neutral carrier ETH 1001. Detection limits are found at pCa=9.2 in Ca²⁺ buffers containing an intracellular background of K⁺ (125 mM). Selectivity studies in mixed solutions show a preference of Ca²⁺ over Na⁺ of 6·10⁵, over K⁺ of 1.6·10⁶, and over Mg²⁺ of 5·10⁶. The microelectrode does not suffer from significant interference by inorganic and organic inhibitors and by lipophilic cations and anions. The low detection limit is unchanged at least during the first eight hours of continuous contact with Ca²⁺ solutions. The EMF drift during the first hour of use is between 5 and 10 mV and is then reduced to about 1 mV/h. The changes in EMF induced between solution of pCa=7 and pCa=8 are reproducible within 24.7±0.4 mV (SD,n=8, about 3 h). These electrode characteristics were found for single-barrelled microelectrodes of one micrometer diameter front-filled with a PVC-containing membrane phase. In the absence of poly(vinyl chloride) in the membrane phase irregular EMF response curves were obtained throughout. Preliminary punctures of ferret ventricular muscle cells indicate that the Ca²⁺ electrode response is not disturbed by the contact of a cytosolic milieu.     

Inositol 1,4,5-trisphosphate formation and ryanodine-sensitive oscillations of cytosolic free Ca2+ concentrations in neuroblastoma×fibroblast hybrid NL308 cells expressing m2 and m4 muscarinic acetylcholine receptor subtypes

Intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) were measured in subclones of NL308 neuroblastoma x fibroblast hybrid cells expressing each of the individual muscarinic acetylcholine receptor (mAChR) subtypes m1, m2, m3 and m4. Application of 100 M acetylcholine (ACh) increased [Ca²⁺]_i in all four subclones. The increased [Ca²⁺]_i levels were significantly higher in m1- and m3-transformed cells than those in m2- and m4-transformed cells. In more than 95% of m2- and m4-transformed cells, [Ca²⁺]_i showed sinusoidal oscillations. ACh-induced increases in [Ca²⁺]_i were not observed in cells treated with an intracellular Ca²⁺ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA). Removal of extracellular Ca²⁺ with ethyleneglycol-bis-(-aminoethyl)-N,N,N,N'-tetraacetate (EGTA) did not affect the initial [Ca²⁺]_i increases, but reduced the late phases of [Ca²⁺]_i in m1- and m3-transformed cells by 20–30%. Oscillations in m2- and m4-transformed cells persisted in EGTA solution (though sometimes slowed in frequency), suggesting that they were of intracellular origin. ACh-induced [Ca²⁺]_i and inositol 1,4,5-trisphosphate formation was completely suppressed by pre-treatment with 50–100 ng ml^–1 Pertussis toxin (PTX) for 12 h in m2- and m4-transformed cells, but not in m1 and m3-transformed cells. In all cells, extracellular application of caffeine and ryanodine, or intracellular application of cyclic adenosine diphosphate ribose (cADPR) produced a rise in [Ca²⁺]_i. ACh-induced [Ca²⁺]_i oscillations were not observed in ryanodine-treated m2-transformed cells. These results show that, while all four mAChRs utilize Ca²⁺ as a common second messenger, m2 and m4 receptors use a different signalling pathway to that used by m1 and m3 receptors.     

Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone,cyclic AMP and phorbol esters

