Blockade of HERG channels expressed in Xenopus laevis oocytes by external divalent cations

We have investigated actions of various divalent cations (Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Zn2+) on human ether-a-go-go related gene (HERG) channels expressed in Xenopus laevis oocytes using the voltage clamp technique. All divalent cations inhibited HERG current dose-dependently in a voltage-dependent manner. The concentration for half-maximum inhibition (Ki) decreased at more negative potentials, indicating block is facilitated by hyperpolarization. Ki at 0 mV for Zn2+, Ni2+, Co2+, Ba2+, Mn2+, and Sr2+ was 0.19, 0.36, 0. 50, 0.58, 2.36, and 6.47 mM, respectively. The effects were manifested in four ways: 1) right shift of voltage dependence of activation, 2) decrease of maximum conductance, 3) acceleration of current decay, and 4) slowing of activation. However, each parameter was not affected by each cation to the same extent. The potency for the shift of voltage dependence of activation was in the order Zn2+ > Ni2+ >/= Co2+ > Ba2+ > Mn2+ > Sr2+, whereas the potency for the decrease of maximum conductance was Zn2+ > Ba2+ > Sr2+ > Co2+ > Mn2+. The kinetics of activation and deactivation were also affected, but the two parameters are not affected to the same extent. Slowing of activation by Ba2+ was most distinct, causing a marked initial delay of current onset. From these results we concluded that HERG channels are nonselectively blocked by most divalent cations from the external side, and several different mechanism are involved in their actions. There exist at least two distinct binding sites for their action: one for the voltage-dependent effect and the other for reducing maximum conductance.     

Mechanisms underlying burst generation of the pyloric muscle in the mantis,shrimp, Squilla oratoria

The pyloric constrictor muscles of the stomach in Squilla can generate spikes by synaptic activation via the motor nerve from the stomatogastric ganglion. Spikes are followed by slow depolarizing afterpotentials (DAPs) which lead to sustained depolarization during a burst of spikes. 1. The frequency of rhythmic bursts induced by continuous depolarization is membrane voltage-dependent. A brief depolarizing or hyperpolarizing pulse can trigger or terminate bursts, respectively, in a threshold-dependent manner. 2. The conductance increases during the DAP response. The amplitude of DAP decreases by imposed depolarization, whereas it increases by hyperpolarization. DAPs from successive spikes sum to produce a sustained depolarizing potential capable of firing a burst. 3. The spike and DAP are reduced in amplitude by decreasing [Ca]o, enhanced by Sr2+ or Ba2+ substituted for Ca2+, and blocked by Co2+ or Mn2+. DAPs are selectively blocked by Ni2+, and the spike is followed by a hyperpolarizing afterpotential. 4. The spike and DAP are prolonged by intracellular injection of the Ca2+ chelator EGTA. A hyperpolarizing afterpotential is abolished by EGTA and enhanced by increasing [Ca]o. The DAP is diminished in Na(+)-free saline and reduced by tetrodotoxin. 5. It is concluded that the muscle fiber is endowed with endogenous oscillatory properties and that the oscillatory membrane events result from changes of a voltage- and time-dependent conductance to Ca2+ and Na+ and a Ca2+ activated conductance to K+.     

Effects of bivalent cations on post-rest adaptation in guinea-pig heart muscle

In isolated papillary muscles of guinea-pig hearts, the inotropic effects of bivalent cations, Ca2+, Ba2+, Sr2+, and Ni2+, were investigated during post-rest adaptation in order to study their individual action on excitation-contraction coupling. Upon exposure to each cation studied, the force of contraction was transiently enhanced, whereas the steady state force was influenced differently: it increased with Ca2+, Ba2+ and Sr2+ and was depressed by Ni2+. The transmembrane action potentials (measured at 90% repolarization) were slightly prolonged by Sr2+ and even more by Ba2+, and were shortened by Ca2+ and Ni2+. After 10 min rest, the post-rest contractions consisted of a late peak (PII) that was enhanced in high Ca2+-solution an by Sr2+. Ni2+ and Ba2+ depressed PII and during adaptation to pre-rest controls an early peak of contraction (PI) prevailed. There was no simple relation between post-rest adaptation of force and the duration of action potential in the presence of the bivalent cations tested. During post-rest adaptation the two components of contraction can be separated. The results are interpreted in terms of a model of excitation-contraction coupling which derives Ca ions for contractile activation from two sources: transmembrane calcium influx and calcium release from cellular stores. From the different effects on post-rest adaptation it is concluded that the individual cations influence excitation-contraction coupling more specifically and not merely by "screening-off" the negative surface charges.     

