Functional assembly and purinergic activation of bestrophins

Proteins of the bestrophin family produce Ca²⁺-activated Cl⁻ currents and regulate voltage-gated Ca²⁺ channels. Bestrophin 1 was first identified in the retinal pigment epithelium. Four human paralogs (hBest1–hBest4) exist, and for some bestrophins, dimeric and heterotetrameric structures have been proposed. Here, we demonstrate that hBest1–hBest4 induce Cl⁻ conductances of different amplitudes when expressed in HEK293 cells and when activated through purinergic stimulation. hBest1 mutants that are known to cause autosomal dominant macular dystrophy (Best disease) did not produce a Cl⁻ current. Bestrophins were colocalized and showed molecular and functional interaction in HEK293 cells, overexpressing hBest1 and hBest2 or hBest4. Interaction was confirmed in airway epithelial cells coexpressing endogenous bestrophins. A fraction of hBest2 and hBest4 was expressed in the membrane, while most of hBest1 was found in the endoplasmic reticulum. Nevertheless, hBest1 has a clear role for the adenosine triphosphate (ATP; or uridine triphosphate)-induced Cl⁻ current in both HEK293 and Calu-3 cells. Since native epithelial tissues typically express several bestrophin paralogs, these proteins may exist as heterooligomeric structures. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Calcium-dependent chloride conductance in epithelia: is there a contribution by Bestrophin?

Although known for more than 20 years, the molecular identity of epithelial Ca²⁺-activated Cl⁻ channels remains obscure. Previous candidate proteins did not hold initial promises, and thus, new hope is put into the recently identified family of bestrophin proteins, as they reflect many of the properties found for native channels. Mutations in the bestrophin gene cause an autosomal form of macular dystrophy of the retina. Bestrophin 1 is assumed to form the basolateral Ca²⁺-activated Cl⁻ channel in the retinal pigment epithelium of the eye. Other data suggest that bestrophin is a regulator of voltage gated Ca²⁺ channels. Structural information on bestrophins is available and a Cl⁻ selective filter has been localized to the second transmembrane domain of bestrophin. It is possible that bestrophins function as physiologically regulated Cl⁻ channels only in association with additional subunits and auxiliary proteins. Little is known about expression of bestrophin in gland acinar cells, which show a pronounced Ca²⁺-activated Cl⁻ secretion. In airways and intestinal epithelia, bestrophins could be particularly important in diseases such as cystic fibrosis and secretory diarrhea.     

Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250–300 pS and 9–14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca²⁺ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca²⁺ action potentials but has a Ca²⁺ sensitivity at –50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca²⁺ at –50 mV, ad its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca²⁺-activated K⁺ tails currents and during Ca²⁺-activated K⁺ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.     

Spontaneous transient inward currents and rhythmicity in canine and guinea-pig tracheal smooth muscle cells

Spontaneous transient inward currents (STICs) were recorded in canine and guinea-pig tracheal myocytes held at negative membrane potentials. STICs were Cl^– selective since their reversal potential was dependent on the Cl^– gradient and they were blocked by the Cl^– channel blocker niflumic acid. STICs were insensitive to Cs⁺, charybdotoxin, and nifedipine. Ca²⁺-activated K⁺ currents often preceded STICs, suggesting that the STICs are Ca²⁺ dependent. In support of this suggestion, we found the Cl^– currents were: (1) abolished by depleting intracellular Ca²⁺ stores using caffeine, acetylcholine, histamine, or substance P; (2) enhanced by increasing external concentrations of Ca²⁺; (3) evoked by voltage-dependent Ca²⁺ influx. The channels responsible for this Cl^– current are of small unitary conductance (<20 pS). Decay of the STICs was described by a single exponential with a time constant of 94±9 ms at –70 mV; the time constant increased considerably at more positive potentials. Using Ca²⁺-dependent Cl^– currents and contractions as indices of internal levels of Ca²⁺, we found that isolated tracheal cells are capable of exhibiting rhythmic behaviour: bursts of currents and contractions with a periodicity of less than 0.1 Hz and which continued for more than 20 min. These rhythmic events were recorded at negative membrane potentials, suggesting that cyclical release of internally sequestered Ca²⁺ is responsible. We conclude that spontaneous release of Ca²⁺ from intracellular stores in tracheal muscle cells leads to transient currents in some cases accompanied by rhythmic contractions. Our studies provide evidence for a cellular mechanism that could underly myogenic oscillations of membrane potential in smooth muscle.     

