Separation of calcium channel current components in mouse chromaffin cells superfused with low- and high-barium solutions

 This study was carried out to characterize the set of voltage-dependent Ca²⁺ channel subtypes expressed by mouse adrenal chromaffin cells superfused with solutions containing low (2 mM) or high (10 mM) Ba²⁺ concentrations. Using 50-ms test pulses at 0 mV from a holding potential of –80 mV, averaged peak current in 10 mM Ba²⁺ was around 1 nA, and in 2 mM Ba²⁺ 0.36 nA. When using 2 mM Ba²⁺ as the charge carrier, nifedipine (3 μM) blocked I _Ba by 40–45%. ω-Conotoxin GVIA (1 μM) caused 26% inhibition, while ω-conotoxin MVIIC (3 μM) produced a 48% blockade. At low concentrations (20 nM), ω-agatoxin IVA caused 5–15% of current inhibition, while 2 μM gave rise to a 35–40% blockade. In 10 mM Ba²⁺, the blocking effects of nifedipine (40%) and ω-conotoxin GVIA (25%) were similar to those seen in 2 mM Ba²⁺. In contrast, blockade by ω-conotoxin MVIIC was markedly reduced in 10 mM Ba²⁺ (20–25%) as compared to 10 mM Ba²⁺ (48%). The blocking actions of ω-agatoxin IVA (2 μM) were also slowed down in 10 mM Ba²⁺, though the final blockade was unaffected. In 2 mM Ba²⁺, I _Ba was quickly inhibited by over 94% with combined nifedipine + ω-conotoxin MVIIC + ω-conotoxin GVIA; in 10 mM Ba²⁺, I _Ba was blocked by 70% with this combination. The data suggest that mouse chromaffin cells express L-type (40%) as well as non-L-type (60%) high-threshold voltage-dependent Ca²⁺ channels. The current carried by non-L-type Ca²⁺ channels consists of about 25% N-type and 35% P/Q-type; P-type channels, if anything, are poorly expressed. The data also indicate that the fraction of current blocked by ω-conotoxin MVIIC and ω-agatoxin IVA might considerably change as a function of the Ba²⁺ concentration of the extracellular solution; taking this fact into consideration, it seems that a residual R-type current is not expressed in mouse chromaffin cells. Received: 21 January 1998 / Received after revision and accepted: 20 February 1998     

Analogies and differences between ω-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells

 The characteristics of the binding sites for the Conus magus toxins ω-conotoxin MVIIC and ω-conotoxin MVIID, as well as their effects on K⁺-evoked ⁴⁵Ca²⁺ entry and whole-cell Ba²⁺ currents (I _Ba), and K⁺-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [¹²⁵I] ω-conotoxin GVIA to bovine adrenal medullary membranes was displaced by ω-conotoxins GVIA, MVIIC and MVIID with IC₅₀ values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [¹²⁵I] ω-conotoxin MVIIC, which was displaced by ω-conotoxins MVIIC, MVIID and GVIA with IC₅₀ values of around 30, 80 and 1.200 nM, respectively. The sites recognized by ω-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC₅₀ values of around 1 nM). Both ω-conotoxin MVIIC and MVIID blocked I _Ba by 70–80%; the higher the [Ba²⁺]_o of the extracellular solution the lower the blockade induced by ω-conotoxin MVIIC. This was not the case for ω-conotoxin MVIID; high Ba²⁺ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba²⁺. A further difference between the two toxins concerns their reversibility; washout of ω-conotoxin MVIIC did not reverse the blockade of I _Ba while in the case of ω-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by ω-conotoxin GVIA, suggesting that it is associated with N-type Ca²⁺ channels. Blockade of K⁺-evoked ⁴⁵Ca²⁺ entry produced results which paralleled those obtained by measuring I _Ba. Thus, 1 μM of each of ω-conotoxin GVIA and MVIIA inhibited Ca²⁺ uptake by 25%, while 1 μM of each of ω-conotoxin MVIIC and MVIID caused a 70% blockade. K⁺-evoked catecholamine secretory responses were not reduced by ω-conotoxin GVIA (1 μM). In contrast, at 1 μM both ω-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca²⁺ channels that contribute to the regulation of secretion and are sensitive to ω-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for ω-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca²⁺ channels or an entirely new subtype of voltage-dependent high-threshold Ca²⁺ channel. Received: 16 April 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997     

Q-type Ca2+ channels are located closer to secretory sites than L-type channels: functional evidence in chromaffin cells

