Opening Of Nonselective Cation Channels Research Articles

Glucose and GTP-binding protein-coupled receptor cooperatively regulate transient receptor potential-channels to stimulate insulin secretion [Review

In pancreatic β-cells, glucose-induced closure of the ATP-sensitive K+ (KATP) channel is an initial process triggering glucose-stimulated insulin secretion (GSIS). This KATP-channel dependent pathway has been believed to be a central mechanism for GSIS. However, since the resting membrane potential of cells is determined by the balance of the net result of current amplitudes in outward and inward directions, it must be taken into consideration that not only KATP channel inhibition but also inward current via the basal opening of non-selective cation channels (NSCCs) plays a crucial role in membrane potential regulation. The basal activity of NSCCs is essential to effectively evoke depolarization in concert with KATP channel closure that is dependent on glucose metabolism. The present study summarizes recent findings regarding the roles of NSCCs in GSIS and GTP-binding protein coupled receptor-(GPCR) operated potentiation of GSIS.

Kisspeptin Directly Excites Anorexigenic Proopiomelanocortin Neurons but Inhibits Orexigenic Neuropeptide Y Cells by an Indirect Synaptic Mechanism

The neuropeptide kisspeptin is necessary for reproduction, fertility, and puberty. Here, we show strong kisspeptin innervation of hypothalamic anorexigenic proopiomelanocortin (POMC) cells, coupled with a robust direct excitatory response by POMC neurons (n > 200) to kisspeptin, mediated by mechanisms based on activation of a sodium/calcium exchanger and possibly opening of nonselective cation channels. The excitatory actions of kisspeptin on POMC cells were corroborated with quantitative PCR data showing kisspeptin receptor GPR54 expression in the arcuate nucleus, and the attenuation of excitation by the selective kisspeptin receptor antagonist, peptide 234. In contrast, kisspeptin inhibits orexigenic neuropeptide Y (NPY) neurons through an indirect mechanism based on enhancing GABA-mediated inhibitory synaptic tone. In striking contrast, gonadotropin-inhibiting hormone (GnIH and RFRP-3) and NPY, also found in axons abutting POMC cells, inhibit POMC cells and attenuate the kisspeptin excitation by a mechanism based on opening potassium channels. Together, these data suggest that the two central peptides that regulate reproduction, kisspeptin and GnIH, exert a strong direct action on POMC neurons. POMC cells may hypothetically serve as a conditional relay station downstream of kisspeptin and GnIH to signal the availability of energy resources relevant to reproduction.

High-Density Presynaptic Transporters Are Required for Glutamate Removal from the First Visual Synapse

Reliable synaptic transmission depends not only on the release machinery and the postsynaptic response mechanism but also on removal or degradation of transmitter from the synaptic cleft. Accumulating evidence indicates that postsynaptic and glial excitatory amino acid transporters (EAATs) contribute to glutamate removal. However, the role of presynaptic EAATs is unclear. Here, we show in the mouse retina that glutamate is removed from the synaptic cleft at the rod to rod bipolar cell (RBC) synapse by presynaptic EAATs rather than by postsynaptic or glial EAATs. The RBC currents evoked by electrical stimulation of rods decayed slowly after pharmacological blockade of EAATs. Recordings of the evoked RBC currents from EAAT subtype-deficient mice and the EAAT-coupled anion current reveal that functional EAATs are localized to rod terminals. Model simulations suggest that rod EAATs are densely packed near the release site and that rods are equipped with an almost self-sufficient glutamate recollecting system.

Cellular mechanisms of nociception in the frog.

