ATX II, a sodium channel toxin, sensitizes skeletal muscle to halothane, caffeine, and ryanodine.

BACKGROUND: The function or expression of subtypes of the sodium ion (Na+) channel is altered in biopsies or cultures of skeletal muscle from many persons who are susceptible to malignant hyperthermia (MH). ATX II, a specific Na+ channel toxin from a sea anemone, causes delayed inactivation of the channel similar to that seen in cell cultures of MH muscle. ATX II was added to skeletal muscle to determine whether altered Na+ channel function could increase the sensitivity of normal skeletal muscle to agents (halothane, caffeine, ryanodine) to which MH muscle is hypersensitive. METHODS: Studies were performed of fiber bundles from the vastus lateralis muscle of persons who were deemed not MH susceptible (MH-) or MH susceptible (MH+) according to the MH diagnostic test and of strips of diaphragm muscle from rats. Preparations in a tissue bath containing Krebs solution were connected to a force transducer. ATX II was introduced 5 min before halothane, caffeine, or ryanodine. RESULTS: ATX II increased the magnitude of contracture to halothane in preparations from most MH-, but not MH+, human participants. After ATX II treatment, preparations from 9 of 24 MH- participants generated contractures to halothane, 3%, that were of the same magnitude as those from MH+ participants. Preparations from four of six ATX II-treated healthy participants also gave responses of the same magnitude as those of MH-susceptible participants to a graded halothane challenge (0.5-3%). The contractures to bolus doses of halothane in specimens from male participants were more than three times larger than the contractures in specimens from female participants. In rat muscle, ATX II increased the magnitude of contracture to caffeine (2 mM) and decreased the time to produce a 1-g contracture to ryanodine (1 microM). CONCLUSIONS: ATX II, which causes delayed inactivation of the Na+ channel in cell cultures similar to that reported in cultures of MH+ skeletal muscle, increased the sensitivity of normal muscle to three agents to which MH+ muscle is hypersensitive. The increased sensitivity to halothane, 3%, occurred in most (79%), but not all, MH- participants, and this effect was most evident in male participants. Therefore, abnormal function of the Na+ channel, even if it is a secondary event in MH, may contribute to a positive contracture test result for MH.

Effects of ethanol on rat somatosensory cortical neurons.

In this study, we characterized the local effects of ethanol (EtOH) on postsynaptic potentials (PSPs) and membrane properties of layer II-III (L2-3) and layer V (L5) somatosensory cortical neurons. Intracellular recordings were done using the in vitro slice preparation of rat somatosensory cortex. Our results show that EtOH exerts local effects on cortical cell membrane at physiologically relevant concentrations. A predominant effect of EtOH was to reduce excitability of L2-3 and L5 neurons by increasing the rheobase, decreasing input resistance and repetitive firing, reducing PSPs amplitude and the probability of evoking action potentials. Early (6 ms) and late (18 ms) PSP components were affected differentially by EtOH, the late components being more suppressed. Overall, EtOH-mediated suppression of PSPs was stronger in L5 neurons. Cortical neurons were divided into three subtypes: regular spiking adapting (RS-A), regular spiking non-adapting (RS-NA) and bursting (D-IB) neurons. PSPs evoked in RS-A neurons were more sensitive to EtOH suppressant effects. EtOH effects on input resistance were distributed differentially among the three groups of neurons. These results support the notion that EtOH disrupts higher processing of somatosensory information via a differential alteration of cortical neuron's membrane properties and synaptic transmission.

Glutamine substitution at alanine1649 in the S4-S5 cytoplasmic loop of domain 4 removes the voltage sensitivity of fast inactivation in the human heart sodium channel.

