Calcium-dependent inactivation of L-type calcium channels in planar lipid bilayers.

Intracellular Ca2+ can inhibit the activity of voltage-gated Ca channels by modulating the rate of channel inactivation. Ca(2+)-dependent inactivation of these channels may be a common negative feedback process important for regulating Ca2+ entry under physiological and pathological conditions. This article demonstrates that the inactivation of cardiac L-type Ca channels, reconstituted into planar lipid bilayers and studied in the presence of a dihydropyridine agonist, is sensitive to Ca2+. The rates and extents of inactivation, determined from ensemble averages of unitary Ba2+ currents, decreased when the calcium concentration facing the intracellular surface of the channel ([Ca2+]i) was lowered from approximately 10 microM to 20 nM by the addition of Ca2+ chelators. The rates and extents of Ba2+ current inactivation could also be increased by subsequent addition of Ca2+ raising the [Ca2+]i to 15 microM, thus demonstrating that the Ca2+ dependence of inactivation could be reversibly regulated by changes in [Ca2+]i. In addition, reconstituted Ca channels inactivated more quickly when the inward current was carried by Ca2+ than when it was carried by Ba2+, suggesting that local increases in [Ca2+]i could activate Ca(2+)-dependent inactivation. These data support models in which Ca2+ binds to the channel itself or to closely associated regulatory proteins to control the rate of channel inactivation, and are inconsistent with purely enzymatic models for channel inactivation.     

Minimal requirements for calcium oscillations driven by the IP3 receptor.

Hormones and neurotransmitters that act through inositol 1,4,5-trisphosphate (IP3) can induce oscillations of cytosolic Ca2+ ([Ca2+]c), which render dynamic regulation of intracellular targets. Imaging of fluorescent Ca2+ indicators located within intracellular Ca2+ stores was used to monitor IP3 receptor channel (IP3R) function and to demonstrate that IP3-dependent oscillations of Ca2+ release and re-uptake can be reproduced in single permeabilized hepatocytes. This system was used to define the minimum essential components of the oscillation mechanism. With IP3 clamped at a submaximal concentration, coordinated cycles of IP3R activation and subsequent inactivation were observed in each cell. Cycling between these states was dependent on feedback effects of released Ca2+ and the ensuing [Ca2+]c increase, but did not require Ca2+ re-accumulation. [Ca2+]c can act at distinct stimulatory and inhibitory sites on the IP3R, but whereas the Ca2+ release phase was driven by a Ca2+-induced increase in IP3 sensitivity, Ca2+ release could be terminated by intrinsic inactivation after IP3 bound to the Ca2+-sensitized IP3R without occupation of the inhibitory Ca2+-binding site. These findings were confirmed using Sr2+, which only interacts with the stimulatory site. Moreover, vasopressin induced Sr2+ oscillations in intact cells in which intracellular Ca2+ was completely replaced with Sr2+. Thus, [Ca2+]c oscillations can be driven by a coupled process of Ca2+-induced activation and obligatory intrinsic inactivation of the Ca2+-sensitized state of the IP3R, without a requirement for occupation of the inhibitory Ca2+-binding site.     

Time and calcium dependence of activation and inactivation of calcium- induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell

Microprocessor-controlled changes of [free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils of a skinned canine cardiac Purkinje cell and aequorin bioluminescence recording were used to study the mechanism of Ca2+-induced release of Ca2+ from the SR. This Ca2+ release is triggered by a rapid increase of [free Ca2+] at the outer surface of the SR of a previously quiescent skinned cell. Ca2+-induced release of Ca2+ occurred under conditions that prevented any synthesis of ATP from ADP, was affected differentially by interventions that depressed the SR Ca2+ pump about equally, and required ionic conditions incompatible with all known Ca2+-releasing, uncoupled, partial reactions of the Ca2+ pump. Increasing the [free Ca2+]trigger up to an optimum increased the amount of Ca2+ released. A supraoptimum increase of [free Ca2+] trigger inactivated Ca2+-induced release of Ca2+, but partial inactivation was also observed at [free Ca2+] below that necessary for its activation. The amplitude of the Ca2+ release induced by a given increase of [free Ca2+] decreased when the rate of this increase was diminished. These results suggest that Ca2+-induced release of Ca2+ is through a channel across the SR membrane with time- and Ca2+-dependent activation and inactivation. The inactivating binding site would have a higher affinity for Ca2+ but a lower rate constant than the activating site. Inactivation appeared to be a first-order kinetic reaction of Ca2+ binding to a single site at the outer face of the SR with a Q10 of 1.68. The removal of inactivation was the slowest step of the cycle, responsible for a highly temperature-dependent (Q10 approximately 4.00) refractory period.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

An estimate of rapid cytoplasmic calcium buffering in a single smooth muscle cell

Cytoplasmic calcium increments in the absence of sarco (endo) plasmic reticulum function were measured with a low-affinity fluorophore Indo-1FF in single isolated smooth muscle cells from guinea-pig urinary bladder. To evaluate the Ca(2+)-buffering properties of the myoplasm, Ca2+ influx, measured as time integral of the Ica (integral of Ica), was compared with corresponding free Ca2+ increments (delta [Ca2+]i) in the cytoplasm. The ratio between integral of ICa and delta [Ca2+]i (integral Ica/delta [Ca2+]i), reflecting the Ca2+ buffering properties of the cytosol, was in the range of 4.9-9.3 pC/microM (mean 6.2 +/- 1.2, n = 12). It remained approximately constant (6.4 +/- 1.4 pC/microM, n = 8) during recordings lasting up to 25 min, suggesting that cytoplasmic Ca2+ binding does not change markedly during cell dialysis and that the endogenous Ca2+ buffer is not significantly washed out of the cell through the patch pipette. Wash-in or wash-out of BAPTA, a mobile high-affinity Ca2+ buffer, into or from the cell markedly changed the relationship between Ca2+ influx through Ca2+ channels and delta [Ca2+]i within minutes. Changes in integral of ICa/delta [Ca2+]i during the sequence of depolarizing steps, which increased free [Ca2+]i up to 5 microM, suggested lower limits for the apparent affinity of a rapid Ca2+ buffer (16 microM) and for the total buffer concentration (530 microM). Introduction of 4 mM DPTA (Kd for Ca2+ = 81 microM) into the cell more than doubled the total cytoplasmic Ca2+ buffer capacity. These results suggest that cytoplasmic Ca2+ buffer in smooth muscle cells has a low affinity for free Ca2+. The Ca(2+)-binding ratio of the cytoplasm in most cells was estimated to be between 30 and 40. The Ca(2+)-binding ratio did not differ markedly between cells isolated from neonatal (< or = 5 days) and adult animals.     

Intracellular calcium fluxes in human platelets

Fluorescence changes and secretory responses have been measured on addition of various excitatory agonists to platelets loaded with the cytosolic Ca2+ probe, Quin 2 or with chlortetracycline as a probe for membrane-associated Ca2+. When extracellular [Ca2+] is decreased to less than 0.1 microM by addition of EGTA a linear correlation is observed between the extent of increase in cytosolic [Ca2+] and the extent of mobilisation of membrane-associated Ca2+ on stimulation by maximal doses of five excitatory agonists. A similar linear correlation between the increase in cytosolic [Ca2+] and the extent of ATP secretion is observed over the thrombin dose/response curve. Similar EC50 values are observed for ATP secretion, the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by thrombin. However, the decrease in chlortetracycline fluorescence shows a sigmoidal relationship with the increase in cytosolic [Ca2+] and a hyperbolic relationship with ATP secretion over this dose/response curve. Addition of prostaglandin D2 prior to thrombin causes parallel inhibition of the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by this agonist. However, addition of prostaglandin D2 after thrombin reverses the increase in cytosolic [Ca2+] induced by this agonist but fails to cause a similar reversal of the decrease in chlortetracycline fluorescence. The data provide further evidence supporting the proposal that chlortatracycline can be used as a probe to monitor mobilisation of membrane-associated Ca2+ but suggest that, in platelets stimulated in the effective absence of extracellular Ca2+, both Ca2+ mobilisation and Ca2+ removal can under some conditions involve sites which are not monitored by this probe.     