Parathyroid hormone (PTH) controls two proximal tubular brush border membrane transport systems, Na⁺/phosphate co-transport and Na⁺/H⁺ exchange. In OK cells, a cell line with proximal tubular transport characteristics, PTH acts via kinase C and kinase A activation to inhibit Na⁺/phosphate co-transport [6, 8, 9, 19, 22]. In the present study, we show that PTH inhibits Na⁺/H⁺ exchange and that this effect can be mimicked by pharmacological activation of kinase A and kinase C. Ionomycin-dependent increases in cytoplasmic Ca²⁺ concentration do not induce inhibition of Na⁺/H⁺ exchange; PTH-dependent inhibition of Na⁺/H⁺ exchange is not prevented by ionomycin or by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (Ca²⁺ clamping). Detailed dose-response curves for the different agonists, given either alone or in combination, suggest that the two regulatory cascades (kinase A and kinase C) are operating independent of each other and reach a common final target, resulting in 40–50% inhibition of Na⁺/H⁺ exchange. An analysis of intracellular pH sensitivity of Na⁺/H⁺ exchange suggests that inhibition is not related to a shift in set point, but is rather explained by a reduced V _max of Na⁺/H⁺ exchange and/or reduced affinity for protons at the internal membrane surface. It is suggested that kinase A as well as kinase C can mediate PTH inhibition of renal proximal tubular Na⁺/H⁺ exchange and that the relative importance of a particular regulatory cascade is determined by the PTH-concentration-dependent rates in the liberation of diacylglycerol (phospholipase C/kinase C) and cAMP (adenylate cyclase/kinase A).Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazineethane-sulphonic acid - EDTA ethylenediaminetetraacetic acid - FCS fetal calf serum - PTH parathyroid hormone - Tris 2-amino-2-hydroxyme-thylpropane-1,3-diol - BCECF 2,7-bis(2-carboxyethyl)-5,6-carboxyfluorescein - P_i inorganic phosphate - pH_i intracellular pH - Ca_i cytosolic free Ca²⁺ - BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - EGTA ethylene glycol-bis(-amino-ethylether)-N,N,N,N-tetraacetic acid - IP₃ inositol 1,4,5-trisphos-phate - DAG 1,2-diacylglycerol     

ADP exerts a protective effect against rundown of the Ca2+ current in bovine chromaffin cells

In isolated chromaffin cells, the high-voltage-activated Ca²⁺ current, recorded using 5 mM Ca²⁺ as the divalent charge carrier, exhibits rundown within 10 min, which is delayed for 1 h at least by the addition of 1 mM adenosine 5-triphosphate (ATP) to the pipette medium. The mechanism of this stabilizing action of ATP has been examined. ATP action is dose dependent; the rundown process, which was delayed at concentrations below 0.4 mM, was totally abolished at higher concentrations. The requirement for ATP was shown to be quite strict: 2 mM inosine 5-triphosphate (ITP) could not replace ATP, whereas guanosine 5-triphosphate (GTP) could, but at higher concentrations. This effect of ATP was shown to require the presence of MgCl₂ and the liberation of a phosphate group since the ATP analogue 5-adenylyl-imidodiphosphate (AMP-PNP) could not act as a substitute for ATP, suggesting an action through either adenosine 5-diphosphate (ADP) or a phosphorylation step. ADP, in the presence of Mg²⁺ only, could replace ATP in the same concentration range. This effect was shown to be specific to ADP; it was maintained after blocking the pathways which convert ADP into ATP, and could not be mimicked by guanosine 5-diphosphate (GDP). Similarly, ATP and ADP effects were abolished at an increased internal Ca²⁺ concentration (pCa 6 instead of pCa 7.7, where pCa = –log₁₀[Ca²⁺]). Nevertheless, the presence of 1 mM Mg-ADP in the bathing solution did not prevent the rundown of the Ca²⁺ channels when going to the inside-out patch recording configuration. In conclusion, the stabilizing effect of ATP may be interpreted by a Mg²⁺-ADP binding site present on high-voltage-activated Ca²⁺ channels. A localization of such an ADP regulatory site on the L-type Ca²⁺ channel itself cannot be excluded, though with an additional requirement since Mg-ADP alone is not able to maintain the corresponding activity on excised patches.     

Copper toxicity in cultured human skeletal muscle cells: the involvement of Na+/K+-ATPase and the Na+/Ca2+-exchanger

