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1.
UV irradiation has multiple effects on mammalian cells, including modification of ion channel function. The present study was undertaken to investigate the response of membrane currents in guinea-pig ventricular myocytes to the type A (355, 380 nm) irradiation commonly used in Ca2+ imaging studies. Myocytes configured for whole-cell voltage clamp were generally held at −80 mV, dialyzed with K+-, Na+-free pipette solution, and bathed with K+-free Tyrode’s solution at 22°C. During experiments that lasted for ≈ 35 min, UVA irradiation caused a progressive increase in slowly-inactivating inward current elicited by 200-ms depolarizations from −80 to −40 mV, but had little effect on background current or on L-type Ca2+ current. Trials with depolarized holding potential, Ca2+ channel blockers, and tetrodotoxin (TTX) established that the current induced by irradiation was late (slowly-inactivating) Na+ current (INa). The amplitude of the late inward current sensitive to 100 μM TTX was increased by 3.5-fold after 20–30 min of irradiation. UVA modulation of late INa may (i) interfere with imaging studies, and (ii) provide a paradigm for investigation of intracellular factors likely to influence slow inactivation of cardiac INa.  相似文献   

2.
The Ca2+-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs2+-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca2+ with Ba2+ slowed the current decay. Increasing the external Ca2+ or Ba2+ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na+ in the absence of extracellular Ca2+. Current carried by Na+ (I Na) was almost completely blocked by the dihydropyridine Ca2+ channel antagonist, nifedipine, suggesting that the I Na is through voltage-dependent L-type Ca2+ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs+-aspartate and 140 mM Na+-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na+ was replaced with an equimolar amount of K+ or Cs+, or 50 mM Ca2+ or Ba2+. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd3+ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca2+-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca2+ channel and stretch-activated Ca2+-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I Ca Ca2+ current - I Na Na+ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier  相似文献   

3.
Freshly dissociated myocytes from nonpregnant, pregnant, and postpartum rat uteri have been studied with the tight-seal patch-clamp method. The inward current contains both INa and ICa that are vastly different from those in tissue-cultured material. INa is abolished by Na+-free medium and by 1 μM tetrodotoxin. It first appears at ∼−40 mV, reaches maximum at 0 mV, and reverses at 84 mV. It activates with a voltage-dependent τ of 0.2 ms at 20 mV, and inactivates as a single exponential with a τ of 0.4 ms. Na+ conductance is half activated at −21.5 mV, and half inactivated at −59 mV. INa reactivates with a τ of 20 ms. ICa is abolished by Ca2+-free medium, Co2+ (5 mM), or nisoldipine (2 μM), and enhanced in 30 mM Ca2+, Ba2+, or BAY-K 8644. It first appears at ∼−30 mV and reaches maximum at +10 mV. It activates with a voltage-dependent τ of 1.5 ms at 20 mV, and inactivates in two exponential phases, with τ''s of 33 and 133 ms. Ca2+ conductance is half activated at −7.4 mV, and half inactivated at −34 mV. ICa reactivates with τ''s of 27 and 374 ms. INa and ICa are seen in myocytes from nonpregnant estrus uteri and throughout pregnancy, exhibiting complex changes. The ratio of densities of peak INa/ICa changes from 0.5 in the nonpregnant state to 1.6 at term. The enhanced role of INa, with faster kinetics, allows more frequent repetitive spike discharges to facilitate simultaneous excitation of the parturient uterus. In postpartum, both currents decrease markedly, with INa vanishing from most myocytes. Estrogen-enhanced genomic influences may account for the emergence of INa, and increased densities of INa and ICa as pregnancy progresses. Other influences may regulate varied channel expression at different stages of pregnancy.  相似文献   

