The role of calmodulin on Ca2+ -dependent K+ transport regulation in the human red cell

Several lipophilic calmodulin antagonists (phenotiazines, butyrophenones and diphenylbutylpiperidines) inhibited Ca2+-induced loss of KC1 from human red cells. However, the Ki values for this effect did not bear good correlation with the Ki values reported for well-known calmodulin-dependent systems. In addition, the inhibition was strongly dependent on the haematocrit and valinomycin-induced KC1 fluxes were also affected. Added calmodulin did not have any effect on Ca2+-dependent 86Rb uptake by inside-out vesicles derived from red cell membranes whereas stimulation of Ca2+-dependent ATPase was apparent. Lipophilic anticalmodulins at high doses had all kinds of effects on 86Rb uptake by inside-out vesicles: increase, decrease or no change of the fraction of activated vesicles reached at submaximal Ca2+ concentrations, with or without modification of the relative rate of 86Rb uptake. The hydrophylic compound 48/80 decreased the fraction of activated vesicles reached at submaximal Ca2+ concentrations without affecting the relative rate of 86Rb uptake, but this effect took place only at concentrations 10-fold higher than the reported Ki for calmodulin-dependent systems. These results suggest that Ca2+-dependent K+ channels of red cells are not regulated by calmodulin.     

Calcium-dependent,swelling-activated K+ conductance in human neuroblastoma cells

In most mammalian cells, regulatory volume decrease (RVD) is mediated by swelling-activated Cl(-) and K(+) channels. Previous studies in the human neuroblastoma cell line CHP-100 have demonstrated that exposure to hypoosmotic solutions activates Cl(-) channels which are sensitive to Ca(2+). Whether a Ca(2+)-dependent K(+) conductance is activated after cell swelling was investigated in the present studies. Reducing the extracellular osmolarity from 290 to 190 mOsm/kg H(2)O rapidly activated 86Rb effluxes. Hypoosmotic stress also increased cytosolic Ca(2+) in fura-2 loaded cells. Pretreatment with 2.5 mM EGTA and nominally Ca(2+) free extracellular solution significantly decreased the hypoosmotically induced rise in cytosolic Ca(2+) and the swelling-activated 86Rb efflux. In cell-attached patch-clamp studies, decreasing the extracellular osmolarity activated a K(+) conductance that was blocked by Ba(2+). In addition, the swelling-activated K(+) channels were significantly inhibited in the presence of nominally free extracellular Ca(2+) and 2.5mM EGTA. These results suggest that in response to hypoosmotic stress, a Ca(2+)-dependent K(+) conductance is activated in the human neuroblastoma cell line CHP-100.     

Induction of 86Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes

Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

Digitonin-permeabilized colonic cell layers. Demonstration of calcium- activated basolateral K+ and Cl- conductances

Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of 86Rb, [3H]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K+ and Cl- currents were generated by imposing transmural ion gradients, and cytosolic free Ca2+ was manipulated by means of a Ca2+-EGTA buffer system in the mucosal bathing solution. Raising the cytosolic free Ca2+ concentration from the nanomolar to the micromolar range activated basolateral conductances for K+ and Cl-. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca2+-induced increases in the K+ and Cl- conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca2+-activated conductive pathways for K+ and Cl-. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca2+-activated channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.     

Calmodulin-sensitive ATP-dependent Ca2+ transport across adipocyte plasma membranes

An ATP-dependent transport system which is active at concentrations of free Ca2+ in the submicromolar range has been identified in adipocyte plasma membranes. The system appears to represent the functional component of the high affinity insulin-sensitive calcium-stimulated, magnesium-dependent adenosine triphosphatase preveiously described in the same preparation (Pershadsingh, H. A., and McDonald, J. M. (1979) Nature 281, 495-497). This ATP-dependent Ca2+ transport pump was stimulated approximately 3-fold by the Ca2+-dependent regulatory protein, calmodulin. This effect was confined to the plasma membrane since a similar effect was undetectable in the fraction enriched in endoplasmic reticulum. Calmodulin stimulation was dose-dependent but saturable with half-maximal activation occurring at 0.72 microgram/ml (43 nM). Calmodulin appeared to stimulate the system primarily by decreasing the apparent half-maximal saturation constant for free Ca2+ from 0.20 +/- 0.04 microM to 0.07 +/- 0.01 microM (n = 3). The Hill coefficient increased from 1.6 +/- 0.2 to 3.2 +/- 0.6 (n = 3), thus showing an increased positive cooperativity which allows the pump to be activated by an exceedingly narrow Ca2+ threshold in the presence of calmodulin. The calmodulin stimulation of the plasma membrane Ca2+ extrusion pump in adipocytes, working in opposition to metabolic signals which increase cytoplasmic Ca2+, could constitute a self-regulating negative feedback device for maintaining a low steady state level of intracellular Ca2+. This feedback system may be of critical importance in regulation of cellular metabolism by insulin.     