The role of bivalent ions in the inactivation of bacteriophage phi X174 by lipopolysaccharide from Escherichia coli C.

The need for Ca2+ in the inactivation of bacteriophage phi X174 by lipopolysaccharide from Escherichia coli C was confirmed. Ca2+ could be replaced almost completely by Na+, but the concentration of Na+ needed was greater by more than an order of magnitude. Other bivalent ions caused inactivation in the same way as Ca2+, and the degree of inactivation varied according to the ion. At 50% inactivation of bacteriophage, the relation between the concentrations of NaCl and of bivalent or tervalent ions (Mx+) fitted the conception that NaCl was neutralizing electrostatic repulsion between virus and lipopolysaccharide by an ionic-strength effect: that is, log[Mx+] varies inversely with square root[NaCl]. The variation in effect of bi- and ter-valent ions and the low concentration needed show that this is not an ionic-strength effect but likely to involve binding to more than one site.     

Divalent ion trapping inside potassium channels of human T lymphocytes

Using the patch-clamp whole-cell recording technique, we investigated the influence of external Ca2+, Ba2+, K+, Rb+, and internal Ca2+ on the rate of K+ channel inactivation in the human T lymphocyte-derived cell line, Jurkat E6-1. Raising external Ca2+ or Ba2+, or reducing external K+, accelerated the rate of the K+ current decay during a depolarizing voltage pulse. External Ba2+ also produced a use-dependent block of the K+ channels by entering the open channel and becoming trapped inside. Raising internal Ca2+ accelerated inactivation at lower concentrations than external Ca2+, but increasing the Ca2+ buffering with BAPTA did not affect inactivation. Raising [K+]o or adding Rb+ slowed inactivation by competing with divalent ions. External Rb+ also produced a use-dependent removal of block of K+ channels loaded with Ba2+ or Ca2+. From the removal of this block we found that under normal conditions approximately 25% of the channels were loaded with Ca2+, whereas under conditions with 10 microM internal Ca2+ the proportion of channels loaded with Ca2+ increased to approximately 50%. Removing all the divalent cations from the external and internal solution resulted in the induction of a non-selective, voltage-independent conductance. We conclude that Ca2+ ions from the outside or the inside can bind to a site at the K+ channel and thereby block the channel or accelerate inactivation.     

The Cytosolic Ca2+ Concentration Gradient of Sinapis alba Root Hairs as Revealed by Ca2+-Selective Microelectrode Tests and Fura-Dextran Ratio Imaging

Using Ca2+-selective microelectrodes and fura 2-dextran ratio imaging, the cytosolic free [Ca2+] was measured in Sinapis alba root hair cells. Both methods yielded comparable results, i.e. values between 158 to 251 nM for the basal [Ca2+] of the cells and an elevated [Ca2+] of 446 to 707 nM in the tip region. The zone of elevated [Ca2+] reaches 40 to 60 [mu]m into the cell and is congruent with the region of inwardly directed Ca2+ net currents measured with an external Ca2+- selective vibrating electrode. The channel-blocker La3+ eliminates these currents, stops growth, and almost completely eliminates the cytosolic [Ca2+] gradient without affecting the basal level of the ion. Growth is also inhibited by pressure-injected dibromo-1,2-bis(o-aminophenoxy)ethane-N,N,N[prime],N[prime]-tetraacetic acid, which causes a decrease in the [Ca2+] in the tip in a concentration-dependent manner. Indole-3-acetic acid, used as a model stimulus, decreases cytosolic free [Ca2+] by 0.2 to 0.3 pCa units in the tip, but only by about 0.1 pCa unit in the shank. Nongrowing root hairs may or may not display a [Ca2+] gradient, but still reversibly respond to external stimuli such as La3+, Ca2+, or indole-3-acetic acid with changes in cytosolic free [Ca2+]. During short time periods, dicyclohexylcarbodiimide inhibition of the plasma membrane H+-ATPase, which stops growth, does not abolish the [Ca2+] gradient, nor does it change significantly the basal [Ca2+] level. We conclude that the cytosolic [Ca2+] gradient and an elevated [Ca2+] in the tip, as in other tip-growing cells, is essential for tip growth in root hairs; however, its presence does not indicate growth under all circumstances. We argue that with respect to Ca2+, tip growth regulation and responses to external signals may not interfere with each other. Finally, we suggest that the combination of the methods applied adds considerably to our understanding of the role of cytosolic free [Ca2+] in signal transduction and cellular growth.     