4.
We studied the peculiarities of permeability with respect to the main extracellular cations, Na+ and Ca2+, of cloned low-threshold calcium channels (LTCCs) of three subtypes, Cav3.1 (α1G), Cav3.2 (α 1H), and Cav3.3 (α1I), functionally expressed in Xenopus oocytes. In a calcium-free solution containing 100 mM Na+ and 5 mM calcium-chelating EGTA buffer (to eliminate residual concentrations of Ca2+) we observed considerable integral currents possessing the kinetics of inactivation typical of LTCCs and characterized by reversion potentials of −10 ± 1, −12 ± 1, and −18 ± 2 mV, respectively, for Cav3.1, Cav3.2, and Cav3.3 channels. The presence of Ca2+ in the extracellular solution exerted an ambiguous effect on the examined currents. On the one hand, Ca2+ effectively blocked the current of monovalent cations through cloned LTCCs (K d = 2, 10, and 18 μM for currents through channels Cav3.1, Cav3.2, and Cav3.3, respectively). On the other hand, at the concentration of 1 to 100 mM, Ca2+ itself functioned as a carrier of the inward current. Despite the fact that the calcium current reached the level of saturation in the presence of 5 mM Ca2+ in the external solution, extracellular Na+ influenced the permeability of these channels even in the presence of 10 mM Ca2+. The Cav3.3 channels were more permeable with respect to Na+ (P Ca/P Na ∼ 21) than Cav3.1 and Cav3.2 (P Ca/P Na ∼ 66). As a whole, our data indicate that cloned LTCCs form multi-ion Ca2+-selective pores, as these ions possess a high affinity for certain binding sites. Monovalent cations present together with Ca2+ in the external solution modulate the calcium permeability of these channels. Among the above-mentioned subtypes, Cav3.3 channels show the minimum selectivity with respect to Ca2+ and are most permeable for monovalent cations. Neirofiziologiya/Neurophysiology, Vol. 38, No. 3, pp. 183–192, May–June, 2006.  相似文献   

5.
K+-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring 86Rb efflux. In crypt cells, basal K+ efflux (rate constant: 0.24 ± 0.044 min−1, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba2+ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K+ efflux, increasing the rate constant to 0.65 ± 0.007 min−1 and the span to 83 ± 3.2%. Ionomycin-induced K+ efflux was inhibited by clotrimazole (IC50 of 25 ± 0.4 μM) and charybdotoxin (IC50 of 65 ± 5.0 nM) and was insensitive to TEA, Ba2+, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca2+-activated intermediate-conductance K+ channels (IKca). Absence of extracellular Ca2+ did neither affect basal nor ionomycin-induced K+ efflux. However, intracellular Ca2+ depletion totally inhibited the ionomycin-induced K+ efflux, indicating that the activation of these K+ channels mainly depends on intracellular calcium liberation. K+ efflux was stimulated by intracellular Ca2+ with an EC50 of 1.1 ± 0.04 μM. In surface cells, K+ efflux (rate constant: 0.17 ± 0.027 min−1; span: 25 ± 3.4%) was insensitive to TEA and Ba2+. However, ionomycin induced K+ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IKCa channels but only crypt cells have TEA- and Ba2+-sensitive conductive pathways, which would determine their participation in colonic K+ secretion.  相似文献   

6.
Low voltage-activated, rapidly inactivating T-type Ca2+ channels are found in a variety of cells, where they regulate electrical activity and Ca2+ entry. In whole-cell patch-clamp recordings from mouse spermatogenic cells, trace element copper (Cu2+) inhibited T-type Ca2+ current (I T-Ca) with IC50 of 12.06 μM. Inhibition of I T-Ca by Cu2+ was concentration-dependent and mildly voltage-dependent. When voltage stepped to −20 mV, Cu2+ (10 μM) inhibited I T-Ca by 49.6 ± 4.1%. Inhibition of I T-Ca by Cu2+ was accompanied by a shift of −2.23 mV in the voltage dependence of steady-state inactivation. Cu2+ upshifted the current–voltage (I-V) curve. To know the change of the gating kinetics of T-type Ca2+ channels, we analyzed the effect of Cu2+ on activation, inactivation, deactivation and reactivation of T-type Ca2+ channels. Since T-type Ca2+ channels are a key component in capacitation and the acrosome reaction, our data suggest that Cu2+ can affect male reproductive function through T-type Ca2+ channels as a preconception contraceptive material.  相似文献   