Normal activation-inactivation coupling in sodium channels insures that inactivation is slow at small but rapid at large depolarizations. M1651Q/M1652Q substitutions in the cytoplasmic loop connecting the fourth and fifth transmembrane segments of Domain 4 (S4-S5/D4) of the human heart sodium channel subtype 1 (hH1) affect the kinetics and voltage dependence of inactivation (Tang, L., R.G. Kallen, and R. Horn. 1996. J. Gen. Physiol. 108:89-104.). We now show that glutamine substitutions NH2-terminal to the methionines (L1646, L1647, F1648, A1649, L1650) also influence the kinetics and voltage dependence of inactivation compared with the wild-type channel. In contrast, mutations at the COOH-terminal end of the S4-S5/D4 segment (L1654, P1655, A1656) are without significant effect. Strikingly, the A1649Q mutation renders the current decay time constants virtually voltage independent and decreases the voltage dependences of steady state inactivation and the time constants for the recovery from inactivation. Single-channel measurements show that at negative voltages latency times to first opening are shorter and less voltage dependent in A1649Q than in wild-type channels; peak open probabilities are significantly smaller and the mean open times are shorter. This indicates that the rate constants for inactivation and, probably, activation are increased at negative voltages by the A1649Q mutation reminiscent of Y1494Q/ Y1495Q mutations in the cytoplasmic loop between the third and fourth domains (O'Leary, M.E., L.Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106:641-658.). Other substitutions, A1649S and A1649V, decrease but fail to eliminate the voltage dependence of time constants for inactivation, suggesting that the decreased hydrophobicity of glutamine at either residues A1649 or Y1494Y1495 may disrupt a linkage between S4-S5/D4 and the interdomain 3-4 loop interfering with normal activation-inactivation coupling.

Chronic cocaine intoxication alters hippocampal sodium channel function.

Repeated daily administration of subconvulsive doses of cocaine results in the appearance and increase in convulsive responsiveness to the drug and its lethal effects. The mechanisms involved in this increased susceptibility to cocaine-induced seizure are yet unknown. In this study, we used whole cell patch-clamp recording techniques to examine the functional changes in voltage-dependent Na+ channels produced by subconvulsive doses of cocaine (45 mg/kg per day, i.p.) in rat hippocampal CA1 pyramidal neurons. Intact animals were injected with cocaine for 5-6 days. Acutely dissociated hippocampal neurons were then recorded in vitro. Our results show that an augmentation of peak Na+ currents and a shift in depolarizing direction of the steady-state inactivation were present in neurons from drug-treated rats. These changes, by making a larger proportion of Na+ channels available for opening, could increase the excitability of CA1 neurons and may contribute to the increase in convulsive responsiveness to cocaine.

Sodium channel in human malignant hyperthermia.

BACKGROUND: The abundance of a specific sodium channel subunit (SkM2) appeared to be altered in vitro in cell cultures from persons susceptible to malignant hyperthermia. This study sought to determine whether these findings are artifacts of cell culture or whether they may be relevant to malignant hyperthermia. METHODS: Regulation of transcript levels of SkM2, a specific sodium channel alpha-subunit, was determined by mRNA analysis. Functional SkM2 protein was estimated in biopsy sections of vastus lateralis muscle by inhibiting the directly elicited muscle twitch by tetrodotoxin, which can differentiate at least three sodium currents in skeletal muscle. RESULTS: The transcript levels of SkM2 were depressed by 115-fold in six of seven persons susceptible to malignant hyperthermia; and the functional expression of the SkM2 protein, based on the tetrodotoxin sensitivity of the directly elicited twitch, was decreased by at least fourfold in muscle from persons susceptible to malignant hyperthermia compared with persons who were not susceptible. CONCLUSIONS: As in previous studies in cell culture, altered mRNA and/or the functional expression of a specific subunit of the sodium channel (SkM2) was found in biopsy sections of muscle from all 12 persons examined who were susceptible to malignant hyperthermia but in none of the 16 nonsusceptible participants. Human malignant hyperthermia is a heterogeneous disorder, and the down-regulation of SkM2 may be involved in the final common pathway through which mutations in any one of several proteins, including the ryanodine receptor, could render a person susceptible.

Similarities and differences in mechanisms of cardiotoxins, melittin and other myotoxins.