Voltage dependence of the Ca2+-activated cation channel TRPM4

TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. At positive potentials, currents are much larger and activate slowly. This voltage-dependent behavior induces a striking outward rectification of the steady state currents. The instantaneous current-voltage relationship, derived from the amplitude of tail currents following a prepulse to positive potentials, is linear. Currents show a Boltzmann type of activation; the fraction of open channels increases at positive potentials and is low at negative potentials. Voltage dependence is not due to block by divalent cations or to voltage-dependent binding of intracellular Ca2+ to an activator site, indicating that TRPM4 is a transient receptor potential channel with an intrinsic voltage-sensing mechanism. Voltage dependence of TRPM4 may be functionally important, especially in excitable tissues generating plateau-like or bursting action potentials.     

Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+

We have studied the modulation by intracellular Ca2+ of the epithelial Ca2+ channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca2+ measurements:1. Currents through ECaC were dramatically inhibited if Ca2+ was the charge carrier. This inhibition was dependent on the extracellular Ca2+ concentration and occurred also in cells buffered intracellularly with 10 mM BAPTA.2. Application of 30 mM [Ca(2)]e induced in non-Ca2+] buffered HEK 293 cells at -80 m V an increase in intracellular Ca2+([Ca2]i) with a maximum rate of rise of 241 +/-15nM/s (n= 18 cells) and a peak value of 891 +/- 106 nM. The peak of the concomitant current with a density of 12.3 +/- 2.6 pA/pF was closely correlated with the peak of the first-time derivative of the Ca2+ transient, as expected if the Ca2+ transient is due to influx of Ca2+. Consequently, no Ca2+] signal was observed in cells transfected with the Ca2+ impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca2+]i by dialyzing the cell with pipette solutions containing various Ca2+] concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Na+ or Ca2+] ions. Half maximal inhibition of Ca(2+)currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Na+ currents in the absence of Ca2+] and Mg2+ were inhibited with an IC50 of 89 nM (n between 6 and 10). Currents through ECaC in the presence of 1 mM Ca2+ and Na+, which are mainly carried by Ca2+, are inhibited by [Ca2]i with an IC50of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca2+impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca2]i (IC50 = 123 nM, n between 7 and 18). 4. The sensitivity of ECaC currents in inside-out patches for [Ca2]i was slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169 nM if Na+ was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca2]e (n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca2+ (whole-cell configuration) or removal of Ca2+ from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 +/- 34 s (n= 15) in whole-cell mode and after 135 +/- 23 s (n = 17) in inside-out patches.6. We conclude that influx of Ca2+ through ECaC and [Ca2]i induce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca2+ in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca( 2+ from an internal Ca2+ binding site at ECaC.     