Regulation of endogenously catalyzed ADP-ribosylation in adipocyte plasma membranes by Ca2+ and calmodulin

The effects of Ca2+ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [32P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca2+. Maximal incorporation of [32P] into the protein was achieved with free Ca2+ concentrations of 90 microM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 microM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [32P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca2+ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca2+ concentrations between 10(-6) - 10(-5) M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca2+ and calmodulin.     

Influence of Ca2+ on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca2+-activated K+ channel

The involvement of Ca2+-activated K+ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3'-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be -57 +/- 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K+ concentration, [K+]o, and were significantly enhanced by exogenous calcium. The apparent Vmax of Na+-dependent glycine uptake was doubled in the presence of calcium, whereas the K0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca2+-activated K+ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K+]o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC50 of 9.4 microM; and (3) cells treated with the Ca2+-activated K+ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca2+-activated K+ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na+-dependent amino acids.     

Effect of shear stress on cytosolic Ca2+ of calf pulmonary artery endothelial cells.

The purpose of the present study was to determine if hemodynamic shear stress increases free cytosolic Ca2+ concentration ([Ca2+]i) of cultured pulmonary artery endothelial cells exposed to steady laminar fluid flow in a parallel plate chamber. Average [Ca2+]i was estimated by measuring cell-associated fura-2 fluorescence using microfluorimetric analysis. To determine [Ca2+]i close to the membrane surface, 86Rb+ efflux via Ca(2+)-dependent K+ channels was measured. Upon initiation of flow or upon step increases in flow, no change in [Ca2+]i was observed using fura-2. However, increases in shear stress produced a large, transient increase in 86Rb+ efflux. The shear stress-dependent increase in 86Rb+ efflux was not blocked by either tetrabutylammonium ions (20 mM) or by charybdotoxin (10 nM), two specific inhibitors of the Ca(2+)-dependent K+ channel of vascular endothelial cells. These results demonstrate that shear stress per se has little effect on either the average cytosolic [Ca2+]i as measured by fura-2 or on [Ca2+]i close to the cytoplasmic surface of the plasmalemma as measured by the activity of Ca(2+)-dependent K+ channels.     

In squid nerve fibers monovalent activating cations are not cotransported during Na+/Ca2+ exchange

Squid axons display a high activity of Na+/Ca2+ exchange which is largely increased by the presence of external K+, Li+, Rb+ and NH+4. In this work we have investigated whether this effect is associated with the cotransport of the monovalent cation along with Ca2+ ions. 86Rb+ influx and efflux have been measured in dialyzed squid axons during the activation (presence of Ca2+i) of Ca2+o/Na+i and Ca2+i/Ca2+o exchanges, while 86Rb+ uptake was determined in squid optic nerve membrane vesicles under equilibrium Ca2+/Ca2+ exchange conditions. Our results show that although K+o significantly increases Na+i-dependent Ca2+ influx (reverse Na+/Ca2+ exchange) and Rb+i stimulates Ca2+o-dependent Ca2+ efflux (Ca2+/Ca2+ exchange), no sizable transport of rubidium ions is coupled to calcium movement through the exchanger. Moreover, in the isolated membrane preparation no 86Rb+ uptake was associated with Ca2+/Ca2+ exchange. We conclude that in squid axons although monovalent cations activate the Na+/Ca2+ exchange they are not cotransported.     