Cellular mechanisms underlying defense reactions induced by noxious heat and acids were studied in frogs (Rana pipiens) by measuring whole cell membrane currents in cultured dorsal root ganglion (DRG) neurons. Seventy-eight of 82 DRG neurons exposed to 3-s ramps of increasing temperature to 48 degrees C exhibited an inward current (I(HEAT)) of 490 +/- 70 pA at -70 mV. I(HEAT) exhibited reversal at approximately 10 mV with a pronounced outward rectification, suggesting opening of nonselective cation channels. In frogs, in contrast to mammals, I(HEAT) was not influenced by capsaicin (5 microM), capsazepine (10 microM), or ruthenium red (10 microM). In a large proportion (approximately 80%) of heat-sensitive DRG neurons, acids produced a large slowly inactivating sodium carried current (I(ACID)) with average pH(50) 5.7. I(ACID) was blocked by 1 mM amiloride (to 22%) and was absent if extracellular Na(+) was substituted by Cs(+). Elevating temperature to 38 degrees C increased I(ACID), whereas temperatures >40 degrees C profoundly inhibited it (by 82 +/- 2%; n = 42). The inhibition was long-lasting (>30 s) but fully reversible. Phorbol ester myristate acetate (PMA, 1 microM) and forskolin (1 microM) inhibited I(ACID) to 37 +/- 5% (n = 5) and 78 +/- 8% (n = 4), respectively. It is suggested that I(HEAT) in frog DRG neurons is carried through capsaicin-insensitive nonselective cation channels distinct from vanilloid receptor in mammals, whereas I(ACID) is carried through amiloride-sensitive sodium channels that are strongly inhibited by noxious heat, possibly due to activation of the intracellular messenger systems.

Angiotensin II induces inward currents in subfornical organ neurones of rats.

The action of angiotensin II on subfornical organ (SFO) neurones was studied using whole-cell current and voltage-clamp recordings in rat slice preparations. In the current-clamp mode, membrane depolarization in response to angiotensin II was accompanied by an increased frequency of action potentials and an increased membrane conductance. In the voltage-clamp mode, angiotensin II elicited inward currents in a dose-dependent manner. The net angiotensin II-induced inward currents were voltage-independent, with a mean reversal potential of -29.8 +/- 6.2 mV. Amplitudes of the angiotensin II-induced inward currents were decreased during perfusion with a low sodium medium. The angiotensin II-induced inward currents were blocked by the AT1 antagonist losartan, and were partially blocked by the AT2 antagonist PD-123319. Neurones which were sensitive to angiotensin II were found in the peripheral region of the SFO, whereas neurones in the central region were less sensitive to angiotensin II. These results suggest that angiotensin II induces inward currents, with opening of nonselective cation channels through mainly AT1 receptors in a subpopulation of SFO neurones of rats.

Neurotensin excites periaqueductal gray neurons projecting to the rostral ventromedial medulla.

Microinjection of neurotensin into the midbrain periaqueductal gray (PAG) produces a potent and naloxone-insensitive analgesic effect. To test the hypothesis that neurotensin induces the analgesic effect by activating the PAG-rostral ventromedial medulla (RVM) descending antinociceptive pathway, PAG neurons that project to RVM (PAG-RVM) were identified by microinjecting DiI(C18), a retrograde tracing dye, into the rat RVM. Subsequently, fluorescently labeled PAG-RVM projection neurons were acutely dissociated and selected for whole cell patch-clamp recordings. During current-clamp recordings, neurotensin depolarized retrogradely labeled PAG-RVM neurons and evoked action potentials. Voltage-clamp recordings indicated that neurotensin excited PAG-RVM neurons by opening the voltage-insensitive and nonselective cation channels. Both SR 48692, a selective NTR-1 antagonist, and SR 142948A, a nonselective antagonist of NTR-1 and NTR-2, failed to prevent neurotensin from exciting PAG-RVM neurons. Neurotensin failed to evoke cationic currents after internally perfusing PAG-RVM projection neurons with GDP-beta-S or anti-G(alpha q/11) antiserum. Cellular Ca(2+) fluorescence measurement using fura-2 indicated that neurotensin rapidly induced Ca(2+) release from intracellular stores of PAG-RVM neurons. Neurotensin-evoked cationic currents were blocked by heparin, an IP(3) receptor antagonist, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a fast chelator of Ca(2+). These results suggest that by activating a novel subtype of neurotensin receptors, neurotensin depolarizes and excites PAG-RVM projection neurons through enhancing Ca(2+)-dependent nonselective cationic conductance. The coupling mechanism via G(alpha q/11) proteins is likely to involve the production of IP(3), and subsequent IP(3)-evoked Ca(2+) release leads to the opening of nonselective cation channels.