Tetraethylammonium-insetisitive,Ca2+-activated whole-cell K+ currents in rat submandibular acinar cells

Using whole-cell patch-clamp techniques, we demonstrate, for the first time, that rat submandibular acinar cells contain a tetraethylammonium (TEA)-insensitive, Ca²⁺-activated K⁺ conductance which is not attributable to large conductance, voltage-sensitive, Ca²⁺-dependent K⁺ channels (maxi-K⁺ channels). Taken together with our recent K⁺ efflux and fluid secretion studies in intact rat submandibular gland, we postulate that the K⁺ conductance reported here may be involved in the basolateral K⁺ efflux pathway activated by cytosolic Ca²⁺ concentration during secretion by this gland.     

T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.     

Potassium transport in epithelial cells isolated from small intestine of the chicken

The transport of potassium has been studied in epithelial cells isolated from chicken small intestine using⁸⁶Rb as a tracer for K⁺. (i) The uptake studies revealed that about 60% of the total K⁺ net flux is inhibited by ouabain and therefore mediated by the Na⁺–K⁺-ATPase. About 20% of the ouabain-insensitive K⁺ net influx was inhibited by furosemide, bumetanide and by either Na⁺ or Cl^– removal from the incubation solution, suggesting that a Na⁺/Cl^–/K⁺ cotransport system might be present in chicken enterocytes. (ii) The efflux of K⁺ was measured from cells preloaded with⁸⁶Rb. K⁺ efflux was inhibited by Ba²⁺, quinine and verapamil; it was stimulated by A23187, and it was unaffected by 3,4-diaminopyridine. Apamin, that has no effect on basal rates of K⁺ efflux, abolished the effect of A23187. These findings suggest that K⁺ efflux appears to occur through Ca²⁺-activated K⁺ channels.     

Calreticulin expression in spinal motoneurons of the rat

We have examined the expression of calreticulin in rat spinal motoneurons in order to reveal the occurrence and distribution of Ca²⁺-storage organelles in these neurons. Calreticulin, the non-muscle equivalent of calsequestrin, is the low-affinity, high-capacity calcium-binding protein responsible for intracompartmental Ca²⁺-storage in a number of different cell types. The results of the present immunohistochemical study show that all spinal motoneurons express calreticulin at approximately the same level; no significant differences in cytoplasmic immunostaining intensity were observed between different motoneuron pools or between small and large spinal motoneurons. Immunoelectron microscopy revealed that the intracellular localization of calreticulin within spinal motoneurons was confined to the endoplasmic reticulum and to spherical or pleiomorphic, frequently ‘coated’ vesicles with a diameter ranging between 120 and 150 nm. Some of these vesicles may represent the so-called calciosomes, the intracellular Ca²⁺-storage vesicles described in liver cells and in cerebellar Purkinje cells. The molecular components responsible for the uptake and release of Ca²⁺ from the Ca²⁺-storage organelles in spinal motoneurons still remain to be identified.     

L-alanine evokes opening of single Ca2+-activated K+ channels in rat liver cells

Cellular uptake of neutral amino acids via Na⁺ cotransporters is known to be associated with an increased membrane K⁺ conductance mediated by an unknown mechanism that is essential for avoiding excessive cell swelling. We now demonstrate by patch-clamp single-channel current recording that exposure of rat liver cells to L-alanine, but not the poorly transported D-stereoisomer, evokes opening of single K⁺ channels and that this effect is reversible upon removal of the amino acid. The nature of the conductance pathways opened in the intact cell by L-alanine has been investigated in cell-free excised membrane patches where it can be shown that the K⁺-selective channels are opened by Ca²⁺ acting from the inside of the membrane at a concentration as low as 0.1 M.     