Inwardly rectifying current-voltage relationship of small-conductance Ca2+-activated K+ channels rendered by intracellular divalent cation blockade

Small conductance Ca2+-activated K+ channels (SK(Ca) channels) are a group of K+-selective ion channels activated by submicromolar concentrations of intracellular Ca2+ independent of membrane voltages. We expressed a cloned SK(Ca) channel, rSK2, in Xenopus oocytes and investigated the effects of intracellular divalent cations on the current-voltage (I-V) relationship of the channels. Both Mg2+ and Ca2+ reduced the rSK2 channel currents in voltage-dependent manners from the intracellular side and thus rectified the I-V relationship at physiological concentration ranges. The apparent affinity of Mg2+ was changed as a function of both transmembrane voltage and intracellular Ca2+ concentration. Extracellular K+ altered the voltage dependence as well as the apparent affinities of Mg2+ binding from intracellular side. Thus, the inwardly rectifying I-V relationship of SK(Ca) channels is likely due to the voltage-dependent blockade of intracellular divalent cations and that the binding site is located within the ion-conducting pathway. Therefore, intracellular Ca2+ modulates the permeation characteristics of SK(Ca) channels by altering the I-V relationship as well as activates the channel by interacting with the gating machinery, calmodulin, and SK(Ca) channels can be considered as Ca2+-activated inward rectifier K+ channels.     

Modulation of insulin release by adenosine A1 receptor agonists and antagonists in INS-1 cells: the possible contribution of 86Rb+ efflux and 45Ca2+ uptake

Due to the lack of specific agonists and antagonists the role of adenosine receptor subtypes with respect to their effect on the insulin secretory system is not well investigated. The A1 receptor may be linked to different 2nd messenger systems, i.e. cAMP, K+- and 45Ca2+ channel activity. Partial A1 receptor agonists are going to be developed in order to improve diabetes (increase in insulin sensitivity, lowering of FFA and triglycerides). In this study newly synthesized selective A1 receptor agonists and antagonists were investigated thereby integrating three parameters, insulin release (RIA), 45Ca2+ uptake and 86Rb+ efflux (surrogate for K+ efflux) of INS-1 cells, an insulin secretory cell line. The presence of A1-receptors was demonstrated by Western blotting. The receptor nonselective adenosine analogue NECA (5-N-ethylcarboxyamidoadenosine) at high concentration (10 microM) had no effect on insulin release and 45Ca2+ uptake which could be interpreted as the sum of effects mediated by mutual antagonistic adenosine receptor subtypes. However, an inhibitory effect mediated by A1 receptor agonism was detected at 10 nM NECA and could be confirmed by adding the A1 receptor antagonist PSB-36 (1-butyl-8-(3-noradamantyl)-3-(3-hydroxy-propyl)xanthine). NECA inhibited 86Rb+ efflux which, however, did not fit with the simultaneous inhibition of insulin secretion. The selective A1 receptor agonist CHA (N6-cyclohexyladenosine) inhibited insulin release; the simultaneously increased Ca2+ uptake (nifedipine dependent) and inhibition of 86Rb+ efflux did not fit the insulin release data. The CHA effect (even the maximum effect at 50 microM) can be increased by 10 microM NECA indicating that CHA and NECA have nonspecific and physiologically non-relevant effects on 86Rb+ efflux in addition to their A1-receptor interaction. Since PSB-36 did not influence the NECA-induced inhibition of 86Rb+ efflux, the NECA effect is not mediated by potassium channel-linked A1 receptors. The nonselective adenosine receptor antagonist caffeine increased insulin release which was reversed by CHA as expected when hypothesizing that both act via A1 receptors in this case. In conclusion, stimulation of A1 receptors by receptor selective and nonselective compounds reduced insulin release which is not coupled to opening of potassium channels (86Rb+ efflux experiments) or inhibition of calcium channels (45Ca2+ uptake experiments). It may be expected that of all pleiotropic 2nd messengers, the cAMP system (not tested here) is predominant for A1 receptor effects and the channel systems (K+ and Ca2+) are of minor importance and do not contribute to insulin release though being coupled to the receptor in other tissues.     

Modulation of ATP-dependent Ca2+ transport in rat parotid basolateral membrane vesicles by K+ + Cl- flux