Electrophysiological evidence for an ATP-gated ion channel in the principal cells of the frog skin epithelium

In the present study we investigated the effects of adenosine 5'-triphosphate (ATP) on Na+ transport in frog skin epithelium. An experimental set-up was constructed to allow simultaneous measurement of Na+ transport, measured as the amiloride-sensitive short circuit current (Isc), and free cytosolic Ca2+ concentration ([Ca2+]i) measured with the Ca(2+)-sensitive dye fura-2. The cell potential (Vsc) was measured with microelectrodes. Addition of ATP (100 micrM) to the basolateral solution resulted in a fast transient decrease in Isc followed by a slower increase and a transient increase in [Ca2+]i. Microelectrode measurements showed that the primary response, i.e. the decline in Isc was accompanied by transient depolarisation, followed by a return to the control value. The decrease in current was Ca2+ independent; i.e. treatment with thapsigargin in Ca(2+)-free solutions abolished the Ca2+ transient but did not influence the current transient. The secondary response, i.e. the slow increase in current, was accompanied by slow depolarisation of the cell. Measurements of apical Na+ permeability showed that this was due to an opening or activation of apical Na+ channels. These data show that ATP causes a fast initial drop and a secondary, long-lasting increase in Na+ absorption. The ability of ATP to cause the initial decline in current is independent of Ca2+, i.e. it is not caused by secondary effects of the P2Y-type receptors present in the tissue. Measurements of intracellular potential indicate that the initial depolarisation is caused by opening of non-selective cation channels, suggesting that this decrease is due to a transient activation of P2X-type ATP receptors.

Actions of prostaglandin E2 on rat supraoptic neurones.

Prostaglandins (PGs) have been implicated in the regulation of vasopressin (VP) and oxytocin (OT) release in response to various stimuli. To examine the site and mechanism of actions of PGs, we studied effects of PGE2 and PG-receptor agonists on supraoptic nucleus (SON) neurones of rat hypothalamic slice preparations using extracellular recording and whole-cell patch-clamp techniques. PGE2 modulated the electrical activity of more than 80% of the neurones studied. The effects of PGE2 on both phasic and non-phasic neurones were mostly excitatory, and dose-dependent. The effects of PGE2 were mimicked by PGF2alpha or the FP agonist, fluprostenol, whereas PGD2 or the selective EP, IP or TP agonist was less effective or had no effect. The effects of PGE2 were unaffected by the EP1 antagonist, SC-51322, but reduced to 80% of control by the EP1/FP/TP antagonist, ONO-NT-012, which reduced the effects of fluprostenol to 32% of control. Moreover, some neurones responsive to PGE2 did not respond to fluprostenol. Patch-clamp analysis in SON slice preparations revealed that PGE2 at 10(-6) M depolarized the membrane potential by 3.9+/-0.3 mV from the resting membrane potential of -58.4+/-2.2 mV in the current-clamp mode. In the voltage-clamp mode, PGE2 induced inward currents at a holding potential of -70 or -80 mV, while it did not affect spontaneous excitatory postsynaptic currents. PGE2 induced currents also in dissociated SON neurones and the reversal potential of the currents was -35.5+/-0.9 mV, which was similar to that of currents induced by fluprostenol. These results suggest that SON neurones possess at least two types of PG receptors, FP receptors and EP receptors of a subclass different from EP1, EP2, or EP3, and that activation of these receptors leads to the opening of nonselective cation channels, membrane depolarization and increase of the action potential discharge.

Patch-clamp analysis of the mechanism of PACAP-induced excitation in rat supraoptic neurones.