Changes in internal ionized Ca2+ and H+ in voltage clamped squid axons

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na+, K+, and some inert cation, at either physiological or zero bath Ca2+ concentration [( Ca2+]o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca2+ level, [( Ca2+]i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K+]o (i.e. approximately -15 mV). In the absence of [Ca2+]o a significant steady depolarization brought about by current flow increased [Ca2+]i (and acidified the axoplasm). Changes in internal hydrogen activity, [H+]i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca2+]i could be made. However, there are effects on [Ca2+]i that can be ascribed to membrane potential. Thus, in the absence of [Ca2+]o, hyperpolarization can reduce [Ca2+]i, implying that a Ca2+ efflux mechanism is enhanced. It is also observed that [Ca2+]i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na+ for Ca2+ in squid giant axon.     

Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.     

Evidence for capacitative and non-capacitative Ca2+ entry pathways coexist in A10 vascular smooth muscle cells

It is generally thought that receptor-operated Ca2+ entry is related to store-operated or capacitative Ca2+ entry mechanism. Recent evidence suggests that non-capacitative Ca2+ entry pathways are also involved in receptor activated Ca2+ influx in many different kinds of cells. In this study, we studied whether alpha1-adrenoreceptor (alpha1-AR)-activated Ca2+ entry is coupled to both capacitative and non-capacitative pathways in A10 vascular smooth muscle cells by fura-2 fluorescence probe and conventional whole-cell patch clamp techniques. We found that both thapsigargin (TG) and phenylephrine (Phe) induced transient increase in cytoplasmic Ca2+ concentration ([Ca2+]i) in Ca2+-free medium, and subsequent addition of Ca2+ evoked a sustained [Ca2+]i rise. When the membrane potential was held at -60 mV, both TG and Phe activated inward currents, which were inhibited by GdCl3(Gd3+), 0Na+/0Ca2+ solution and 1-{beta[3-(4-mehtoxyphenyl)propoxy]-4-methoxypheneth-yl}-1H- imidazole hydro-chloride (SK&F96365), but not by nifedipine. When Ca2+ store was depleted by TG in Ca2+-free solution, Phe failed to further evoke [Ca2+]i rise. However, when capacitative Ca2+ entry was activated by TG in the medium containing Ca2+, 10 microM Phe further increased [Ca2+]i. At the same concentration, TG activated an inward cation current, subsequent addition of Phe also further induced an inward cation current. Furthermore, the amplitudes of [Ca2+]i increase and current density induced by Phe in the presence of TG were less than that induced by Phe alone. Our results suggest that both capacitative and non-capacitative Ca2+ entry pathways are involved in Ca2+ influx induced by activation of alpha1-AR in A10 vascular smooth muscle cells.     

Evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors

The Ca2+ indicator photoprotein, aequorin, was used to estimate and monitor intracellular Ca2+ levels in Limulus ventral photoreceptors during procedures designed to affect Na+/Ca2+ exchange. Dark levels of [Ca2+]i were estimated at 0.66 +/- 0.09 microM. Removal of extracellular Na+ caused [Ca2+]i to rise transiently from an estimated 0.5-0.6 microM in a typical cell to approximately 21 microM; [Ca2+]i approached a plateau level in 0-Na+ saline of approximately 5.5 microM; restoration of normal [Na+]o lowered [Ca2+]i to baseline with a time course of 1 log10 unit per 9 s. The apparent rate of Nao+-dependent [Ca2+]i decline decreased with decreasing [Ca2+]i. Reintroduction of Ca2+ to 0-Na+, 0-Ca2+ saline in a typical cell caused a transient rise in [Ca2+]i from an estimated 0.36 microM (or lower) to approximately 16.5 microM. This was followed by a decline in [Ca2+]i approaching a plateau of approximately 5 microM; subsequent removal of Cao2+ caused [Ca2+]i to decline slowly (1 log unit in approximately 110 s). Intracellular injection of Na+ in the absence of extracellular Na+ caused a transient rise in [Ca2+]i in the presence of normal [Ca2+]o; in 0-Ca2+ saline, however, no such rise in [Ca2+]i was detected. Under constant voltage clamp (-80 mV) inward currents were measured after the addition of Nao+ to 0-Na+ 0-Ca2+ saline and outward currents were measured after the addition of Cao2+ to 0-Na+ 0-Ca2+ saline. The results suggest the presence of an electrogenic Na+/Ca2+ exchange process in the plasma membrane of Limulus ventral photoreceptors that can operate in forward (Nao+-dependent Ca2+ extrusion) or reverse (Nai+-dependent Ca2+ influx) directions.     