Selectivity of the Ca binding site in synaptosome Ca channels. Inhibition of Ca influx by multivalent metal cations

K-stimulated (voltage-dependent) influx of 45Ca was measured in synaptosomes (isolated presynaptic nerve terminals) from rat brain. Influx was terminated at 1 s with a rapid-filtration technique, so that most of the Ca uptake was mediated by inactivating ("fast") Ca channels (Nachshen, D. A., and Blaustein, M. P., 1980, J. Gen. Physiol., 76:709- 728). This influx was blocked by multivalent cations with half- inhibition constants (K1) that clustered in three distinct groups: (a) K1 greater than 1 mM (Mg2+, Sr2+, and Ba2+); (b) K1 = 30-100 microM (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Hg2+); (c) K1 less than 1 micro M (Cd2+, Y3+, La3+ and the trivalent lanthanides, and Pb2+). Most of these ions had very little effect on synaptosome steady state membrane potential, which was monitored with a voltage-sensitive fluorescent dye, or on the voltage dependence of Ca influx, which was assessed by measuring voltage-dependent Ca uptake at two levels of depolarization. The blockers inhibited Ca influx by competing with Ca for the channel site that is involved in the transport of divalent cations. Onset of fast channel inhibition by Mg, Co, Ni, Cu, Zn, Cd, La, Hg, and Pb was rapid, occurring within 1 s; inhibition was similar after 1 s or 30 min of exposure to these ions. The inhibition produced by Co, Cu, Zn, Cd, La, and Pb could be substantially reversed within 1 s by removing the inhibitory cation. The relative efficacies of the lanthanides as fast channel blockers were compared; there was a decrease in inhibitory potency with decreasing ionic radius. A model of the Ca channel binding site is considered, in which inhibitory polyvalent cation selectivity is determined primarily by coulombic interactions between the binding site and the different cations. The site is envisaged as consisting of two anions (radius 1 A) with a separation of 2 A between them. Small cations are unable to bind effectively to both anions. The selectivity sequences predicted for the alkaline earth cations, lanthanides, and transition metals are in substantial agreement with the selectivity sequences observed for inhibition of the fast Ca channel.     

Stimulation of non-selective cation channels providing Ca2+ influx into platelets by platelet-activating factor and other aggregation inducers.

To elucidate the mechanism of the receptor-stimulated Ca2+ entry into human platelets, the influence of Ca(2+)-mobilizing agonists on plasma membrane potential (Em) has been studied. Em changes were registered using potentiometric probe 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide. The agonist effect on Em varied from hyperpolarization to slight and slow rise. On the contrary, after loading of platelets with intracellular Ca2+ indicator quin2, platelet-activating factor (PAF), thrombin, vasopressin, ADP and thromboxane-A2-mimetic U46619 cause substantial transient membrane depolarization. Similar effects were observed after platelet loading with other Ca2+ chelators fura-2 and indo-1. Agonist-induced depolarization considerably reduced if quin2-loaded platelets were suspended in isoosmotic choline-containing medium. Using Ba2+ as a substitute of Ca2+, we have demonstrated that in choline-containing medium PAF-induced Ba2+ entry into platelets results in membrane depolarization. Dependence on Ba2+ concentration and depolarization kinetics correlates with the dose dependence and kinetics of Ba2+ entry detected by quin2 fluorescence. The agonists also stimulate considerable Na+, Li+ and Cs+ inward currents into platelets. Na(+)-dependent depolarization is 2-5-fold suppressed by extracellular Ca2+ [median inhibitory concentration (IC50) approximately 0.3 mM]. Ni2+ and Cd2+ at similar concentrations block Ca2+ entry and agonist-induced Na2+ current (IC50 for both cations approximately 50 microM). Agonist-induced depolarization is blocked by the adenylate cyclase stimulator prostaglandin E1 and the protein kinase C stimulator phorbol ester. It is concluded that agonists stimulate Ca2+ entry into human platelets via receptor-operated channels which are not strictly selective toward divalent cations and are permeable to Na+, Li+ and Cs+.     