Myonecrosis induced in vivo by cardiotoxin, melittin, and Asp49 and Lys49 phospholipase A2 (PLA2) myotoxins involves rapid lysis of the sarcolemma, myofibril clumping, and hypercontraction of sarcomeres. In contrast, skeletal muscle necrosis induced by crotamine and myotoxin a is much slower, consisting of mitochondrial and sarcoplasmic reticulum swelling, myofibril degeneration, and lack of sarcolemma or transverse tubule damage. The mechanisms contributing to the myonecrosis induced by these peptides were evaluated. Two cardiotoxins and two Lys49 PLA2 myotoxins lysed primary cultures of human skeletal muscle within 24 hr at a concentration of 0.25 microM, while melittin, crotamine, and myotoxin a, and an Asp49 PLA2 myotoxin were non-cytolytic at concentrations up to 5.0 microM, suggesting that cytolysis is not a good measure of myotoxicity. Crotamine and the Lys49 PLA2 myotoxin altered Ca2+ ion flux in human heavy sarcoplasmic reticulum by opening the ryanocine receptor. Whole-cell patch-clamp studies demonstrated that administrating crotamine intracellularly increased Na+ currents. Free fatty acids, liberated by activation of tissue phospholipase C or by the PLA2 activity of the myotoxins, were monitored for crotamine, myotoxin a and a Lys49 PLA2 myotoxin in cell cultures in which the lipids had been radiolabeled. Only the Lys49 myotoxin produced significant amounts of fatty acid in cell cultures, supporting a potential role for fatty acid production only in the mechanism of sarcolemma-destroying myotoxins. These findings, coupled with those in the literature, support a hypothesis in which the myotoxins and/or products of lipase activity (e.g. fatty acids) are acting at a site existing on both the Na+ channel and a protein involved in Ca2+ release and probably serving a modulatory function for ion regulation. Based on the similarities in mechanisms between the toxins and fatty acids, the most likely site would be a fatty acid binding site on the protein (either similar to that on fatty acid binding proteins, or an acylated cysteine residue) or in the membrane.

Modulation of human muscle sodium channels by intracellular fatty acids is dependent on the channel isoform.

Free fatty acids (FFAs), including arachidonic acid (AA), are implicated in the direct and indirect modulation of a spectrum of voltage-gated ion channels. Skeletal muscle sodium channels can be either activated or inhibited by FFA exposure; the response is dependent on both FFA structure and site of exposure. Recombinant human skeletal muscle sodium channels (hSkM1) were transfected into heterologous human renal epithelium HEK293t cells. Cytoplasmic delivery of 5 microM AA augmented the voltage-activated sodium current of hSkM1 channels by 190% (+/-54 S.E., n = 7) over a 20-min period. Similar results were seen with 5 microM oleic acid. Sodium currents in HEK293t cells transfected with human cardiac muscle sodium channels (hH1) were insensitive to AA treatment, and exposure to oleic acid inhibited the hH1 currents over a 20-min period by 29% (+/-13 S.E., n = 5). The increase in hSkM1 current was not accompanied by shifts in voltage dependence of activation, steady-state inactivation, or markedly altered kinetics of inactivation of the macroscopic current. The FFA-induced increase in sodium currents was not dependent on protein kinase C activity. In contrast, both isoforms were reversibly inhibited by external application of unsaturated FFA. Thus, the differential effects of FFA on skeletal muscle sodium channels first noted in cultured muscle cells can be reproduced by expressing recombinant sodium channels in epithelial cells. Although the responses to applied FFAs could be direct or indirect, we suggest that: 1) SkM1 has two classes of response to FFA, one which produces augmentation of macroscopic currents with intracellular FFA, and a second which produces inhibition with extracellular FFA; 2) H1 has only one class of response, which produces inhibition with extracellular FFA. A testable hypothesis is that the presence or absence of each response is due to a specific structure in SkM1 or H1. These specific structures may directly interact with FFA or may interact with intermediate components.

Altered sodium current response to intracellular fatty acids in halothane-hypersensitive skeletal muscle.

Biopsies of human skeletal muscle were analyzed by an in vitro contracture test (IVCT) for responsiveness to a halothane challenge: noncontracting (nonresponsive; IVCT-) and contracting (IVCT+). A muscle biopsy that is IVCT+ indicates potential malignant hyperthermia (MH) susceptibility. Primary cultures were grown from portions of the skeletal muscle biopsies, and voltage-activated currents were measured by whole cell recording in the presence or absence of 2-5 microM intracellular arachidonic or oleic acids. In untreated IVCT- cells, Na+ currents were predominantly tetrodotoxin (TTX) insensitive, indicating that most of the current was carried through the embryonic SkM2 isoform of the Na+ channel. Inclusion of fatty acids in the recording pipette of IVCT- cells produced an increase in voltage-activated Na+ currents during 20 min of recording. Approximately 70% of currents in fatty acid-treated cells were TTX sensitive, indicating activation of the adult SkM1 isoform of the Na+ channel. In contrast to IVCT- cells, IVCT+ cells expressed Na+ currents that were predominantly TTX sensitive even in the absence of added fatty acid, thus showing a relatively large baseline functional expression of SkM1 channels. Addition of fatty acids to the recording pipette produced little further change in the magnitude or TTX sensitivity of the whole cell currents in IVCT+ cells, suggesting altered functional regulation of Na+ channels in MH muscle.