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.     

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.     

Survival of mammalian B104 cells following neurite transection at different locations depends on somal Ca2+ concentration

We report that cell survival after neurite transection in a mammalian neuronal model (cultured B104 cells) critically depends on somal [Ca2+]i, a novel result that reconciles separate long-standing observations that somal survival decreases with more-proximal axonal transections and that increased somal Ca2+ is cytotoxic. Using fluorescence microscopy, we demonstrate that extracellular Ca2+ at the site of plasmalemmal transection is necessary to form a plasmalemmal barrier, and that other divalent ions (Ba2+, Mg2+) do not play a major role. We also show that extracellular Ca2+, rather than injury per se, initiates the formation of a plasmalemmal barrier and that a transient increase in somal [Ca2+]i significantly decreases the percentage of cells that survive neurite transection. Furthermore, we show that the increased somal [Ca2+]i and decreased cell survival following proximal transections are not due to less frequent or slower plasmalemmal sealing or Ca2+ entry through plasmalemmal Na+ and Ca2+ channels. Rather, the increased somal [Ca2+]i and lethality of proximal neurite injuries may be due to the decreased path length/increased diameter for Ca2+ entering the transection site to reach the soma. A ryanodine block of Ca2+ release from internal stores before transection has no effect on cell survival; however, a ryanodine- or thapsigargin-induced buildup of somal [Ca2+]i before transection markedly reduces cell survival, suggesting a minor involvement of Ca2+-induced release from internal stores. Finally, we show that cell survival following proximal injuries can be enhanced by increasing intracellular Ca2+ buffering capacity with BAPTA to prevent the increase in somal [Ca2+]i.     

Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.     

Ca2+-dependent potentiation of the nonselective cation channel TRPV4 is mediated by a C-terminal calmodulin binding site

Most Ca2+-permeable ion channels are inhibited by increases in the intracellular Ca2+ concentration ([Ca2+]i), thus preventing potentially deleterious rises in [Ca2+]i. In this study, we demonstrate that currents through the osmo-, heat- and phorbol ester-sensitive, Ca2+-permeable nonselective cation channel TRPV4 are potentiated by intracellular Ca2+. Spontaneous TRPV4 currents and currents stimulated by hypotonic solutions or phorbol esters were reduced strongly at all potentials in the absence of extracellular Ca2+. The other permeant divalent cations Ba2+ and Sr2+ were less effective than Ca2+ in supporting channel activity. An intracellular site of Ca2+ action was supported by the parallel decrease in spontaneous currents and [Ca2+]i on removal of extracellular Ca2+ and the ability of Ca2+ release from intracellular stores to restore TRPV4 activity in the absence of extracellular Ca2+. During TRPV4 activation by hypotonic solutions or phorbol esters, Ca2+ entry through the channel increased the rate and extent of channel activation. Currents were also potentiated by ionomycin in the presence of extracellular Ca2+. Ca2+-dependent potentiation of TRPV4 was often followed by inhibition. By mutagenesis, we localized the structural determinant of Ca2+-dependent potentiation to an intracellular, C-terminal calmodulin binding domain. This domain binds calmodulin in a Ca2+-dependent manner. TRPV4 mutants that did not bind calmodulin lacked Ca2+-dependent potentiation. We conclude that TRPV4 activity is tightly controlled by intracellular Ca2+. Ca2+ entry increases both the rate and extent of channel activation by a calmodulin-dependent mechanism. Excessive increases in [Ca2+]i via TRPV4 are prevented by a Ca2+-dependent negative feedback mechanism.