7.
Summary Smooth muscle cells normally do not possess fast Na2+ channels, but inward current is carried through two types of Ca2+ channels: slow (L-type) Ca2+ channels and fast (T-type) Ca2+ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pusles, applied from a holding potential of –90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]0, and was inhibited by TTX (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]0, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na2+ channel current, and that the slow inward current is a Ca2+ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihudropuridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation [9]. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na2+ channels, and it suggested that the fast Na+ current may be involved in spread of excitation. The Ca2+ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 of 12 mM) and nifedipine (K0.5 of 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extect. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of ICa(s).  相似文献   

8.
Excitatory effect of ATP on rat area postrema neurons   总被引:1,自引:0,他引:1  
ATP-induced inward currents and increases in the cytosolic Ca2+ concentration ([Ca]in) were investigated in neurons acutely dissociated from rat area postrema using whole-cell patch-clamp recordings and fura-2 microfluorometry, respectively. The ATP-induced current (I ATP) and [Ca]in increases were mimicked by 2-methylthio-ATP and ATP-γS, and were inhibited by P2X receptor (P2XR) antagonists. The current–voltage relationship of the I ATP exhibited a strong inward rectification, and the amplitude of the I ATP was concentration-dependent. The I ATP was markedly reduced in the absence of external Na+, and the addition of Ca2+ to Na+-free saline increased the I ATP. ATP did not increase [Ca]in in the absence of external Ca2+, and Ca2+ channel antagonists partially inhibited the ATP-induced [Ca]in increase, indicating that ATP increases [Ca]in by Ca2+ influx through both P2XR channels and voltage-dependent Ca2+ channels. There was a negative interaction between P2XR- and nicotinic ACh receptor (nAChR)-channels, which depended on the amplitude and direction of current flow through either channel. Current occlusion was observed at V hs between −70 and −10 mV when the I ATP and ACh-induced current (I ACh) were inward, but no occlusion was observed when these currents were outward at a V h of +40 mV. The I ATP was not inhibited by co-application of ACh when the I ACh was markedly decreased either by removal of permeant cations, by setting V h close to the equilibrium potential of I ACh, or by the addition of d-tubocurarine or serotonin. These results suggest that the inhibitory interaction is attributable to inward current flow of cations through the activated P2XR- and nAChR-channels.  相似文献   

9.
With the help of a standard voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In Ca2+-dependent K+ current, we identified and studied the properties of an apamin-sensitive voltage-independent component carried through the channels of low conductance (in many publications called small conductance,I SK(Ca)). This component did not show the temporal inactivation;I SK(Ca) was insensitive to the action of 4 mM tetraethylammonium, but was completely blocked by 500 nM of apamin. It was shown thatI SK(Ca) is very sensitive to changes in the intracellular Ca2+ concentration ([Ca2+] i ): a decrease in [Ca2+] i up to 50 nM resulted in the almost complete blockade of the current. The entry of Ca ions into a cell from the external solution through the voltage-operated Ca2+ channels of L-type was not an obligatory condition for activation ofI SK(Ca). The current-voltage relationship forI SK(Ca) had a maximum within the voltage range of +20 to +50 mV. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 87–94, March–April, 2000.  相似文献   