Transmembrane potential responses during HL-60 promyelocyte differentiation.

Myeloid cells, including granulocytes and monocyte/macrophages, are important in disease-associated inflammatory reactions. These cells come from a common progenitor, the promyelocyte. The human promyelocytic cell line, HL-60, can be induced to terminally differentiate into granulocytes or monocyte/ macrophages in a controlled fashion providing a model to study various aspects of myelomonocytic differentiation. The expression of several ion channels is controlled in HL-60 cells in a differentiation specific pattern. The purpose of this study was to determine if lineage-specific ion channel expression during HL-60 differentiation resulted in differences in functional responses to external stimuli. This was investigated by examining transmembrane potential responses in HL-60 promyelocytes, HL-60-derived polymorphonuclear cells (PMNs), and monocytes to various stimuli using the transmembrane potential sensitive dye, diSBAC2-(3). Exposure of HL-60 promyelocytes to ionomycin or ATP produced a membrane hyperpolarization. Studies using ion substitutions and ion channel blockers indicate that the hyperpolarization was mediated by KCa channels. During HL-60 promyelocyte differentiation to PMNs, the membrane potential response to ionomycin and ATP shifted from a hyperpolarization to a depolarization over 7 days. Conversely, HL-60-derived monocytes exhibited a membrane hyperpolarization in response to ionomycin and ATP. HL-60-derived monocytes also exhibit a Cl- conductance specifically induced by ATP. Lineage-specific expression of ion channels during HL-60 cell differentiation is important in determining the transmembrane potential response of these cells. This may be translated into functional responses of various myelomonocytic cells during disease-associated inflammatory reactions.

Modulation of a potassium conductance in developing skeletal muscle.

K+ conductances dominate and potentially modulate the resting potential of skeletal muscle cells. The expression and modulation of a major K+ conductance were examined during in vitro differentiation of the mouse myoblast cell line C2C12. The inwardly rectifying K+ conductance (IKi) increased from unmeasurable levels in undifferentiated myoblasts to approximately 1.56 +/- 0.51 nA (n = 17) in myoballs derived from myotubes at 5 days after induction of differentiation. The inward rectifier was subject to modulation by intracellular signals. Exposure of cytoplasm to guanosine 5'-O-(3-thiotriphosphate) during whole cell recording produced a concentration (5-100 microM)- and time (1-20 min)-dependent inhibition of the mean conductance. Elevation of intracellular free Ca2+ (> 200 nM) also inhibited IKi. These findings demonstrate a potential mechanism for modulation of the resting potential of muscle fibers via the control of skeletal muscle IKi.

Mitogen-activated Ca++ channels in human B lymphocytes.

Two complementary experimental methods have been used to examine mitogen-induced transmembrane conductances in human B cells using the Daudi cell line as a model for human B cell activation. Spectrofluorometry was used to investigate mitogen-induced changes in [Ca++]i and transmembrane potential. Activation of human B cells with anti-mu antibodies resulted in a biphasic rise in [Ca++]i, the second phase being mediated by the influx of extracellular Ca++. Ca++ influx was inhibited by high [K+]e, suggesting that this influx was transmembrane potential sensitive. Membrane currents of Daudi cells were investigated using voltage clamp techniques. Before mitogenic stimulation, the cells were electrically quiet. Within several minutes of the addition of anti-mu antibodies to the bath solution, inward currents were observed at negative voltages. Whole-cell currents changed instantly with voltage steps and were transmembrane potential sensitive in that at potentials more positive than -40 mV no currents were detectable. A similar conductance was also activated by the introduction of IP3 into the intracellular solution, suggesting that IP3 generation after surface IgM crosslinking is involved in the activation of this conductance. Both anti-mu and IP3 induced currents were blocked by 1 mM La , which is known to block Ca++ channels. These results strongly support the presence of membrane Ca++ channels in human B cells that function in the early stages of activation. Changes in transmembrane potential appear to be important in regulating Ca++ influx. These mechanisms work in concert to regulate the level of [Ca++]i during the early phases of human B cell activation.