In neurosecretory cells of the supraoptic nucleus (SON) of rats, pituitary adenylate cyclase activating polypeptide (PACAP) causes an increase in [Ca2+]i, and stimulates somatodendritic vasopressin (VP) release. In this report, to elucidate the ionic mechanism of the action of PACAP, membrane potentials and ionic currents were measured from SON neurones in slice preparations or from dissociated SON neurones. In the current clamp mode, PACAP depolarized membrane potentials of both phasic and non-phasic neurones and increased the firing rate. Moreover, simultaneous measurements of membrane potentials and [Ca2+]i revealed that the membrane depolarization correlated well with increases in [Ca2+]i. In the voltage-clamp mode, PACAP induced inward currents at a holding potential of -70 or -80 mV in a dose-dependent manner and the time course of the currents was similar to that of the PACAP-induced membrane depolarization. The averaged reversal potential of the PACAP-induced currents obtained from dissociated SON neurones was -33 mV, which was close to the reversal potential of non-selective cation currents in SON neurones. The currents were rapidly and reversibly inhibited by a cation-channel blocker, gadolinium. Analysis of synaptic inputs into SON neurones in slice preparations revealed that PACAP had little or no effects on the frequency of spontaneous excitatory and inhibitory postsynaptic currents. These results suggest that pituitary adenylate cyclase activating polypeptide (PACAP) activates PACAP receptors in the postsynaptic membrane of the supraoptic nucleus (SON) neurones, and that the activation of PACAP receptors leads to opening of non-selective cation channels, depolarization of the membrane potential, and increase in the firing rate in SON neurones. Such mechanisms may account for the PACAP-induced increase in [Ca2+]i and vasopressin (VP) release observed in SON neurones.

Metabotropic glutamate receptor enhancement of spontaneous IPSCs in neocortical interneurons.

Metabotropic glutamate receptor enhancement of spontaneous IPSCs in neocortical interneurons. J. Neurophysiol. 78: 2287-2295, 1997. Using neocortical layer I neurons as a model for GABAergic interneurons, we have studied gamma-aminobutyric acid-A (GABAA) receptor-mediated spontaneous inhibitory postsynaptic currents (IPSCs) and modulation by metabotropic glutamate receptors (mGluRs). In the presence of 0.5 mu M tetrodotoxin (TTX) and ionotropic glutamate receptor antagonists and under symmetrical Cl- conditions, the mean amplitude of miniature IPSCs (mIPSCs) was approximately 50 pA at a holding potential of -70 mV with individual events ranging from 10 to 400 pA. Averaged mIPSCs had a 10-90% rise time of approximately 0.6 ms. The decay was double exponential. The fast component had a time constant of approximately 4 ms and comprised approximately 40% of the total amplitude. The slow component had a time constant of approximately 22 ms. The frequency of spontaneous IPSCs (sIPSCs), recorded in the absence of TTX, was increased by bath application of the mGluR agonist 1S,3R-1-aminocyclopentane-1, 3-dicarboxylic acid (ACPD; 10-100 mu M) or the group I mGluR selective agonist quisqualic acid (Quis; 0.5-1 mu M). Under identical conditions, mIPSCs were not affected. The kinetics of sIPSCs and mIPSCs were not altered by ACPD or Quis. Quis (1 mu M) induced an inward current of approximately 70 pA at a holding potential of -70 mV, whereas ACPD (40-200 mu M) induced a smaller inward current. This current was linear over the voltage range -70 to +30 mV and reversed polarity near 0 mV. In current-clamp recordings, both Quis and ACPD induced a depolarization and action potential firing in layer I and deeper layer interneurons. We conclude that neocortical layer I neurons receive GABAA receptor-mediated inhibitory synaptic inputs. Activation of mGluRs, possibly mGluR1 and/or mGluR5, causes an enhancement of inhibitory synaptic transmission by directly depolarizing corticalGABAergic interneurons through the opening of nonselective cation channels.

M2 receptor activation of nonselective cation channels in smooth muscle cells: calcium and Gi/G(o) requirements.