10.
Summary T-type calcium channels (I T channels) were studied in cell-attached patch electrode recordings from the ventricular cell membrane of 14-day embryonic chick heart. All experiments were performed in the absence of Ca2+ with Na+ (120mm) as the charge carrier.I T channels were distinguished from L-type calcium channels (I L) by their more negative activation and inactivation potential ranges; their smaller unitary slope conductance (26 pS), and their insensitivity to isoproterenol or D600. Inactivation kinetics were voltage dependent. The time constant of inactivation was 37 msec when the membrane potential was depolarized 40 mV from rest (R+40 mV), and 20 msec atR+60 mV. The frequency histogram of channel open times 0 was fit by a single-exponential curve while that of closed times c was biexponeintial. o was the same atR+40 mV andR+60 mV whereas c was shortened atR+60 mV. The open-state probability (P o) increased with depolarization: 0.35 atR+40 mV, 0.8 atR+60 mV and 0.88 atR+80 mV. This increase inP o at depolarized potentials could be accounted for by the decrease in c.  相似文献   

11.
Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K+ channels combining high conductance (170 pS) with high selectivity for K+, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K+>Rb+>NH 4 + Na+ and Cl. Divalent cytosolic ions reduce the K+ conductance of this channel and alter its K+ gating in a voltage-dependent manner. The order of blocking potency is Ba2+>Sr2+>Ca2+>Mg2+. The channel is activated by micromolar cytosolic Ca2+, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca2+ needed to open the channel. The possible role of the Ca2+-activated K+ channel in the tonoplast ofChara is discussed.  相似文献   

12.
Summary The relative contributions of the Na+/Ca2+ exchange and the plasma membrane Ca2+ pump to active Ca2+ efflux from stimulated rat pancreatic acini were studied. Na+ gradients across the plasma membrane were manipulated by loading the cells with Na+ or suspending the cells in Na+-free media. The rates of Ca2+ efflux were estimated from measurements of [Ca2+] i using the Ca2+-sensitive fluorescent dye Fura 2 and45Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca2+ efflux is described by a single exponential function under control, Na+-loaded, and Na+-depleted conditions. Manipulation of Na+ gradients had no effect on the hormone-induced increase in [Ca2+] i . The results indicate that Ca2+ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca2+ pump. The effects of several cations, which were used to substitute for Na+, on cellular activity were also studied. Choline+ and tetramethylammonium+ (TMA+) released Ca2+ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na+, TMA+, choline+, or N-methyl-d-glucamine+ (NMG+) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG+, choline+, and TMA+ inhibited amylase release by 55, 40 and 14%, respectively. NMG+ also increased the Ca2+ permeability of the plasma membrane. Thus, to study Na+ dependency of cellular function, TMA+ is the preferred cation to substitute for Na+. The stimulatory effect of TMA+ can be blocked by atropine.  相似文献   

13.
The Ca2+-extruding ATPase pump of the human platelet was studiedin situ by measuring Ca2+ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988.J. Membrane Biol. 104:147–163). Cytoplasmic pH (pHcyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca2+ to 2.5 μM and monitoring active Ca2+ extrusion into a Ca2+-free medium. The pump was shown to perturb pHcyt, to not respond to changes in membrane potential and to respond to imposed changes in pHcyt in a manner consistent with the Ca2+ pump acting as a 2 Ca2+/nH+ exchanger. (i) Raising the external pH (pHext) from 7.40 to 7.60 lowers the Vmax of the pump in basal condition (Vmax,1) from 110±18 to 73±12 μM/min (=μmol/liter cell volume/min). (ii) Lowering pHext to 7.13 raised Vmax,1 to 150±15 μM/min. (iii) In an N-methyl-d-glucamine (NMDG+) medium, the pump operation against high [Ca2+]cyt acidifies the cytoplasm by −0.36±0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pHcyt down to 6.23 reduces the Vmax,1 to 18±11 μM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na+ raises the Vmax,1 (basal state withK m,1=80 nM) to 136±24 μM/min, but also activates the pump fourfold (Vmax,2=280±28 μM/min;K m,2=502±36 nM). (vi) Transient elevation of pHcyt by NH4Cl at high [Ca2+]cyt activates the pump eightfold (Vmax,2≥671±350 μM/min). The large activation by alkaline pHcyt at high [Ca2+]cyt can be explained by Ca2+-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992.J. Membrane Biol. 130:63–82) and by increased Ca2+ affinity of calmodulin at high pH.  相似文献   

