The role of K+ in the regulation of the increase in intracellular Ca2+ mediated by the T lymphocyte antigen receptor

The regulation of the increase in intracellular calcium ([Ca2+]i) occurring in cytolytic T lymphocytes (CTLs) upon their interaction with antigen was examined. This [Ca2+]i increase and lytic function were insensitive to verapamil, a Ca channel blocker. An antigen-independent increase in [Ca2+]i was not induced by depolarization of CTLs with excess extracellular K+, suggesting that Ca2+ influx is not mediated by the ubiquitous voltage-gated Ca channel. The antigen-induced [Ca2+]i increase was inhibited by prior membrane hyperpolarization with valinomycin. Hyperpolarization occurred under normal circumstances in CTLs exposed to antigen-receptor-specific antibodies. This potential change was Ca2+-dependent and inhibited by K channel blockade. Conversely, K channel blockade augmented the antigen-specific [Ca2+]i increase while markedly decreasing the K+ efflux associated with CTL lytic function. Therefore, either membrane potential or intracellular K+ regulates the antigen-specific [Ca2+]i increase in CTLs.     

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

Caffeine activates a Ca(2+)-permeable, nonselective cation channel in smooth muscle cells

The effects of caffeine on cytoplasmic [Ca2+] ([Ca2+]i) and plasma membrane currents were studied in single gastric smooth muscle cells dissociated from the toad, Bufo marinus. Experiments were carried out using Fura-2 for measuring [Ca2+]i and tight-seal voltage-clamp techniques for recording membrane currents. When the membrane potential was held at -80 mV, in 15% of the cells studied caffeine increased [Ca2+]i without having any effect on membrane currents. In these cells ryanodine completely abolished any caffeine induced increase in [Ca2+]i. In the other cells caffeine caused both an increase in [Ca2+]i and activation of an 80-pS nonselective cation channel. In this group of cells ryanodine only partially blocked the increase in [Ca2+]i induced by caffeine; moreover, the change in [Ca2+]i that did occur was tightly coupled to the time course and magnitude of the cation current through these channels. In the presence of ryanodine, blockade of the 80-pS channel by GdCl3 or decreasing the driving force for Ca2+ influx through the plasma membrane by holding the membrane potential at +60 mV almost completely blocked the increase in [Ca2+]i induced by caffeine. Thus, the channel activated by caffeine appears to be permeable to Ca2+. Caffeine activated the cation channel even when [Ca2+]i was clamped to below 10 nM when the patch pipette contained 10 mM BAPTA suggesting that caffeine directly activates the channel and that it is not being activated by the increase in Ca2+ that occurs when caffeine is applied to the cell. Corroborating this suggestion were additional results showing that when the membrane was depolarized to activate voltage-gated Ca2+ channels or when Ca2+ was released from carbachol- sensitive internal Ca2+ stores, the 80-pS channel was not activated. Moreover, caffeine was able to activate the channel in the presence of ryanodine at both positive and negative potentials, both conditions preventing release of Ca2+ from stores and the former preventing its influx. In summary, in gastric smooth muscle cells caffeine transiently releases Ca2+ from a ryanodine-sensitive internal store and also increases Ca2+ influx through the plasma membrane by activating an 80- pS cation channel by a mechanism which does not seem to involve an elevation of [Ca2+]i.     

Synaptically activated increases in Ca2+ concentration in hippocampal CA1 pyramidal cells are primarily due to voltage-gated Ca2+ channels.

Changes in intracellular Ca2+ concentration ([Ca2+]i) in the soma and dendrites of hippocampal CA1 pyramidal neurons were measured using intracellularly injected fura-2. A large component of the [Ca2+]i elevation caused by high frequency stimulation of the Schaffer collaterals was correlated with the Na+ spikes triggered by the excitatory postsynaptic potentials (EPSPs). These spikes were generated in the soma and proximal dendrites and stimulated Ca2+ entry through voltage-gated Ca2+ channels. Suppressing spikes by hyperpolarizing the soma or by injecting QX-314 revealed a smaller nonspike component of Ca2+ entry. A substantial fraction of this component was mediated by the action of the EPSPs on voltage-gated Ca2+ channels, because it persisted in 2-amino-5-phosphonovaleric acid and because it was usually reduced when Ca2+ channel activity was suppressed by hyperpolarization. Ca2+ entry through the N-methyl-D-aspartate receptor channel could not be detected with certainty, perhaps because it was highly localized.     

Somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry through voltage-dependent Ca2+ channels in the beta cell.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.     

Membrane potential as a modulator of the free intracellular Ca2+ concentration in agonist-activated endothelial cells.

We have used combined patch clamp and fura-2 fluorescence to elucidate the role of membrane potential in the regulation of the cytosolic Ca2+ concentration ([Ca2+]i) in a human umbilical vein derived endothelial cell-line, EA.hy926 (EA cells) stimulated with vasoactive agonists, such as ATP, histamine and bradykinin. This stimulation caused hyperpolarization and sustained Ca2+ plateau in nonclamped cells. Clamping agonist-stimulated cells at negative potentials enhanced the amplitude of this plateau, whereas it was smaller at more depolarized potentials, indicating that Ca2+ influx follows its driving force. Depolarization of the membrane by increasing extracellular K+ or by applying charybdotoxin, a blocker of big conductance Ca2+-dependent K+ channels during agonist stimulation diminished the plateau rise in [Ca2+]i. It is concluded that the membrane potential is an efficient regulator of Ca2+ influx during the plateau phase of agonist-mediated Ca2+ signals. In addition, the modulating effects on Ca2+ signals should be interpreted with caution if the membrane potential of the cells is not controlled.     

Effects of insulin and phosphatase on a Ca2(+)-dependent Cl- channel in a distal nephron cell line (A6)

A Cl- channel with a small single-channel conductance (3 pS) was observed in cell-attached patches formed on the apical membrane of cells from the distal nephron cell line (A6) cultured on permeable supports. The current-voltage (I-V) relationship from cell-attached patches or inside-out patches with 1 microM cytosolic Ca2+ strongly rectified with no inward current at potentials more negative than ECl. However, the rectification decreased (i.e., inward current increased) when the cytosolic Ca2+ concentration ([Ca2+]i) was increased above 1 microM. If [Ca2+]i is increased to 800 microM, the I-V relationship became linear. Besides the change in the I-V relationship, an increase in [Ca2+]i also increases the open probability of the channel. Regardless of the recording condition, the channel has one open and one closed state. Both closing and opening rates were dependent on [Ca2+]i; an increase of [Ca2+]i decreased the closing rate and increased the opening rate. The Ca2+ dependence of transition rates at positive membrane potentials (cell interior with respect to external surface) were much larger than the dependence at negative intracellular potentials. The I-V relationship of chloride channels in inside-out patches from cells pretreated with insulin was linear even with 1 microM [Ca2+]i, while channel currents from cells under similar conditions but without insulin still strongly rectified. Alkaline phosphatase applied to the intracellular surface of inside-out patches altered the outward rectification of single channels in a manner qualitatively similar to that of insulin pretreatment. These observations suggest that phosphorylation/dephosphorylation of the channel modulates the sensitivity of the Cl- channel to cytosolic Ca2+ and that insulin produces its effect by promoting dephosphorylation of the channel.     