Inhibitory neuromuscular transmission mediated by the P2Y1 purinergic receptor in guinea pig small intestine

ATP is a putative inhibitory neurotransmitter responsible for inhibitory junction potentials (IJPs) at neuromuscular junctions (IJPs) in the intestine. This study tested the hypothesis that the purinergic P2Y(1) receptor subtype mediates the IJPs. IJPs were evoked by focal electrical stimulation in the myenteric plexus and recorded with "sharp" intracellular microelectrodes in the circular muscle coat. Stimulation evoked three categories of IJPs: 1) purely purinergic IJPs, 2) partially purinergic IJPs, and 3) nonpurinergic IJPs. Purely purinergic IJPs were suppressed by the selective P2Y(1) purinergic receptor antagonist MRS2179. Purely purinergic IJPs comprised 26% of the IJPs. Partially purinergic IJPs (72% of the IJPs) consisted of a component that was abolished by MRS2179 and a second unaffected component. The MRS2179-insensitive component was suppressed or abolished by inhibition of formation of nitric oxide by N(omega)-nitro-l-arginine methyl ester (l-NAME) in some, but not all, IJPs. An unidentified neurotransmitter, different from nitric oxide, mediated the second component in these cases. Nonpurinergic IJPs were a small third category (4%) of IJPs that were abolished by l-NAME and unaffected by MRS2179. Exogenous application of ATP evoked IJP-like hyperpolarizing responses, which were blocked by MRS2179. Application of apamin, which suppresses opening of small-conductance Ca(2+)-operated K(+) channels in the muscle, decreased the amplitude of the purinergic IJPs and the amplitude of IJP-like responses to ATP. The results support ATP as a neurotransmitter for IJPs in the intestine and are consistent with the hypothesis that the P2Y(1) purinergic receptor subtype mediates the action of ATP.     

Leukotriene and purinergic receptors are involved in the hyperpolarizing effect of glucagon in liver cells

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K+ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba2+ (BaCl2, 5 mmol/l) and the blocker of the Na+/K+ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B4 receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 micromol/l). ATPgammaS (10 micromol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 micromol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPgammaS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane.     

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca²⁺-activated K⁺ channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current (I_VNSCC) and a relatively high-threshold voltage-activated (L-type) Ca²⁺ current (I_L). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect I_VNSCC or I_L in dialyzed cells. ATP (1 mM) increased I_VNSCC and decreased I_L in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on I_VNSCC. ADP decreased I_L but had no effect on I_VNSCC. The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on I_VNSCC. ATP stimulation of I_VNSCC was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y₄ is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates I_VNSCC and depresses I_L via binding of P2Y₄ receptors and stimulation of the phospholipase C/PKC pathway. inhibitory junction potentials; smooth muscle; enteric nervous system     

Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore

We studied the blocking actions of external Ca2+, Mg2+, Ca2+, and other multivalent ions on single Ca channel currents in cell-attached patch recordings from guinea pig ventricular cells. External Cd or Mg ions chopped long-lasting unitary Ba currents promoted by the Ca agonist Bay K 8644 into bursts of brief openings. The bursts appear to arise from discrete blocking and unblocking transitions. A simple reaction between a blocking ion and an open channel was suggested by the kinetics of the bursts: open and closed times within a burst were exponentially distributed, the blocking rate varied linearly with the concentration of blocking ion, and the unblocking rate was more or less independent of the blocker concentration. Other kinetic features suggested that both Cd2+ and Mg2+ lodge within the pore. The unblocking rate was speeded by membrane hyperpolarization or by raising the Ba concentration, as if blocking ions were swept into the myoplasm by the applied electric field or by repulsive interaction with Ba2+. Ca ions reduced the amplitude of unitary Ba currents (50% inhibition at approximately 10 mM [Ca]o with 50 mM [Ba]o) without detectable flicker, presumably because Ca ions exit the pore very rapidly following Ba entry. However, Ca2+ entry and exit rates could be resolved when micromolar Ca blocked unitary Li+ fluxes through the Ca channel. The blocking rate was essentially voltage independent, but varied linearly with Ca concentration (rate coefficient, 4.5 X 10(8) M-1s-1); evidently, the initial Ca2+-pore interaction is outside the membrane field and much faster than the overall process of Ca ion transfer. The unblocking rate did not vary with [Ca]o, but increased steeply with membrane hyperpolarization, as if blocking Ca ions were driven into the cell. We suggest that Ca is both an effective permeator and a potent blocker because it dehydrates rapidly (unlike Mg2+) and binds to the pore with appropriate affinity (unlike Cd2+). There appears to be no sharp dichotomy between "permeators" and "blockers," only quantitative differences in how quickly ions enter and leave the pore.     