In basolateral membrane vesicles (BLMV) isolated from rat parotid glands, the initial rate of ATP-dependent Ca2+ transport, in the presence of KCl, was approx. 2-fold higher than that obtained with mannitol, sucrose or N-methyl-D-glucamine (NMDG)-gluconate. Only NH4+, Rb+, or Br- could effectively substitute for K+ or Cl-, respectively. This KCl activation was concentration dependent, with maximal response by 50 mM KCl. An inwardly directed KCl gradient up to 50 mM KCl had no effect on Ca2+ transport, while equilibration of the vesicles with KCl (greater than 100 mM) increased transport 15-20%. In presence of Cl-, 86Rb+ uptake was 2.5-fold greater than in the presence of gluconate. 0.5 mM furosemide inhibited 86Rb+ flux by approx. 60% in a Cl- medium and by approx. 20% in a gluconate medium. Furosemide also inhibited KCl activation of Ca2+ transport with half maximal inhibition either at 0.4 mM or 0.05 mM, depending on whether 45Ca2+ transport was measured with KCl (150 mM) equilibrium or KCl (150 mM) gradient. In a mannitol containing assay medium, potassium gluconate loaded vesicles had a higher (approx. 25%) rate of Ca2+ transport than mannitol loaded vesicles. Addition of valinomycin (5 microM) to potassium gluconate loaded vesicles further stimulated (approx. 30%) the Ca2+ transport rate. These results suggest that during ATP dependent Ca2+ transport in parotid BLMV, K+ can be recycled by the concerted activities of a K+ and Cl- coupled flux and a K+ conductance.     

Characterisation of an ATP-dependent Ca2+ transport system in a plasma membrane enriched fraction from rat parotid

A membrane fraction enriched in plasma membrane marker enzymes K+-dependent p-nitrophenyl phosphatase, 5'-nucleotidase and alkaline phosphatase was prepared from rat parotid glands using Percoll self-forming gradient. This fraction contained an ATP-dependent CA2+ transport system which was distinct from those located on the endoplasmic reticulum and mitochondria of parotid glands. The Km for ATP was 0.57 +/- 0.07 mM (n = 3). Nucleotides other than ATP such as ADP, AMP, GTP, CTP, UTP or ITP were unable to support significant Ca2+ uptake. ATP-dependent Ca2+ uptake displayed sigmoidal kinetics with respect to free Ca2+ concentration with a Hill coefficient of 2.02. The K0.5 for Ca2+ was 44 +/- 3.1 nM (n = 3) and the average Vmax was 13.5 +/- 1.1 nmol/min per mg of protein. The pH optimum was 7.2. Trifluorperazine inhibited Ca2+ transport with half maximal inhibition observed at 30.8 microM. Complete inhibition was observed at 70 microM trifluorperazine. Exogenous calmodulin however had no effect on the rate of transport. Na+ and K+ ions activated Ca2+ transport at 20 to 30 mM ion concentrations. Higher concentrations of Na+ or K+ were inhibitory.     

Properties of the apamin-sensitive Ca2+-activated K+ channel in PC12 pheochromocytoma cells which hyper-produce the apamin receptor

Undifferentiated PC12 cell produce high levels of apamin receptors (measured with 125I-apamin) after 7 days in culture. These levels are at least 50 times higher than those found in other cellular types which are also known to have apamin receptors and apamin-sensitive Ca2+-activated K+ channels in their membranes. Treatment of undifferentiated PC12 cells with nerve growth factor maintains these cells in a state having a low level (10 times less after 7 days of culture) of apamin receptors. Ca2+ injection into PC12 cells with the calcium ionophore A23187 has been used to monitor the activity of the Ca2+-activated K+ channel following 86Rb+ efflux. A large component of this Ca2+-activated 86Rb+ efflux is inhibited by apamin. Half-maximum inhibition by apamin of both 86Rb+ efflux and 125I-apamin binding was observed at 240 pM apamin. Another component of 86Rb+ efflux is due to another type of Ca2+-activated K+ channel which is resistant to apamin and sensitive to tetraethylammonium. The Ca2+ channel activator Bay K8644 also triggers an apamin-sensitive Ca2+-dependent 86Rb+ efflux. Bay K8644 has been used to analyze the internal Ca2+ concentration dependence of the apamin-sensitive channel activity. Under normal conditions, the internal Ca2+ concentration is 109 +/- 17 nM, and the apamin-sensitive channel is not activated. The channel is fully activated at an internal Ca2+ concentration of 320 +/- 20 nM.     