Developmental differences in Ca2+-activated K+ channel activity in ovine basilar artery

A primary determinant of vascular smooth muscle (VSM) tone and contractility is the resting membrane potential, which, in turn, is influenced heavily by K+ channel activity. Previous studies from our laboratory and others have demonstrated differences in the contractility of cerebral arteries from near-term fetal and adult animals. To test the hypothesis that these contractility differences result from maturational changes in voltage-gated K+ channel function, we compared this function in VSM myocytes from adult and fetal sheep cerebral arteries. The primary current-carrying, voltage-gated K+ channels in VSM myocytes are the large conductance Ca2+-activated K+ channels (BKCa) and voltage-activated K+ (KV) channels. We observed that at voltage-clamped membrane potentials of +60 mV in perforated whole cell studies, the normalized outward current densities in fetal myocytes were >30% higher than in those of the adult (P < 0.05) and that these were predominantly due to iberiotoxin-sensitive currents from BKCa channels. Excised, insideout membrane patches revealed nearly identical unitary conductances and Hill coefficients for BKCa channels. The plot of log intracellular [Ca2+] ([Ca2+]i) versus voltage for half-maximal activation (V(1/2)) yielded linear and parallel relationships, and the change in V(1/2) for a 10-fold change in [Ca2+] was also similar. Channel activity increased e-fold for a 19 +/- 2-mV depolarization for adult myocytes and for an 18 +/- 1-mV depolarization for fetal myocytes (P > 0.05). However, the relationship between BKCa open probability and membrane potential had a relative leftward shift for the fetal compared with adult myocytes at different [Ca2+]i. The [Ca2+] for half-maximal activation (i.e., the calcium set points) at 0 mV were 8.8 and 4.7 microM for adult and fetal myocytes, respectively. Thus the increased BKCa current density in fetal myocytes appears to result from a lower calcium set point.     

Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium

Voltage-gated n-type K(V) and Ca(2+)-activated K+ [K(Ca)] channels were studied in cell-attached patches of activated human T lymphocytes. The single-channel conductance of the K(V) channel near the resting membrane potential (Vm) was 10 pS with low K+ solution in the pipette, and 33 pS with high K+ solution in the pipette. With high K+ pipette solution, the channel showed inward rectification at positive potentials. K(V) channels in cell-attached patches of T lymphocytes inactivated more slowly than K(V) channels in the whole-cell configuration. In intact cells, steady state inactivation at the resting membrane potential was incomplete, and the threshold for activation was close to Vm. This indicates that the K(V) channel is active in the physiological Vm range. An accurate, quantitative measure for Vm was obtained from the reversal potential of the K(V) current evoked by ramp stimulation in cell-attached patches, with high K+ solution in the pipette. This method yielded an average resting Vm for activated human T lymphocytes of -59 mV. Fluctuations in Vm were detected from changes in the reversal potential. Ionomycin activates K(Ca) channels and hyperpolarizes Vm to the Nernst potential for K+. Elevating intracellular Ca2+ concentration ([Ca2+]i) by ionomycin opened a 33-50-pS channel, identified kinetically as the CTX-sensitive IK-type K(Ca) channel. The Ca2+ sensitivity of the K(Ca) channel in intact cells was determined by measuring [Ca2+]i and the activity of single K(Ca) channels simultaneously. The threshold for activation was between 100 and 200 nM; half-maximal activation occurred at 450 nM. At concentrations > 1 microM, channel activity decreased. Stimulation of the T-cell receptor/CD3 complex using the mitogenic lectin, PHA, increased [Ca2+]i, and increased channel activity and current amplitude resulting from membrane hyperpolarization.     

Protein phosphorylation activates the guard cell Ca2+ channel and is a prerequisite for gating by abscisic acid