14.
Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate that luminal and basolateral ATP via P2Y2 receptors stimulate luminal Ca2+-activated Cl channels and inhibit Na+ transport. Here we address the mechanism of ATP-mediated inhibition of Na+ transport. M-1 cells had a transepithelial voltage (V te ) of −31.4 ± 1.3 mV and a transepithelial resistance (R te ) of 1151 ± 28 Ωcm2. The amiloride-sensitive short circuit current (I sc ) was −28.0 ± 1.1 μA/cm2. The ATP-mediated activation of Cl channels was inhibited when cytosolic Ca2+ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca2+]i increase was paralleled by a rapid and transient R te decrease (297 ± 51 Ωcm2). In the presence of CPA, basolateral ATP led to an R te increase by 144 ± 17 Ωcm2 and decreased V te from −31 ± 2.6 to −26.6 ± 2.5 mV. I sc dropped from −28.6 ± 2.4 to −21.6 ± 1.9 μA/cm2. Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated inhibition of Na+ absorption. Lowering [Ca2+]i by removal of extracellular Ca2+ did not alter the ATP effect. PKC inhibition or activation were without effect. Na+ absorption was activated by pHi alkalinization and inhibited by pHi acidification. ATP slightly acidified M-1 cells by 0.05 ± 0.005 pH units, quantitatively not explaining the ATP-induced effect. In summary this indicates that extracellular ATP via luminal and basolateral P2Y2 receptors inhibits Na+ absorption. This effect is not mediated via [Ca2+]i, does not involve PKC and is to a small part mediated via intracellular acidification. Received: 9 February 2001/Revised: 17 May 2001  相似文献   

15.
Membrane potential changes accompanying Ca2+ influx stimulated by release of Ca2+ from intracellular stores (store-regulated Ca2+ uptake) were monitored in BAPTA-loaded rat thymic lymphocytes using the fluorescent indicator bis(1,3-diethylthiobarbituric acid)trimethine oxonol. Depletion of [Ca2+] i stores by the application of thapsigargin, ionomycin or cyclopiazonic acid induced a depolarization which was (i) dependent upon BAPTA-loading, (ii) dependent upon extracellular Ca2+, (iii) independent of extracellular Na+ and (iv) abolished by 5 mm extracellular Ni2+. This depolarization was followed by a charybdotoxin-sensitive repolarization and subsequent hyperpolarization to values approximating the K+ equilibrium potential, consistent with secondary activation of a K+ conductance. These membrane potential changes temporally correlated with Ca2+ influx from the extracellular medium as measured fluorimetrically with indo-1. The divalent cation permeability sequence was investigated by monitoring the magnitude of the depolarization observed following the addition of 4 mm Ca2+, Mn2+, Ba2+ or Sr2+ to cells pretreated with doses of thapsigargin or ionomycin known to activate the store-regulated calcium uptake pathway. On the basis of these experiments, we conclude that the store-regulated Ca2+ uptake pathway has the following permeability sequence: Ca2+ > Mn2+ Ba2+, Sr2+ with Mn2+ displaying significant permeability relative to Ca2+. This pathway is distinguishable from other divalent cation uptake pathways reported in other cells types on the basis of its activation by thapsigargin and its high Mn2+ permeability.This work is supported by grants from the American Heart Association, Louisiana Affiliate (LA-92-6-28), Louisiana Education Quality Support Fund (LEQSF(1993-96)-RD-A-31) and Tulane University Graduate Program in Molecular and Cellular Biology.  相似文献   