 This study uses a new strategy to investigate the hypothesis that, of the various Ca²⁺ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca²⁺ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca²⁺ concentrations ([Ca²⁺]_o). So, at low [Ca²⁺]_o the K⁺-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60–70% by 2 μM ω-agatoxin IVA (P/Q-type Ca²⁺ channel blockade), by 3 μM ω-conotoxin MVIIC (N/P/Q-type Ca²⁺ channel blockade), or by 3 μM lubeluzole (N/P/Q-type Ca²⁺ channel blockade); in high [Ca²⁺]_o these blockers inhibited the responses by only 20–35%. At 1–3 μM ω-conotoxin GVIA (N-type Ca²⁺ channel blockade) or 3 μM furnidipine (L-type Ca²⁺ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca²⁺]o. Combined furnidipine plus ω-conotoxin MVIIC, ω-agatoxin IVA or ω-conotoxin GVIA exhibited additive blocking effects at both Ca²⁺ concentrations. The results suggest that Q-type Ca²⁺ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca²⁺ that enters the cell through a Ca²⁺ channel located near to chromaffin vesicles will saturate the K⁺ secretory response at both [Ca²⁺]_o, i.e. 0.5 mM and 5 mM. In contrast, Ca²⁺ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca²⁺]_o. Received: 23 September 1997 / Received after revision: 29 October 1997 / Accepted: 30 October 1997     

Two types of ω-agatoxin IVA-sensitive Ca2+ channels are coupled to adrenaline and noradrenaline release in bovine adrenal chromaffin cells

 To clarify the role of P-type Ca²⁺ channels in catecholamine release from adrenal chromaffin cells we examined the concentration dependence of the effect of ω-agatoxin IVA on the release both of adrenaline and noradrenaline induced by a K⁺-evoked depolarization. ω-Agatoxin IVA caused a biphasic dose-dependent inhibition of secretion with a high-potency component (IC₅₀<1 nM), responsible for 10–15% of catecholamine release evoked by 70 mM K⁺, and a low-potency component that accounted for about 40% of release, with IC₅₀ values of 57 nM and 48 nM for noradrenaline and adrenaline release, respectively. The release of catecholamines from chromaffin cells was also inhibited dose dependently by ω-conotoxin MVIIC with IC₅₀ values of 182 and 218 nM for noradrenaline and adrenaline release, respectively. The effects of 3 nM ω-agatoxin IVA and 3 μM ω-conotoxin MVIIC were additive, indicating that at the concentrations used the toxins were acting at independent sites, presumably, P- and Q-type Ca²⁺ channels. The blockade of Q-type channels inhibited the release of adrenaline (72 ± 4.1%) significantly more than the release of noradrenaline (50 ± 2.7%), suggesting a higher density or a closer coupling of these channels to exocytosis in adrenergic chromaffin cells. The blockade of P-type channels caused a greater inhibition of catecholamine secretion at low levels of K⁺-evoked depolarization and shorter times of stimulation than that observed at higher levels of stimulation. The contribution of Q-type channels to catecholamine secretion did not change significantly with the intensity of stimulation. The data show that two types of ω-agatoxin IVA-sensitive Ca²⁺ channels are coupled to catecholamine release in chromaffin cells, and that the contribution of P-type channels to secretion is larger at low levels of depolarization. Received: 6 March 1997 / Received after revision and accepted: 28 April 1997     

Calcium channel subtypes in porcine adrenal chromaffin cells

 The effects of nifedipine, ω-conotoxin GVIA (ω-CgTx) and ω-agatoxin IVA (ω-AgTx) on Ca²⁺ currents, a 60-mM-K⁺-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and catecholamine secretion were examined to clarify the subtypes of Ca²⁺ channels in cultured adrenal chromaffin cells from the pig. Nifedipine, ω-CgTx, and ω-AgTx inhibited Ca²⁺ currents in a dose-dependent manner, suggesting the presence of L-, N- and P-type Ca²⁺ channels. The maximal doses of nifedipine (10 μM), ω-CgTx (1 μM), and ω-AgTx (0.1 μM) inhibited Ca²⁺ currents to 85%, 22%, and 94% of control currents, respectively. The inhibitory effects of these three blockers were observed in the same cell, indicating that at least three subtypes of Ca²⁺ channels are present in porcine chromaffin cells. The increase in [Ca²⁺]_i and catecholamine secretion induced by 60 mM K⁺ were inhibited equally by nifedipine (10 μM) and ω-CgTx (1 μM), but not by ω-AgTx (0.1 μM). These results suggest that L-, N- and P-type Ca²⁺ channels are present in porcine adrenal chromaffin cells, and that the major pathways of Ca²⁺ entry evoked by a high concentration of K⁺ are L- and N-type Ca²⁺ channels. Received: 6 September 1996 / Received after revision: 3 February 1997 / Accepted: 18 February 1997     

Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells

 Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba²⁺ (holding potential, V _h=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca²⁺ channels; T-type Ca²⁺ channels were not seen. By using supramaximal concentrations of selective Ca²⁺ channel blockers, the whole-cell I _Ba could be fractionated into various subcomponents. Thus, I _Ba had a 25% fraction sensitive to 1 μM nifedipine (L-type channels), 21% sensitive to 1 μM ω-conotoxin GVIA (N-type channels), and 60% sensitive to 2 μM ω-agatoxin IVA (P/Q-type channels). The activation of I _Ba was considerably slowed down, and the peak current was inhibited upon superfusion with 10 μM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of I _Ba was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca²⁺ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba²⁺. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca²⁺ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5–50%. The results also provide strong support for the hypothesis that Ca²⁺ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates. Received: 1 May 1998 / Received after revision and accepted: 21 May 1998     