3-Isobutyl-1-methylxanthine (IBMX) affects potassium permeability in rat sensory neurones via pathways that are sensitive and insensitive to [Ca2+]in

The effects of externally applied 3-isobutyl-1-methylxanthine (IBMX), in millimolar concentrations, on the membrane currents in dorsal root ganglia (DRG) neurones isolated from newborn rats were investigated using the amphotericin-based perforated patch-clamp technique. In some experiments, simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_in) were performed using fura-2 microfluorimetry. Applications of IBMX induced elevation of [Ca²⁺]_in resulting from Ca²⁺ release from caffeine-ryanodine-sensitive internal stores. In addition to Ca²⁺ release, IBMX produced a biphasic membrane current response comprised of an inward current transiently interrupted by outward current. The onset of the inward current slightly preceded the onset of the [Ca²⁺]_in transient, while the interrupting outward current developed synchronously with the [Ca²⁺]_in rise. The development of IBMX-induced outward current ultimately needed the [Ca²⁺]_in elevation. After the depletion of Ca²⁺ stores by IBMX or caffeine exposure, the subsequent IBMX challenge failed to produce both the [Ca²⁺]_in transient and outward membrane current, although the inward current remained unchanged. Both components of the IBMX-induced membrane current response had a reversal potential close to the K⁺ equilibrium potential and the IBMX-induced membrane current response disappeared while dialysing the cell interior with K⁺-free, Cs⁺-containing solutions suggesting their association with K⁺ channel activity. External administration of 10 mM tetraethylammonium chloride (TEA-Cl) evoked an inward current similar to that observed in response to IBMX; in the presence of TEA-Cl, IBMX application was almost unable to induce additional inward current. IBMX (5 mM) effectively (50%) inhibited K⁺ currents evoked by step depolarizations of membrane potential. We suggest that IBMX affects membrane permeability via activation of Ca²⁺-regulated K⁺ channels and direct inhibition of TEA-sensitive K⁺ channels.     

Electrophysiological properties of neurones in cultures from postnatal rat dentate gyrus

Electrophysiological properties of neurofilament-positive neurones in dissociated cell cultures were prepared at postnatal days 4–5 from rat dentate gyrus and studied using the whole-cell patch-clamp technique. These cells expressed a fast-inactivating, 0.5 M tetrodotoxin-sensitive Na⁺ current; a high-voltage-activated (HVA) Ca²⁺ current, which was 30 M Cd²⁺- and partially 2 M nicardipine-sensitive; and an inward rectifier current, which was sensitive to extracellularly applied 1 mM Cs⁺. The outward current pattern was composed of a delayed rectifier-like outward current sensitive to 20 mM tetraethylammonium (TEA) and a fast-inactivating, Ca²⁺-dependent outward current. This transient Ca²⁺-dependent K⁺ outward current was identified by a subtraction procedure. K⁺ currents recorded under conditions of blocked Ca²⁺ currents (after rundown of the HVA Ca²⁺ current or blocked by extracellularly applied Cd²⁺) were subtracted from control currents. By comparison with the current pattern of identified dentate granule cells, it is concluded that the investigated cell type originated from interneurones or projection neurones of the dentate hilus.     

The pathway for refilling intracellular Ca2+ stores passes through the cytosol in human leukaemia cells

The pathway for refilling the intracellular Ca²⁺ stores of HL60 and U937 human leukaemia cells loaded with fura-2 has been investigated. On addition of external Ca²⁺ to cells with empty stores there was an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which preceded the refilling of the stores. The increase in [Ca²⁺]_i was faster than the refilling, by 3-to 15-fold, depending on the cell type. In measurements in single HL60 cells we found that the refilling of the stores correlated with the extent of the [Ca²⁺]_i increase on addition of external Ca²⁺. The cells showing no [Ca²⁺]_i increase were unable to refill their stores. The addition of Ni²⁺ to the extracellular medium prevented both the [Ca²⁺]_i increase and the refilling of the stores. These results indicate that the limiting step for store refilling is the entry of Ca²⁺ from the extracellular medium to the cytosol. Hence, we conclude that extracellular Ca²⁺ cannot gain access directly to the intracellular Ca²⁺ stores in these cells, but must first enter the cytosol and be taken up from there into the stores.     