Protein phosphorylation and cytosolic-free [Ca2+] ([Ca2+]i) contribute to signalling cascades evoked by the water-stress hormone abscisic acid (ABA) that lead to stomatal closure in higher-plant leaves. ABA activates an inward-rectifying Ca2+ channel at the plasma membrane of stomatal guard cells, promoting Ca2+ entry by shifting the voltage-sensitivity of the channels. Because many of these effects could be mediated by kinase/phosphatase action at the membrane, we examined a role for protein (de-)phosphorylation in plasma membrane patches from Vicia guard cells. Ca2+ channel activity decayed rapidly in excised patches, and recovered on adding ATP (K1/2, 1.3 +/- 0.7 mm) but not the non-hydrolyzable analog ATPgammaS. ABA activation of the channel required the presence of ATP and like ABA, the 1/2 A-type protein phosphatase antagonists okadaic acid (OA) and calyculin A (CA) enhanced Ca2+ channel activity by increasing the open probability and number of active channels. Neither ATP nor the antagonists affected the mean open lifetime of the channel, suggesting an action through changes in closed lifetime distributions. Like ABA, OA and CA shifted the voltage-sensitivities of the Ca2+ current and [Ca2+]i increases in intact guard cells towards positive voltages. OA and CA also augmented the [Ca2+]i rise evoked by hyperpolarization and delayed its recovery. These results demonstrate a membrane-delimited interaction between 1/2 A-type protein phosphatase(s) and the Ca2+ channel or associated proteins, and they are consistent with a role for protein (de-)phosphorylation in ABA signalling mediated directly through Ca2+ channel gating that leads to [Ca2+]i increases in the guard cells.     

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.     

Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.     

Dopamine inhibits cytosolic Ca2+ increases in rat lactotroph cells. Evidence of a dual mechanism of action

Single rat lactotroph cells were studied after loading with the cytosolic free Ca2+ concentration ([Ca2+]i) indicator fura-2 either 1 or 3 days after cell dispersion. Under unstimulated conditions, two groups of lactotrophs were observed, the first (predominant at day 1) with large [Ca2+]i fluctuations (peaks up to 300 nM) probably due to spontaneous action potentials and the second (predominant at 3 days) with stable [Ca2+]i (values variable between 65 and 200 nM). The effect of dopamine on the resting [Ca2+]i was different in the two groups. Even at high dopamine concentrations, no change occurred in the second group; whereas in the first, disappearance of fluctuations and marked decrease of [Ca2+]i were observed. These effects of dopamine appear to be due to hyperpolarization that was demonstrated by the use of a specific fluorescent indicator, bis(oxonol). Two types of triggered [Ca2+]i transients were studied in detail: those due to redistribution of Ca2+ from the intracellular stores (induced by thyrotropin-releasing hormone) and those due to Ca2+ influx through voltage-gated Ca2+ channels (induced by high [K+]). Dopamine (1 microM) markedly inhibited both these transients by the action of D2 receptors (blocked by 1-sulpiride and domperidone). All effects of dopamine were prevented by treatment of the cells with pertussis toxin, indicating the involvement of one (or more) GTP-binding protein(s). Another consequence of D2 receptor activation is the inhibition of adenylate cyclase. Treatments (cholera toxin, forskolin), known to raise cAMP levels, were found to dissociate the effects of dopamine on [Ca2+]i inasmuch as they markedly relieved the inhibition of the redistributive transients by thyrotropin-releasing hormone but left hyperpolarization and inhibition of K+ transients unaffected. The spectrum of intracellular signals elicited by the activation of D2 receptors is therefore complex and includes at least two mechanisms that involve [Ca2+]i, one related and the other independent of the decrease of cAMP levels.     

Thyrotropin-releasing hormone-induced rise in cytosolic calcium and activation of outward K+ current monitored simultaneously in individual GH3B6 pituitary cells

Thyrotropin-releasing hormone (TRH) acts on pituitary cells to raise the cytosolic free Ca2+ concentration ([Ca2+]i) and causes simultaneously a transient hyperpolarization of the plasma membrane. The combination of the microfluorimetric monitoring of [Ca2+]i with electrophysiological recordings obtained using the patch clamp technique in its whole cell configuration, allows the analysis of the correlation between changes in [Ca2+]i and the alterations in ionic currents at the plasma membrane. It was shown that in the absence of hormone stimulation, a depolarization-induced change in steady state [Ca2+]i, as well as the internal perfusion with Ca2+ at microM levels at constant membrane potential led to the activation of outward K+ current. TRH stimulation resulted in a marked but transient rise in [Ca2+]i; concomitantly, there was an increase in membrane conductance and an enhancement of outward current. During the time course of an individual response, an excellent correlation between the changes in [Ca2+]i and those in conductance or current was observed. The relative changes of current and conductance during the TRH response were consistent with the activation of a single type of ionic current, the apparent reversal potential of which coincided with the equilibrium potential for K+. A strong correlation between the TRH-induced changes in [Ca2+]i and K+, conductance was demonstrated in a large number of cells with varied kinetic features: significant correlation coefficients were found both for the transition time from basal to maximal values (r = 0.85, p less than 0.001) as well as for the total duration of the responses (r = 0.68, p less than 0.002). It is concluded that during the early phase of TRH action, the hormone-induced rise in [Ca2+]i is the principal cause of enhanced K+ channel activation.     