Modulation of Ca2+-mediated K+-gating of erythrocyte ghosts by external Ca-EGTA

Using 86Rb+ as a marker for K+ permeability, we find that extracellular Ca-EGTA influences the rate of 86Rb+ efflux from erythrocyte ghosts preloaded with 86Rb+ and "buffered" Ca2+. At an internal free Ca2+, where the rate of 86Rb+ efflux is minimal and uninfluenced by either external EGTA or external Ca2+, external Ca-EGTA at 0.2-0.5 mM can raise the flux rate to as high as can be attained by raising internal Ca2+, in the presence of an excess externally either of Ca2+ or of EGTA. Higher concentrations of Ca-EGTA (up to 1-2 mM) diminish the flux rate. External Ca-EDTA or Mg-EDTA can substitute for Ca-EGTA in enhancing and suppressing flux rate. The peak rate is insensitive to external free Ca2+ but depends on internal Ca2+; internal Mg-EDTA does not substitute for internal Ca-EGTA. Thus, the erythrocyte membrane is asymmetric with respect to its interaction with Ca2+ and Ca-EGTA. Also, 22Na+ does not substitute for 86Rb+. The peak rate of 86Rb+ flux produced by external Ca-EGTA is diminished by chlorpromazine (0.1 mM) and augmented by 1-propranolol (25 microM), in the same way as the rate produced by increasing internal Ca2+. The results suggest that external Ca-EGTA enhances the affinity of internal Ca2+ for its receptor(s) which operate the K+-gate at the inner surface of the membrane. At external concentrations of Ca-EGTA above 1-2 mM, 86Rb+ flux rate again rises with increase of Ca-EGTA. This phenomenon does not depend upon internal Ca2+, is not affected by chlorpromazine or by 1-propranolol, and is associated with an enhanced permeability to 22Na+, inulin, and haemoglobin.     

Characterization of increased K+ permeability associated with the stimulation of receptors for immunoglobulin E on rat basophilic leukemia cells.

Aggregation of immunoglobulin E-receptor complexes on the surface of rat basophilic leukemia cells stimulates an increase in plasma membrane K+ permeability that is monitored as an increase in the rate of efflux of preloaded 86Rb+. A major component of this stimulated 86Rb+ efflux appears to be due to a Ca(2+)-activated K+ channel because it is inhibited by quinidine in parallel with the inhibition of degranulation and membrane potential repolarization, it is blocked by 0.1 mM La3+, and it is dependent on external Ca2+. Depolarization of the plasma membrane by carbonyl cyanide 3-chlorophenylhydrazone inhibits stimulated Ca2+ influx and prevents antigen-induced 86Rb+ efflux, and increased external Ca2+ partially restores 86Rb+ efflux under these conditions. In addition, potentiation of antigen-stimulated Ca2+ influx by pretreatment with cholera toxin increases the initial rate of stimulated 86Rb+ efflux. Another component of antigen-stimulated K+ efflux appears to be mediated by a guanine nucleotide-binding protein because pretreatment of rat basophilic leukemia cells with pertussis toxin decreases the initial rate of antigen-stimulated 86Rb+ efflux to 40% of that for the untreated cells. Stimulated 86Rb+ efflux is also observed when ionomycin is used to increase cytoplasmic Ca2+ and to trigger membrane depolarization. The efflux stimulated by ionomycin is inhibited by quinidine but not by pertussis toxin pretreatment; thus, it appears to occur through the Ca(2+)-activated K+ efflux pathway. It is proposed that these K+ efflux pathways serve to sustain the Ca2+ influx that is necessary for receptor-mediated triggering of cellular degranulation.     

The role of calmodulin on Ca2+-dependent K+ transport regulation in the human red cell

Several lipophilic calmodulin antagonists (phenotiazines, butyrophenones and diphenylbutylpiperidines) inhibited Ca²⁺-induced loss of KCl from human red cells. However, the K_i values for this effect did not bear good correlation with the K_i values reported for well-known calmodulin-dependent systems. In addition, the inhibition was strongly dependent on the haematocrit and valinomycin-induced KCl fluxes were also affected. Added calmodulin did not have any effect on Ca²⁺-dependent ⁸⁶Rb uptake by inside-out vesicles derived from red cell membranes whereas stimulation of Ca²⁺-dependent ATPase was apparent. Lipophilic anticalmodulins at high doses had all kinds of effects on ⁸⁶Rb uptake by inside-out vesicles: increase, decrease or no change of the fraction of activated vesicles reached at submaximal Ca²⁺ concentrations, with or without modification of the relative rate of ⁸⁶Rb uptake. The hydrophylic compound 48/80 decreased the fraction of activated vesicles reached at submaximal Ca²⁺ concentrations without affecting the relative rate of ⁸⁶Rb uptake, but this effect took place only at concentrations 10-fold higher than the reported K_i for calmodulin-dependent systems. These results suggest that Ca²⁺-dependent K⁺ channels of red cells are not regulated by calmodulin.