Muscarinic stimulation of fura 2-loaded smooth muscle cells evoked a rapidly inactivating Ca(2+)-activated Cl- current [ICl(Ca)] and a sustained nonselective cation current (Icat) as well as a transient (delta Ca(tran)) and a sustained (delta Ca(sus)) elevation of cytosolic Ca2+ concentration ([Ca2+]i). Caffeine and inositol 1,4,5-trisphosphate induced delta Ca(tran) and ICl(Ca) but not Icat or delta Ca(sus). M2 receptor antagonism blocked muscarinic activation of Icat and delta Ca(sus) but not ICl(Ca) and delta Ca(tran). M3 antagonism blocked activation of ICl(Ca) and Icat and a rise in [Ca2+]i, but application of caffeine with methacholine restored Icat and delta Ca(sus). After depletion of intracellular Ca2+ stores, methacholine failed to induce Icat or a [Ca2+]i increase and, in pertussis toxin-treated cells, ICl(Ca) and delta Ca(tran) but not Icat or delta Ca(sus) were evoked. Anti-G alpha i-1/G alpha i-2 antibodies and anti-G alpha i-3/ G(o) alpha antibodies blocked Icat but did not affect ICl(Ca). Anti-Gq alpha/ G alpha 11 antibodies greatly inhibited ICl(Ca) but did not affect Icat. Activation of M2 receptors leads to the opening of nonselective cation channels through Gi/G(o) proteins in smooth muscle cells, resulting in a sustained rise in [Ca2+]i. Arise in [Ca2+]i is necessary but not sufficient for activation of nonselective cation channels.

The excitatory effect of cholecystokinin on rat neostriatal neurons: ionic and molecular mechanisms

Whole-cell patch-clamp recordings were performed to study ionic and molecular mechanisms by which cholecystokinin (CCK) peptides modulate the membrane excitability of acutely dissociated rat neostriatal neurons. Immunohistochemical staining studies indicated that about 95% of acutely isolated neostriatal neurons were GABA(γ-aminobutyric acid)ergic medium-sized cells. During current-clamp recordings, sulfated cholecystokinin octapeptide (CCK-8) depolarized neostriatal neurons and evoked action potentials. During voltage-clamp recordings, CCK-8 induced inward currents at negative membrane potentials by increasing the voltage-insensitive and non-selective cationic conductance. Cholecystokinin tetrapeptide (CCK-4), a selective CCK B receptor agonist, also evoked cationic currents. The CCK-8-induced cation currents were antagonized by PD135,158 (4-&{[2-[[3-(1H-indol-3yl)-2-mehtyl-1-oxo-2-[[[1.7.7.-tri-methyl-bicyclo [2.2.1]hept-2-yl)oxy]carbonyl]amino]propyl]amino]-1-phenylethyl]amino-4-oxo-[1S-1α, 2β[S ∗(S ∗)]4α]}-butanoate N-methyl- d-glucamine). a highly specific and potent CCK b receptor antagonist. The CCK-8-evoked inward currents were blocked by the internal perfusion of 1 mM GDP-β-S. In neostriatal neurons dialyzed with 0.5 mM GTP-γ-S. the cationic currents produced by CCK-8 became irreversible. Pretreating neostriatal neurons with 500 ng/ml pertussis toxin did not prevent CCK-8 from evoking cationic currents. Internal administration of heparin (2 mg/ml), an inositol 1,4,5-trisphosphate (IP 3) receptor antagonist, and buffering of intracellular calcium with the Ca 2+-chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid. 10 mM), suppressed CCK-8-evoked cationic currents. These findings suggest that, by activating CCK B receptors, CCK-8 excites rat neostriatal neurons through enhancing a non-selective cationic conductance and that pertussis toxin-insensitive G-proteins mediate CCK-8 enhancement of the cationic conductance. The coupling mechanism via G-proteins is likely to involve the production of IP 3 and the subsequent IP 3-evoked Ca 2+ release leads to the opening of non-selective cation channels.