16.
Many organic anions bind free Ca2+, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (Kapp) describing the Ca2+ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca2+ electrodes coupled to either a 3 M KCl or a Na+ selective electrode were used to redetermine Kapp. All solutions contained 130 mM Na+, whereas the concentration of the studied anion was varied from 15 to 120 mM, replacing Cl that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3 M KCl reference electrode, leading to a systematic underestimation of Kapp if left uncorrected. Because the Na+ concentration in all solutions was constant, a Na+ electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5 mV but twice those predicted by the Henderson equation. Determination of Kapp either after correction for these LJP changes or via direct reference to a Na+ electrode showed that SCFAs do not bind Ca2+ and that the Kapp for the binding of Ca2+ to gluconate at pH 7.4, ionic strength 0.15 M, and 23 °C was 52.7 mM.  相似文献   

17.
The cytoplasmic Ca2+ concentration ([Ca2+]cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca2+ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca2+]cyt. We measured changes of [Ca2+]cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca2+] from 1.3 mM to 5 mM induced an incrase in [Ca2+]cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd3+, 10 μM La3+ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca2+ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca2+) resulted in a decrease of [Ca2+]cyt from 105±10 nM to 88.5±10 nM with La3+, from 103±12 to 89±12 nM with Gd3+ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca2+ entry beside its function in Ca2+ signaling contributes to the regulation of resting [Ca2+]cyt.  相似文献   

18.
The epithelial Na+ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na+ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na+ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na+ conductance was Li+ > Na+ >> K+ = N-methyl-d-glucamine+ (NMDG+). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na+ channel current, was found to be ∼5 and 8 pS when Na+ and Li+ were used as a charge carrier, respectively. K+ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P o), was increased by membrane hyperpolarization. Both whole-cell Na+ current and conductance were saturated with increased extracellular Na+ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP o) was significantly decreased when cytosolic Na+ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na+ conductance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca2+ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca2+ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca2+ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na+ and Ca2+.  相似文献   

19.
To clarify the transmural heterogeneity of action potential (AP) time course, we examined the regulation of L-type Ca2+ current (ICa,L) by voltage and Ca2+-dependent mechanisms. Currents were recorded using patch clamp of single rat subepicardial (EPI) and subendocardial (ENDO) of left ventricular, right ventricular (RV) and septal (SEP) cardiomyocytes. Voltage clamp commands were derived from ENDO and EPI APs or rectangular voltage pulses.During rectangular pulses, peak ICa,L was significantly greater in EPI than in other cells. The inactivation of ICa,L by Ca2+-dependent mechanisms (suppressed by ryanodine and BAPTA) was present in all cells but greater in extent in ENDO and SEP cells. Activation and inactivation curves for all regions show subtle differences that are Ca2+ sensitive, with Ca2+ inactivation shifting the activation variables negative by ∼ 7 mV and inactivation variables positive by 2-7 mV (EPI being least, RV greatest). In AP-clamps, the peak ICa,L was significantly smaller in ENDO than in EPI cells, while the integrated current was significantly larger in ENDO than in EPI cells. The results are discussed with regard to the interplay of AP time course and net Ca2+ influx.  相似文献   

20.
The effect of amidiniums on high-threshold Ca2+ channel currents (I Ca) was studied in chick dorsal root ganglion neurons. Guanidinium reduced I Ca in a dose-dependent fashion. The block was relieved by increasing the concentration of the permeant ions, Ba2+ or Ca2+, suggesting a competition for a common binding site within the channel. Formamidinium and methyl-guanidinium suppressed I Ca with similar potencies, whereas l-arginine had no effect. A neutral amidine, urea, increased I Ca. In Ca2+-free solutions guanidinium and Na+ permeated through the Ca2+ channel equally well. Structure-activity relationship obtained for blocking efficacies of different amidiniums are used to discuss possible configurations of the selectivity filter in the Ca2+ channel.The author wishes to thank Ms. S. Engers for the cell isolation and making of the electrodes.  相似文献   

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