Generation of nonadrenergic inhibitory junction potentials in the smooth muscle of the guinea-pig gastrointestinal tract after replacing potassium ions with cesium ions

Nonadrenergic inhibitory junction potentials (IJPs), evoked by intramural nerve stimulation, were studied in the smooth muscle of the guinea-pig stomach, cecum, and colon, using a modified sucrose-gap technique. After incubating smooth muscle preparations for 4–9 h in potassium-free Krebs solution, IJPs were abolished, but reappeared when cesium ions (6 mM) were added to the Krebs solution. Under these conditions, in the majority of cases the amplitude of the IJP was half as small, and the latency and duration were significantly longer, than in normal conditions; also ATP, but not adenosine, caused hyperpolarization of the smooth muscle membrane. The amplitude of the IJP depended on the extracellular concentration of cesium. In all types of preparation, in cesium-containing Krebs solution, apamin usually abolished the IJP and responses to ATP. These results are consonant with the purinergic hypothesis of inhibitory neuromuscular transmission. The generation of the IJP in these potassium-free conditions depends on cesium ions, which pass through the small-conductance apamin-sensitive, calcium-dependent potassium channels.A. A. Bogomoletz Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 634–641, September–October, 1990.     

Properties of receptors for the Ca2+-channel blocker verapamil in transverse-tubule membranes of skeletal muscle. Stereospecificity, effect of Ca2+ and other inorganic cations, evidence for two categories of sites and effect of nucleoside triphosphates

The verapamil receptor associated with the voltage-dependent calcium channel of rabbit skeletal muscle transverse tubule membranes has the following properties. (i) This receptor is stereospecific and discriminates between the different stereoisomers of verapamil, gallopamil and diltiazem. (ii) Inorganic divalent cations inhibit the binding of [3H]verapamil to its receptor in an apparently non-competitive fashion. The rank order of potency is: Ca2+ = Mn2+ greater than Mg2+ greater than Sr2+ greater than Ba2+ much greater than Co2+ much greater than Ni2+. Ca2+ and Mn2+ have inhibition constants of 0.3 mM. Binding of [3H]verapamil is also sensitive to monovalent cations such as Cs+, K+, Li+ and Na+. The most active of these cations (Cs+ and K+) have inhibition constants in the range of 30 mM. (iii) Binding of [3H]verapamil is pH-dependent and reveals the presence on the verapamil receptor of an essential ionizable group with a pKa of 6.5. (iv) A low-affinity binding site for verapamil and for some other Ca2+ channel blockers is detected by studies of dissociation kinetics of the [3H]verapamil receptor in the presence of high concentrations of verapamil, gallopamil, bepridil and diltiazem. (v) GTP and nucleoside analogs change the properties of [3H]verapamil binding to verapamil binding sites. High-affinity binding sites seem to be transferred into low-affinity sites. Dissociation constants obtained from inhibition studies of [3H]verapamil binding are in the range of 0.1-0.3 mM for GTP, ATP and Gpp(NH)p.     

Voltage-dependent calcium channels in pituitary cells in culture. II. Participation in thyrotropin-releasing hormone action on prolactin release