Differential modulation of a sodium conductance in skeletal muscle by intracellular and extracellular fatty acids.

Voltage-activated sodium channels of cultured skeletal muscle show diametrically divergent responses to intracellular vs. extracellular exposure to free fatty acids. Intracellular exposure to 1-20 microM arachidonic acid increased the magnitude of voltage-activated sodium currents, but not potassium currents, in whole cell recordings of human primary muscle cells and in the C2C12 mouse cell line. Oleic and stearic acids also stimulated increased sodium currents. In contrast, extracellular exposure to 5-10 microM arachidonic acid reversibly inhibited inward currents. Externally applied oleic acid was a less effective inhibitor, and stearic acid (up to 20 microM) produced no inhibition. The difference in sodium current responses to intracellular vs. extracellular exposure indicates that fatty acids can modulate skeletal muscle sodium channel function by at least two different pathways.

A lineage-specific Ca(2+)-activated K+ conductance in HL-60 cells.

Cells of the human promyelocytic cell line HL-60 can be controllably induced to terminally differentiate into either granulocytes or monocyte/macrophages. HL-60 promyelocytes and terminally differentiated macrophages express a K(+)-selective ion channel which is activated by intracellular free Ca2+ concentrations above 10(-7) M. Because of its voltage independence, this channel can be distinguished from the voltage- and Ca(2+)-activated family of outward-rectifying channels. The channel is selective for K+ against Na+ and is blocked by Ba2+, thus it may be similar to the Ca(2+)-activated K+ channel previously described in human macrophages. In its sensitivity to block by charybdotoxin, this channel also resembles a Ca(2+)-activated K+ channel of lymphocytes, which plays a role in activation-dependent hyperpolarization. In contrast to promyelocytes and macrophages, functional expression of the Ca(2+)-activated K+ channel is suppressed to nearly undetectable levels in granulocytes derived from HL-60 cells by retinoic acid-induced differentiation. These data suggest that signals which produce elevation of intracellular Ca2+ will hyperpolarize promyelocytes and differentiated macrophages by activating this conductance; however, signals which elevate free Ca2+ in granulocytes must act on other effectors, which may produce a different final influence on membrane potential.

Modulation of Ca2+ release and Na+ channel function in skeletal muscle by fatty acids.

Ca(2+)- and halothane-induced Ca(2+) release and Na+ currents are modulated by free fatty acids (FAs). FA modulation of ion currents may have important implications for general muscle physiology and skeletal muscle disorders, including malignant hyperthermia (MH).

Ca(2+)-insensitive modulation of a K+ conductance by inositol polyphosphates.

Macrophages derived from phorbol ester-induced human leukemic (HL-60) cells exhibit a voltage-activated inward rectifying potassium conductance which was modulated by macrophage colony-stimulating factor (Wieland, S. J., Chou, R. H., and Gong, Q. H. (1990) J. Cell. Physiol. 142, 643-651). Roles of intracellular messengers in this regulatory mechanism were investigated. Intracellular dialysis with inositol 1,3,4,5-tetrakisphosphate (IP4) or inositol 1,4,5-trisphosphate during tight-seal whole cell recording produced a rapid increase in the inward rectifying conductance. Changes in intracellular Ca2+ levels alone did not reproduce the stimulatory effect of these modulators. Intracellular dialysis with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) resulted in profound inhibition of this conductance. These data suggest a novel cellular function for inositol polyphosphates, particularly IP4, and show antagonistic modulation with GTP gamma S on a human macrophage inward rectifier.

Effects of a cardiotoxin from Naja naja kaouthia venom on skeletal muscle: involvement of calcium-induced calcium release, sodium ion currents and phospholipases A2 and C.