Calcium is permeable through a maitotoxin-activated nonselective cation channel in mouse L cells.

The shellfish poison maitotoxin causes the irreversible opening of nonselective cation channels in mouse L cell fibroblasts, consistent with the action of this toxin in other cell types and the previously demonstrated existence of 28-pS voltage-insensitive nonselected cation channels that are activated by platelet-derived growth factor in these cells. Toxin-induced opening of these nonselective cation channels led to increases of intracellular calcium and secondary activation of calcium-activated potassium channel. These effects were completely dependent on influx of extracellular calcium, supporting the conclusion that the maitotoxin-activated nonselective cation channels are permeable to calcium as well as to sodium and potassium. The implication of this finding is that calcium signaling through this channel underlies its links into the growth factor response.

Activation of a cAMP-regulated Ca2+-Signaling Pathway in Pancreatic β-Cells by the Insulinotropic Hormone Glucagon-like Peptide-1

Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone that is currently under investigation for use in the treatment of diabetes mellitus. To investigate the Ca2+ signaling pathways by which GLP-1 may stimulate the secretion of insulin from pancreatic beta-cells, we examined its effects on the concentration of free intracellular Ca2+ ([Ca2+]i) while simultaneously determining what action it exerts on ion channel function. Measurements of [Ca2+]i were obtained from single rat beta-cells and from beta TC6 and HIT-T15 insulinoma cells loaded with the Ca2+ indicator fura-2, and changes in membrane potential and current were monitored using the perforated patch clamp technique. We report a previously undocumented action of GLP-1 and analogs of cAMP (8-bromo-cAMP, Sp- or Rp-adenosine 3',5'-cyclic monophosphothionate triethylamine) to raise [Ca2+]i that is attributable to the activation of a prolonged inward current designated here as IcAMP. Activation of IcAMP is associated with an increased membrane conductance, membrane depolarization, and triggers large increases of [Ca2+]i. IcAMP is primarily a Na+ current that is blocked by extracellularly applied La3+ or by intracellular administration of Ca2+ chelators (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl, EGTA) and which exhibits a reversal potential of about -26 mV. We propose that IcAMP results from the opening of nonselective cation channels that are activated by intracellular Ca2+ and cAMP and which might play an important role in the regulation of insulin secretion from pancreatic beta-cells.

Effects of excitatory neurotransmitters on Ca2+ channel current in smooth muscle cells isolated from guinea‐pig urinary bladder

1. A whole-cell voltage clamp technique was used to examine the effects of purinoceptor and muscarinic receptor agonists on voltage-sensitive Ca2+ channels in guinea-pig isolated urinary bladder cells. 2. When the cell membrane was clamped at the holding potential, rapid application of ATP elicited a large inward current in normal solution containing 2.5 mM Ca2+, and reduced the subsequent Ca2+ channel current evoked by a depolarizing pulse (0 mV). Carbachol (CCh) elicited little membrane current, but similarly reduced the Ca2+ current. 3. When purinoceptor agonists were rapidly applied during conditioning depolarizations at +80 mV, an outward current was elicited, and the Ca2+ channel current evoked by the subsequent test potential of 0 mV was not affected. Application of CCh at +80 mV also elicited an outward current, but it reduced the subsequently evoked Ca2+ current. 4. The inhibitory effect of muscarinic agonists on the Ca2+ channel current was attenuated by caffeine (10 mM). 5. In Ca(2+)-free, low-Mg2+ solution, a Na+ current flowing through voltage-dependent Ca2+ channels was evoked by depolarization. This current was not reduced by bath application of purinoceptor agonists (ATP and alpha,beta-methylene ATP). 6. These results suggest that the main effect of purinoceptor stimulation is opening of non-selective cation channels, and that muscarinic stimulation triggers Ca2+ release from intracellular stores. Voltage-sensitive Ca2+ channels are inactivated through an increase in intracellular Ca2+ induced by either activation of purinoceptor or muscarinic receptors.