Depolarization of membrane potential by high external K+ activates Ca2+ influx via voltage-dependent Ca2+ channels in GH4C1 cells (Tan, K.-N., and Tashjian, A. H., Jr. (1983) J. Biol. Chem. 258, 418-426). The involvement of this channel in thyrotropin-releasing hormone (TRH) action on prolactin (PRL) release was assessed by comparing the pharmacological characteristics of TRH-induced PRL release with PRL release due to high K+. Two components of TRH-stimulated PRL release were detected. The major component (approximately equal to 75%) was dependent on external Ca2+ concentration and was inhibited by voltage-dependent Ca2+ channel blockers in a manner quantitatively similar to high K+-stimulated PRL release. The minor component (approximately equal to 25%) of TRH-stimulated PRL release was insensitive to voltage-dependent Ca2+ channel blockers and could occur in the presence of low external Ca2+ (10(-5)-10(-7) M). Neither voltage-dependent Ca2+ channel blockers nor depletion of medium Ca2+ prevented the action of TRH on mobilizing cell-associated 45Ca2+ from GH4C1 cells. Divalent cations that permeate voltage-dependent Ca2+ channels (Sr2+ and Ba2+) substituted for Ca2+ in supporting high K+- and TRH-stimulated PRL release while Mg2+, a nonpermeant cation, did not. We conclude that TRH stimulates PRL release by increasing [Ca2+]i through at least two mechanisms: one requires only low [Ca2+]o, the second involves Ca2+ influx via voltage-dependent Ca2+ channels. This latter mechanism accounts for approximately equal to 75% of maximum TRH-induced PRL release.     

The role of ATP and divalent cations in the regulation of a cardiac phosphorylase phosphatase (phosphoprotein phosphatase) of Mr = 35,000.

The effects of ATP and divalent cations on a divalent cation-independent phosphorylase phosphatase of Mr = 35,000 (phosphatase S) purified from canine cardiac muscle have been studied. The enzyme can be rapidly inactivated by ATP or other nucleoside di- and triphosphates and PPi, but not by AMP, adenosine, adenine, Pi, EDTA, ethylene glycol bis(beta-aminoethyl ether)N,N' -tetraacetic acid, 1,10-phenanthroline, or 8-hydroxyquinoline. After removing the inactivating agent, such as ATP or PPi, by gel filtraiton followed by exhaustive dialysis, the inactivated enzyme (apophosphatase S) can be reactivated by preincubating with Mn2+ or Co2+, but not with Mg2+, Ca2+, Ni2+, Zn2+, Fe2+, Cu2+, Ba2+, Hg2+, Pb2+, or Cd2+. The Mn2+ -reactivated enzyme, which is less active than the Co2+ -reactivated enzyme, can be again inactivated by preincubating with ATP. The present findings indicate that phosphatase S contains a tightly bound divalent cation, probably Mn2+, in the active site. ATP and PPi, due to their structural similarity to the phosphoprotein substrate and their ability to chelate metal ions, can readily enter the active site to remove the divalent cation(s) essential for the catalytic function. The present findings also indicate that phosphatase S, a common catalytic subunit of several larger molecular forms of nospecific phosphoprotein phosphatase in cardiac muscle, can exist in two interconvertible forms, a metallized form (active) and a demetallized form (inactive). ATP and metal ions may regulate this class of isozymes by mediating the interconversions.     

Electrophysiology of phagocytic membranes. Role of divalent cations in membrane hyperpolarizations of macrophage polykaryons

The electrophysiological properties of the membrane of mouse peritoneal macrophage polykaryons are studied. Slow hyperpolarizations can be elicited by iontophoretic injections of either Ca2+ or Sr2+ into the cytoplasm. The effect of both cations is identical, since: it is invariably triggered by the cation injection, the amplitude is dependent on the K+ gradient, quinine blocks reversibly the response to both cation injections. Mg2+, Ba2+ and Mn2+ did not elicit responses when injected into the cytoplasm. Ca2+ induced slow hyperpolarizations were reversibly blocked by the addition of Ba2+ to the external saline, but were not affected by the presence of external tetraethylammonium chloride. Cells maintained in saline containing high concentrations of Ca2+, Sr2+ or Mn2+ exhibited sustained hyperpolarizations. Quinine blocked the hyperpolarization induced by high Ca2+ or Sr2+, but was ineffective for the case of Mn2+. Cells hyperpolarized by external Mn2+ frequently exhibited nonlinear, voltage-current characteristics. Similar patterns could also be observed in a small fraction (less than 10%) of the cells in control conditions. Current-induced shifts between two stable membrane potentials were seen either in high Ca2+ or normal medium. The great variability of the responses described for this phagocytic membrane is discussed. The evidence supports the assumption that Ca2+ and Sr2+ can induce transient or persistent hyperpolarized states by activating a potassium permeability. External Mn2+ may act in part by reducing impalement-related current leakage from the phagocytic membrane.     