Snake venom cardiotoxin (CTX) fractions induce contractures of skeletal muscle and hemolysis of red blood cells. The fractions also contain trace amounts of venom-derived phospholipase A2 (PLA2) contamination and activate tissue phospholipase C (PLC) activity. The present study examines the mechanisms of action of a CTX fraction from Naja naja kaouthia venom in skeletal muscle. Sphingosine competitively antagonized CTX-induced red blood cell hemolysis, but not skeletal muscle contractures. CTX rapidly lowered the threshold for Ca(2+)-induced Ca2+ release in heavy sarcoplasmic reticulum fractions, as monitored with arsenazo III. There was also a slower time-dependent reduction of Na+ currents, as assessed by whole cell patch-clamp techniques. The CTX fractions elevated levels of free fatty acids and diacylglycerol for 2 hr in primary cultures of human skeletal muscle by a combined action of venom-derived PLA2 contamination in the fraction and activation of endogenous PLC activity. The activation of tissue PLC activity could be readily distinguished from the contribution of the venom PLA2 by p-bromophenacyl bromide treatment of CTX fractions. The mechanism of action involved in contractures of skeletal muscle appears to be related to the immediate and specific effect of CTX (Ca2+ release by the sarcoplasmic reticulum), while the mechanisms involved in hemolysis of red blood cells and decreased Na+ currents in skeletal muscle most likely relate to long-term effects on lipid metabolism.

Macrophage-colony-stimulating factor (CSF-1) modulates a differentiation-specific inward-rectifying potassium current in human leukemic (HL-60) cells.

A voltage-activated inward-rectifying K+ conductance (lKi) appears in human promyelocytic leukemia (HL-60) cells during phorbol ester-induced differentiation into macrophages. This conductance was detected in the cells 24 hours after exposure to phorbol-12-myristate-13-acetate (PMA), as the cells began to express the macrophage phenotype, and continued to increase for 4 days after PMA exposure. The magnitude of inward current was a function of external K+; current was blocked by extracellular or intracellular Cs+ and by extracellular Ba++. Hyperpolarization produced activation at membrane potentials more negative than -80 mV, and a slower, partial inactivation also occurred at potentials more negative than -100 mV. This conductance was not detected in proliferating cells nor in granulocytes derived from HL-60 cells which were induced to differentiate with retinoic acid (RA). Exposure of differentiated macrophages to recombinant human CSF-1 produced inhibition of the lKi beginning within 1 minute after exposure. CSF-1 inhibition of lKi channels in cell-attached patches indicated that channel modulation was via intracellular mediators. The rapid inhibition of the inward rectifier by the macrophage-specific CSF-1 appears to be one of the earliest cellular responses to this factor.

Malignant hyperthermia: slow sodium current in cultured human muscle cells.

Voltage-activated ion currents were measured in cultured skeletal muscle myoballs. Cultures were generated from biopsies from patients referred for diagnosis of susceptibility to malignant hyperthermia (MH); diagnosis of susceptibility (MH+) or nonsusceptibility (MH-) was made on the basis of in vitro halothane-induced contracture of a separate piece of biopsy. Measurements of ion currents were made at room temperature in the absence of anesthetic agents, using tight-seal whole-cell recording. Fast transient Na+ currents and delayed outward K+ currents were similar in magnitude and kinetics in cells from MH+ and MH- patients. An additional slowly inactivating inward current component was commonly observed in cells from MH+ patients. This current was blockable by tetrodotoxin and was carried by Na+ but not by Ba2+. The component was less frequently observed and was of a lower magnitude in cells from MH- patients. The increased magnitude of the slow inward current observed in cultured muscle cells from MH+ patients may be a manifestation of the lesion that causes MH.

Effect of a phospholipase A2 with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents.

The action of a 16,300 mol. wt phospholipase A2 with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage- and Ca2(+)-activated K+ current; the sensitivity for voltage activation of the K+ current was enhanced by increasing free Ca2+ in the recording pipette from 10(-8) M to 2 x 10(-6) M. In contrast, peripheral blood lymphocytes possessed voltage-activated K+ currents that were inhibited by increasing intracellular Ca2+. Exposure of either preparation to B. fasciatus toxin (0.2-5 x 10(-6) M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of -140 mV to -40 mV. However, the sensitivity for voltage activation of the K+ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K- currents in human lymphocytes, suggesting a specific increase in intracellular Ca2- levels in both cell types. The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca2+ levels. Exposure of either lymphocytes or epithelial cells to toxin (10(-6) M) resulted in a transient increase in Ca2+. However, while the Ca2+ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca2(+)-regulated K+ channels may reflect an irreversible action of the B. fasciatus phospholipase A2 on a Ca2+-dependent regulatory process.