Growth hormone-releasing factor reduces voltage-gated Ca2+ channel current in Rat GH3 cells

Summary The action of GRF on GH₃ cell membrane was examined by patch electrode techniques. Under current clamp with patch elecrtrode, spontaneous action potentials were partially to totally eliminated by application of GRF. In the case of partial elimination, the duration of remaining spontaneous action potentials was prolonged and the amplitude of afterhyperpolarization was decreased. The evoked actiion potential in the cells which did not show spontaneous action potentials was also eliminated by GRF. In order to examine what channels were affected by GRF, voltage-clamp analysis was performed. It was revealed that voltage-gated Ca²⁺ channel current and Ca²⁺-induced K⁺ channels current were decreased by GRF, while voltage-gated Na⁺ channel and delayed K⁺ channel current was considered to be a consequence of he decrease of voltage-gated Ca²⁺ channels current. Therefore it is likely that the effect of GRF on GH₃ cells was due to the block of voltage-gated Ca²⁺ channels. The elimination of action potential under current clamp corresponded to the block of voltage-gated Ca²⁺ channels and the prolongation of action potential could be explained by the decrease of Ca²⁺-induced K⁺ channel current. The amplitude decrease of afterhyperpolarization could also be explained by the reduction of Ca²⁺-induced K⁺ channel current. Thus the results under current clamp well coincide with the results under voltage clamp. Hormone secretion from GH₃ cells was not stimulated by GRF. However, the finding that GRF solely blocked voltage-gated Ca²⁺ channel suggested the specific action of GRF on GH₃ cell membranes.     

Voltage-dependent activation and inactivation of calcium channels in PC12 cells. Correlation with neurotransmitter release

The existence and mechanisms of inactivation of voltage-gated Ca2+ channels are important, but still debatable, physiological problems. By using the Ca2+ indicators quin2 and fura-2, we demonstrate that in PC12 cells voltage-gated Ca2+ channels undergo inactivation dependent on both voltage and [Ca2+]i. Inactivation, however, is never complete and a small number of channels remains open during prolonged depolarization, explaining the steady state elevation of [Ca2+]i observed in cells depolarized with high KCl. A close parallel exists between Ca2+ channel inactivation and the transient nature of neurotransmitter release: secretion is rapidly stimulated during the first 30 s of depolarization, when a transient overshoot in [Ca2+]i can be demonstrated, while it is negligible during the following period, despite the persistence of an elevated [Ca2+]i; predepolarization in Ca2+-free medium and subsequent addition of Ca2+ (a condition which allows the development of the voltage inactivation) abolishes the fast phase of secretion, while not modifying the steady state [Ca2+]i eventually attained; and increases in the intracellular Ca2+ buffering decreases the amplitude of the fast secretion phase induced by KCl without altering the steady state [Ca2+]i. We suggest that localized [Ca2+]i gradients form close to the plasma membrane shortly after depolarization and that the [Ca2+]i reached in these regions is the relevant parameter in the regulation of secretion.     