Characterization of a Ca2+ uniporter from Bacillus subtilis by partial purification and reconstitution into phospholipid vesicles

Ca2+ was accumulated by right-side-out membrane vesicles of Bacillus subtilis following imposition of a diffusion potential, inside-negative, owing to K+-efflux via valinomycin. Uptake was dependent on the magnitude of the membrane potential. This voltage-dependent Ca2+ uptake was inhibited by Ca2+ channel blockers such as nitrendipine, verapamil and LaCl3, and was competitively inhibited by Ba2+ and Sr2+. The system showed saturation kinetics with an apparent Km for Ca2+ of about 250 microM. Proteins responsible for the voltage-dependent Ca2+ uptake were partially purified by preparative isoelectric focusing in a Sepharose bed. A fraction at pH 5.28-5.33 contained the activity. The characteristics of Ca2+ uptake in reconstituted proteoliposomes were the same as those in membrane vesicles (sensitive to Ca2+ channel blockers; inhibited by Ba2+ and Sr2+). In addition, uptake was not influenced by a pH gradient imposed on the vesicles. The apparent Km for Ca2+ in the reconstituted system was about 260 microM. The specific activity was increased about 50-fold by purification with isoelectric focusing.     

Voltage-dependent Ba2+ permeation through store-operated CRAC channels: implications for channel selectivity

Store-operated Ca2+ entry through CRAC channels is a major route for Ca2+ influx in non-excitable cells. Studies on store-operated channel selectivity using fluorescent dyes have found that the channels are impermeable to Ba2+. Furthermore, in such studies, agonists have been reported to increase Ba2+ influx, leading to the conclusion that additional Ca2+ entry pathways (permeable to Ba2+) co-exist with the Ba2+-impermeable store-operated channels. However, patch clamp experiments demonstrate that CRAC channels are permeable to Ba2+. We have addressed this paradox using fluorescence measurements and whole cell patch clamp recordings of ICRAC. In store-depleted cells loaded with fura 2, Ba2+ application results in a slower and smaller rise in fluorescence than is the case with Ca2+. Ba2+, unlike Ca2+, depolarises the membrane potential by approximately 40 mV, due to rapid block of an inwardly rectifying K+ current. Although Ba2+ permeates CRAC channels at very negative potentials in patch clamp recordings, Ba2+ permeation is steeply voltage-dependent. This combination of Ba2+-dependent depolarisation and voltage-dependent Ba2+ permeation accounts for the apparent lack of Ba2+ permeation through store-operated channels seen in fluorescence experiments. Our findings identify major limitations with the use of Ba2+ as a surrogate for Ca2+ in probing Ca2+ entry pathways and raise the possibility that some of the previous reports proposing multiple Ca2+ entry pathways based on Ba2+ entry into fura 2-loaded cells could be explained by voltage-dependent Ba2+ permeation through CRAC channels.     

Sodium-pump potentials and currents in guinea-pig ventricular muscles and myocytes.

When guinea-pig papillary muscles were depolarized to ca. -30 mV by superfusion with K+-free Tyrode's solution supplemented with Ba2+, Ni2+, and D600, addition of Cs+ transiently hyperpolarized the membrane in a reproducible manner. The size of the hyperpolarization (pump potential) depended on the duration of the preceding K+-free exposure; peak amplitudes (Epmax) elicited by 10 mM Cs+ after 5-, 10-, and 15-min K+-free exposures were 12.9, 17.7, and 23.2 mV, respectively. Pump potentials were unaffected by external Cl- but suppressed by cardiac glycosides, hyperosmotic conditions, and low-Na+ solution. Using Epmax as an indicator of Na+ pump activation, the half-maximal concentration for activation by Cs+ was 12-16.3 mM. At 6 mM, Cs+ was three times less potent than Rb+ or K+ and five times more potent than Li+. From these findings, and correlative voltage-clamp data from myocytes, we calculate that (i) a pump current of 7.8 nA/cm2 generates an Epmax of 1 mV and (ii) resting pump current in normally polarized muscle (approximately 0.16 microA/cm2) is five times smaller than previously estimated.