Changes in membrane ionic conductances and excitability characteristics of rat skeletal muscle during aging

Membrane electrical properties,component ionic conductances and excitability characteristics of extensor digitorum longus muscle from 3–4, 16 and 29 months old rats were measured “in vitro”. Fiber diameter, membrane resistance(Rm) and membrane capacitance, increased with aging,and the increase was significant at 29 months. The increase of Rm was mostly due to a decrease of chloride conductance(GCl),whereas potassium conductance(GK) increased only slightly, at 16 and 29 months. Due to the lowered GCl, the latency of action potential increased at both ages with a consequent prolongation of the duration of action potential. Nevertheless, a decrease in the firing capability was recorded in the aged fibers. Our results indicate,that during aging, the most affected parameter of skeletal muscle fibers is GCl, although changes of this passive conductance alone cannot entirely account for the changes in the excitability characteristics recorded.     

Cholinergic agonists increase cell calcium in rat medullary collecting tubules

The intracellular free calcium concentration [Ca²⁺]_i of rat medullary collecting tubules was calculated from microscope fluorescence measurements in single pieces of fura-2-loaded tubules superfused at 37 °C. When carbachol (10^–4–10^–3M) was added in the superfusate, a biphasic increase in [Ca²⁺]_i was generally obtained, which included an early peak phase and a sustained plateau thereafter; sometimes, the peak phase was not apparent; the plateau was maintained as long as the agonist was applied. Several responses could be induced successively without a fall in responsiveness. From dose/response curves, K_1/2 values of about 10^–5M for carbachol and 10^–6M for acetylcholine were obtained. The effects of the agonists were suppressed with 10^–4M of atropine or pirenzepine, indicating the presence of muscarinic receptors of the M₁ type. In the absence of external calcium, the peak phase of the response was preserved while the plateau phase was suppressed; thus, the peak involves the release of calcium stored in organelles, whereas the plateau involves the entry of external calcium through calcium channels which were voltage independent and insensitive to the usual calcium blockers.     

Blocking actions of Ca2+ antagonists on the Ca2+ channels in the smooth muscle cell membrane of rabbit small intestine

Actions of Ca²⁺ antagonists, verapamil, nicardipine and diltiazem, were investigated on the Ca²⁺ inward current in the fragmented smooth muscle cell membrane (smooth muscle ball; SMB) obtained from the longitudinal muscle layer of the rabbit ileum, by enzymatic dispersion. All Ca²⁺ antagonists inhibited the inward current, in a dose-dependent manner. The ID₅₀ value on the maximum amplitude of the inward current of nicardipine was 24 nM, and this value was roughly 50 times lower than values obtained with verapamil and diltiazem, when the inward current was provoked by 0 mV command pulse from the holding potential of –60 mV. Lowering the holding potential to –80 mV shifted the dose-response curve to the right. When depolarizing pulses (100 ms, stepped up to 0 mV from –60 mV or –80 mV) were applied every 20 s, the peak amplitude of the inward current remained unchanged, but nicardipine immediately, and diltiazem and verapamil slowly reduced the peak amplitude. These slow inhibitions by the latter two drugs depended on the frequency or number of stimulations. Nicardipine but not diltiazem and verapamil shifted the voltage-dependent inactivation curve to the left (3 s duration of the conditioning pulse). However, with a longer conditioning pulse (10 s) verapamil and diltiazem shifted the voltage-dependent inactivation curves to the left. Therefore, the inhibitory actions of these Ca²⁺ antagonists differ. Namely, diltiazem and verapamil inhibit the Ca²⁺ channels, mainly in a frequency-or use-dependent manner while nicardipine does so in a voltage-dependent manner.     

High-frequency fatigue of skeletal muscle: role of extracellular Ca2+

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.     