Thrombin and NaF, but not epinephrine, raise cytosolic free Na + in human platelets

We have investigated changes in [Na +] i in SBFI-loaded platelets stimulated at 37°C with thrombin, epinephrine, and NaF. Basal [Na +] i was 4.9 ± 1.3 mM ( n = 70). Stimulation of platelets with thrombin (0.1 U / ml) in the presence of 1 mM extracellular Ca 2+ rapidly raised [Na +] i by 27.3 ± 6 mM ( n = 16). Part of this increase (approx. 20–30%) is caused by Na + H + exchange, the rest is predominantly due to Na + influx. Epinephrine (20 μM) failed to change [Na +] i both in the absence and presence of fibrinogen. This is in agreement with earlier reports showing that epinephrine also fails to activate Na + H + exchange in human platelets. NaF which activates platelets via a direct effect on GTP-binding proteins induced a slow rise in [Na +] i to 9.5 ± 2.5 mM ( n = 4) and 33.0 ± 3.6 mM ( n = 12) at 10 and 20 mM NaF, respectively. This effect was completely blocked by SK&F 96365, a blocker of receptor-mediated Ca 2+ entry. Hence, the NaF-induced increase in [Na +] i is exclusively due to the opening of non-selective cation channels. This latter finding agrees with earlier observations which showed that NaF does not induce activation of Na + H + exchange in platelets.

Influence of internal calcium stores on calcium-activated membrane currents in smooth muscle.

Agonist-induced activation of second messenger systems that increase inositol 1,4,5-trisphosphate concentration plays an important role in the mobilisation of stored Ca2+ in smooth muscle. Release of Ca2+ which occurs spontaneously or by agonist stimulation causes activation of Ca(2+)-sensitive currents. Experiments using selective modulators of the Ca2+ uptake and release mechanisms of the sarcoplasmic reticulum provide evidence that depletion of the internal Ca2+ store triggers an influx of Ca2+ from the extracellular space. Recent data suggest that pool depletion causes the opening of non-selective cation channels which are possibly similar to those previously designated 'receptor-operated Ca2+ channels'. Ca2+ entry through these channels not only refills the internal Ca2+ pool, but also modulates membrane excitability by stimulating Ca(2+)-activated currents.

Ca2+ induces charybdotoxin-sensitive membrane potential changes in rat lymphocytes

There is disagreement regarding the existence of Ca2+-activated K+ channels in lymphocytes. Depolarization, hyperpolarization, or little change in membrane potential (Em) has been reported following elevation of free cytosolic Ca2+ concentration ([Ca2+]i). Patch-clamping studies have demonstrated inhibition of voltage-gated K+ channels, but Ca2+-activated K+ channels have not been detected. We used charybdotoxin (CTX), a potent inhibitor of Ca2+-activated K+ channels, to assess their presence in rat thymic lymphocytes. Fluorescent probes were used to measure Em and [Ca2+]i in cell suspensions treated with ionomycin. At basal [Ca2+]i, CTX had no effect on Em, suggesting that Ca2+-activated K+ channels do not contribute importantly to the resting potential. Elevation of [Ca2+]i in the submicromolar range induced a hyperpolarization that was dependent on the outward K+ gradient. The shape and duration of the Em change closely followed the elevation of [Ca2+]i. This hyperpolarization was inhibited by nanomolar concentrations of CTX. When [Ca2+]i approached or exceeded 1 microM, a biphasic Em change was recorded. A transient, CTX-sensitive hyperpolarization was followed by a sustained depolarization. The latter was greatly reduced when external Na+ was omitted. The data suggest that thymic lymphocytes possess Ca2+-sensitive K+ channels, which are activated by moderate increases in [Ca2+]i, resulting in hyperpolarization. At higher [Ca2+]i, the effect of K+ channels on Em is superseded by opening of nonselective cation channels, producing depolarization. Variations in the level of [Ca2+]i attained in earlier studies can explain existing discrepancies.