Copper (Cu²⁺) intoxication has been shown to induce pathological changes in various tissues. The mechanism underlying Cu²⁺ toxicity is still unclear. It has been suggested that the Na⁺/K⁺-ATPase and/or a change of the membrane permeability may be involved. In this study we examined the effects of Cu²⁺ on the Na⁺ and Ca²⁺ homeostasis of cultured human skeletal muscle cells using the ion-selective fluorescent probes Na⁺-binding benzofuran isophtalate (SBFI) and Fura-2, respectively. In addition, we measured the effect of Cu²⁺ on the Na⁺/K⁺-ATPase activity. Cu²⁺ and ouabain increase the cytoplasmic free Na⁺ concentration ([Na⁺]_i). Subsequent addition of Cu²⁺ after ouabain does not affect the rate of [Na⁺]_i increase. Cu²⁺ inhibits the Na⁺/K⁺-ATPase activity with an IC₅₀ of 51 M. The cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) remains unaffected for more than 10 min after the administration of Cu²⁺. Thereafter, [Ca²⁺]_i increases as a result of the Na⁺/Ca²⁺-exchanger operating in the reversed mode. The effects of Cu²⁺ on the Na⁺ homeostasis are reversed by the reducing and chelating agent dithiothreitol and the heavy metal chelator N,N,N,N,-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). In conclusion, SBFI is a good tool to examine Na⁺ homeostasis in cultured human skeletal muscle cells. Under the experimental conditions used, Cu²⁺ does not modify the general membrane permeability, but inhibits the Na⁺/K⁺-pump leading to an increase of [Na⁺]_i. As a consequence the operation mode of the Na⁺/Ca²⁺-exchanger reverses and [Ca²⁺]_i rises.The authors thank staff and coworkers of the Department of Neurology of the University Hospital Nijmegen, Nijmegen for their kind cooperation in obtaining muscle biopsies. Mr. Arie Oosterhof is gratefully acknowledged for culturing of the human muscle cells. The Prinses Beatrix Fonds and the Dutch-Chinese scientific exchange program contributed financial support for this study.     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

Protein-protein interaction and functionTRPC channels

Since their identification in the concluding years of the last century, the mammalian transient receptor potential (canonical) (TRPC) channels have remained in the limelight as the primary candidates for the Ca²⁺ entry pathway activated by the hormones, growth factors, and neurotransmitters that exert their effect through activation of PLC. Although TRPC channels have been shown clearly to mediate, at least in part, receptor-activated Ca²⁺ entry in literally all cell types, several of their central characteristics, as recorded in expression systems using recombinant channels, differ from those of the native receptor-dependent Ca²⁺ influx channels. The present review attempts to highlight the interaction of TRPC channels with other proteins, which may explain the variability of TRPC channel activation and regulatory mechanisms observed with the native and recombinant channels. These include the homologous and heterotopous interactions of TRPC channel isoforms, the interaction of TRPC channels with calmodulin, PLC, IP₃ receptors, and with scaffolding proteins like InaD, EBP50/NEHRF, caveolin, Janctate and Homers.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

Kinetics of receptor-mediated endocytosis of albumin in cells derived from the proximal tubule of the kidney (opossum kidney cells): influence of Ca2+ and cAMP

In this study we investigated the effects of Ca²⁺ and cyclic adenosine monophosphate (cAMP) on the kinetic of receptor-mediated (RME) and fluidphase (FPE) endocytosis in opossum kidney (OK) cells, derived from the proximal tubule of the kidney. We used fluorescein isothiocyanate (FITC)-labelled albumin and FITC-labelled dextran as endocytotic substrates for RME and FPE, respectively. Removal of extracellular Ca²⁺ led to a dramatic decrease of the apparent affinity of RME, but did not influence the maximum endocytotic uptake rate (J _max). Reduction of extracellular Ca²⁺ to 1 mol/l had no effect. Apparent affinity of specific binding of albumin to the plasma membrane was increased to 200% of control in the absence of extracellular Ca²⁺, whereas maximum binding capacity was slightly decreased. FPE was not affected by removal of extracellular Ca²⁺. Additional removal of cytoplasmic Ca²⁺, using ionomycin, had no further effect on RME and did not affect FPE. Increases of cytoplasmic (using ionomycin at extracellular Ca²⁺ concentrations of 1 mol/l or 1.2 mmol/l) or extracellular Ca²⁺ did not alter the kinetics of RME or FPE. Dibutyryl-cAMP reduced J _max but left the apparent affinity of RME unchanged. FPE and albumin binding to the plasma membrane were not changed in the presence of cAMP. Removal of extracellular Ca²⁺ and addition of cAMP led to an alkalinization of endocytotic vesicles. Yet the alkalinization induced by removal of Ca²⁺ was significantly greater as compared to the alkalinization in the presence of cAMP. Endosomal alkalinization with bafilomycin A₁ had no further effect in the absence of Ca²⁺, but reduced RME in the presence of cAMP. From these results we conclude that cAMP and extracellular Ca²⁺ influence the kinetics of RME in proximal tubular cells. The action of Ca²⁺ removal seems to be mediated partially by changes of endosomal pH. Ca²⁺ seems to be a prerequisite for RME but not a regulator, whereas cAMP may act as physiological inhibitor of RME.