Changes in the structure and functions of membranes in erythrocytes and Ehrlich ascites carcinoma cells under the influence of a new generation hybrid antioxidant IKhFAN-10

We studied the effect of a new generation hybrid antioxidant IKhFAN-10 on the structure and function of cell membranes (organization of the lipid bilayer and proteins and activity of ion channels). The test preparation proposed for the therapy of neurodegenerative diseases modified properties of membranes in erythrocytes and Ehrlich ascites carcinoma cells and affected functional activity of cells. We determined the doses of this antioxidant, which did not cause side effects. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 402–406, April, 2007     

Influence of Ca2+-binding proteins and the cytoskeleton on Ca2+-dependent inactivation of high-voltage activated Ca2+ currents in thalamocortical relay neurons

Ca²⁺-dependent inactivation (CDI) of high-voltage activated (HVA) Ca²⁺ channels was investigated in acutely isolated and identified thalamocortical relay neurons of the dorsal lateral geniculate nucleus (dLGN) by combining electrophysiological and immunological techniques. The influence of Ca²⁺-binding proteins, calmodulin and the cytoskeleton on CDI was monitored using double-pulse protocols (a constant post-pulse applied shortly after the end of conditioning pre-pulses of increasing magnitude). Under control conditions the degree of inactivation (34±9%) revealed a U-shaped and a sigmoid dependency of the post-pulse current amplitude on pre-pulse voltage and charge influx, respectively. In contrast to a high concentration (5.5 mM) of EGTA (31±3%), a low concentration (3 µM) of parvalbumin (20±2%) and calbindin_D28K (24±4%) significantly reduced CDI. Subtype-specific Ca²⁺ channel blockers indicated that L-type, but not N-type Ca²⁺ channels are governed by CDI and modulated by Ca²⁺-binding proteins. These results point to the possibility that activity-dependent changes in the intracellular Ca²⁺-binding capacity can influence CDI substantially. Furthermore, calmodulin antagonists (phenoxybenzamine, 22±2%; calmodulin binding domain, 17±1%) and cytoskeleton stabilizers (taxol, 23±5%; phalloidin, 15±3%) reduced CDI. Taken together, these findings indicate the concurrent occurrence of different CDI mechanisms in a specific neuronal cell type, thereby supporting an integrated model of this feedback mechanism and adding further to the elucidation of the role of HVA Ca²⁺ channels in thalamic physiology.     

Ionic and osmotic dependence of secretion from permeabilised acini of the rat pancreas

Many types of secretory granule have been observed to swelll as a result of cell stimulation implying a degree of osmotic control, although the regulation of granule fusion with the apical plasma membrane is not clearly understood. In the present study we have investigated the ionic and osmotic dependency of basal and stimulated³H-protein release from rat pancreatic acini, permeabilised by either digitonin or high voltage electric discharge. Acini were stimulated with either cholecystokinin-pancreozymin octapeptide (CCK-8), carbachol (CCh), or with phorbol ester (TPA) plus cAMP. Stimulated secretion was significantly reduced when 130 mmol/l Cl^– in the buffer was replaced by I^–, NO ₃ ^– , SCN^– or cyclamate^–. Secretion in Cl^– buffers was inhibited by the anion transport inhibitor 4,4-diisothiocyanatostibene-2,2-disulfonic acid (DIDS), by 40% of the control response. Neither Na⁺ nor N-methyl-d-glucamine^– could replace K⁺ in the buffer. Ba²⁺ and quinine, which block K⁺ conductance pathways, inhibited stimulated secretion by 50%. Finally, stimulated secretion from leaky cells was nearly abolished by doubling buffer osmolarity. The data suggest that when the cell is stimulated, a Cl^– and a K⁺ permeability appear in the zymogen granule membrane and the ions enter down their electrochemical gradients. The increased intragranular osmolarity results in granular swelling which is intimately associated with secretion.Parts of this study have been previously presented at the XIXth Meeting of the European Pancreatic Club (1987) and at the 65th Meeting of the Deutsche Physiologische Gesellschaft (1988)