Mitogen-induced changes in Ca2+ permeability are not mediated by voltage-gated K+ channels

Binding of mitogenic lectins to T lymphocytes results in elevated cytoplasmic Ca2+ concentrations ([Ca2+]i). This change in [Ca2+]i is thought to be essential for cellular proliferation. In addition, the lectins increase the conductance to K+ through voltage-sensitive channels. Based on the inhibitory effect of K+ channel blockers on lectin-induced mitogenesis, it has been suggested that Ca2+ could enter the cells through these activated K+ channels (Chandy, K. G., De Coursey, T. E., Cahalan, M. D., McLaughlin, C., and Gupta, S. (1984) J. Exp. Med. 160, 369-385; Chandy, K. G., De Coursey, T. E., Cahalan, M. D., and Gupta, S. (1985) J. Clin. Immunol. 5, 1-5). This hypothesis was tested experimentally by measuring the effect of activation or blockade of K+ channels on [Ca2+]i using quin-2 and indo-1 and by determining the effect of K+ channel blockers on lectin-induced proliferation. We found that: depolarization of the membrane, which is expected to open the K+ channels, failed to increase [Ca2+]i, K+ channel blockers such as tetraethylammonium and 4-aminopyridine had only a marginal effect on the lectin-induced increase in [Ca2+]i, and the inhibitory effect of K+ channel blockers on proliferation was found to be nonspecific, occurring also when proliferation was triggered by phorbol esters under conditions where [Ca2+]i is not elevated. It is concluded that the lectin-induced changes in [Ca2+]i are not mediated by the opening of voltage-gated K+ channels.     

Spontaneous electrical and calcium oscillations in unstimulated pituitary gonadotrophs.

Single pituitary cells often fire spontaneous action potentials (APs), which are believed to underlie spiking fluctuations in cytosolic calcium concentration ([Ca2+]i). To address how these basal [Ca2+]i fluctuations depend on changes in plasma membrane voltage (V), simultaneous measurements of V and [Ca2+]i were performed in rat pituitary gonadotrophs. The data show that each [Ca2+]i spike is produced by the Ca2+ entry during a single AP. Using these and previously obtained patch-clamp data, we develop a quantitative mathematical model of this plasma membrane oscillator and the accompanying spatiotemporal [Ca2+]i oscillations. The model demonstrates that AP-induced [Ca2+]i spiking is prominent only in a thin shell layer neighboring the cell surface. This localized [Ca2+]i spike transiently activates the Ca2(+)- dependent K+ current resulting in a sharp afterhyperpolarization following each voltage spike. In accord with experimental observations, the model shows that the frequency and amplitude of the voltage spikes are highly sensitive to current injection and to the blocking of the Ca(2+)-sensitive current. Computations also predict that leaving the membrane channels intact, the firing rate can be modified by changing the Ca2+ handling parameters: the Ca2+ diffusion rate, the Ca2+ buffering capacity, and the plasma membrane Ca2+ pump rate. Finally, the model suggests reasons that spontaneous APs were seen in some gonadotrophs but not in others. This model provides a basis for further exploring how plasma membrane electrical activity is involved in the control of cytosolic calcium level in unstimulated as well as agonist-stimulated gonadotrophs.     

Participation of transient-type Ca2+ channels in the sustained increase of Ca2+ level in GH3 cells

Participation of two types of Ca2+ channels (T- and L-types) in the sustained increase of cytosolic-free Ca2+ concentration [( Ca2+]i) was studied in thyrotropin-releasing hormone (TRH)-stimulated clonal GH3 pituitary cells. The effects of Ca2+ channel blockers were analyzed by measuring Ca2+ channel current and [Ca2+]i, using whole-cell voltage-clamp and Fura-2 fluorometry, respectively. Phenytoin (100 microM) and Ni2+ (100 microM) selectively blocked T-type Ca2+ channels and suppressed the TRH-induced sustained [Ca2+]i increase in single cells. Synthetic omega-conotoxin (omega-CgTX, 2 microM) preferentially blocked L-type Ca2+ channels, but it did not suppress the TRH-induced sustained [Ca2+]i increase. The present results suggest that the sustained elevations of [Ca2+]i triggered by TRH may be mediated by T-type Ca2+ channels in GH3 cells.