Multiple calcium channel subtypes in isolated rat chromaffin cells

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca²⁺ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of –80 mV, using 10 mM Ba²⁺ as the charge carrier, only high-voltage-activated (HVA) Ca²⁺ channels were found. Ba²⁺ currents (I _Ba) snowed variable sensitivity to dihydropyridine (DHP) Ca²⁺ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 M and 10 M respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca²⁺ channel agonist Bay K 8644 (1 M) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of I _Ba was accompanied by a 20-mV shift in the activation curves for Ca²⁺ channels towards more hyperpolarizing potentials. Application of the conus toxin -conotoxin GVIA (GVIA; 1 M) blocked 31% of I _Ba; blockade was irreversible upon removal of the toxin from the extracellular medium, -Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of I _Ba. -Conotoxin MVIIC (MVIIC; 5 M) produced a 36% blockade of I _Ba; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca²⁺ channels. The sequential addition of supramaximal concentrations of furnidipine (10 M), GVIA (1 M), IVA (100 nM) and MVIIC (3 M) produced partial inhibition of I _Ba, which were additive. Our data suggest that the whole cell I _Ba in rat chromaffin cells exhibits at least four components. About 50% of I _Ba is carried by L-type Ca²⁺ channels, 30% by N-type Ca²⁺channels and 15% by P-type Ca²⁺ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca²⁺channels account for 45% of the current, N-type carry 35% and L-type Ca²⁺ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.     

Calcium channel types contributing to excitatory and inhibitory synaptic transmission between individual hypothalamic neurons

The contribution of L-, N-, P- and Q-type Ca²⁺ channels to excitatory and inhibitory synaptic transmission and to whole-cell Ba²⁺ currents through Ca²⁺ channels (Ba²⁺ currents) was investigated in rat hypothalamic neurons grown in dissociated cell culture. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were evoked by stimulating individual neurons under whole-cell patch-clamp conditions. The different types of high-voltage-activated (HVA) Ca²⁺ channels were identified using nifedipine, ω-Conus geographus toxin VIA (ω-CTx GVIA), ω-Agelenopsis aperta toxin IVA (ω-Aga IVA), and ω-Conus magus toxin VIIC (ω-CTx MVIIC). N-, but not P- or Q-type Ca²⁺ channels contributed to excitatory as well as inhibitory synaptic transmission together with Ca²⁺ channels resistant to the aforementioned Ca²⁺ channel blockers (resistant Ca²⁺ channels). Reduction of postsynaptic current (PSC) amplitudes by N-type Ca²⁺ channel blockers was significantly stronger for IPSCs than for EPSCs. In most neurons whole-cell Ba²⁺ currents were carried by L-type Ca²⁺ channels and by at least two other Ca²⁺ channel types, one of which is probably of the Q-type and the others are resistant Ca²⁺ channels. These results indicate a different contribution of the various Ca²⁺ channel types to excitatory and inhibitory synaptic transmission and to whole-cell currents in these neurons and suggest different functional roles for the distinct Ca²⁺ channel types. Received: 15 November 1995/Accepted: 30 January 1996     

Calcium channel current facilitation in porcine adrenal chromaffin cells

 The mechanisms of depolarizing-prepulse-induced facilitation of Ca²⁺ channel current were investigated in a study of porcine chromaffin cells. The Ba²⁺ current evoked by a pulse to 0 mV was increased by a strong depolarizing prepulse (conditioning pulse), termed ”facilitation”. This facilitation increased with an increase in either the duration or the voltage of the conditioning pulse, and decreased with an increase in the interpulse interval. For example, the Ba²⁺ current was increased to 1.14 times the control (facilitation ratio) by a 150-ms conditioning pulse to +100 mV followed by a 10-ms interpulse interval. Forskolin, 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-bromo-cAMP) and Rp-adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) did not affect the facilitation of the Ba²⁺ current, suggesting that a cAMP-dependent mechanism is not involved. Intracellular guanosine 5′-O-(3-thiotriphosphate) (GTPγS) decreased the Ba²⁺ current to 0.59 times the control and GDPβS increased it to 1.19. However, neither GTPγS nor guanosine 5′-O-(2-thiodiphosphate) (GDPβS) changed the amplitude of the Ba²⁺ current that was facilitated by the conditioning pulse. Thus, GTPγS increased the facilitation ratio to 2.05 and GDPβS decreased it to 1.05. Furthermore, the facilitation of the Ba²⁺ current was abolished by ω-conotoxin GVIA but not by either ω-agatoxin IVA or nifedipine. These results suggest that, in porcine chromaffin cells, there is a ω-conotoxin GVIA-sensitive N-type Ca²⁺ channel that is under the inhibitory control of a G protein, which can be relieved by a conditioning pulse. Received: 25 September 1997 / Received after revision: 14 November 1997 / Accepted: 16 December 1997     

P/Q-type calcium channels activate neighboring calcium-dependent potassium channels in mouse motor nerve terminals

 The identity of the voltage-dependent calcium channels (VDCC), which trigger the Ca²⁺-gated K⁺ currents (I _K(Ca)) in mammalian motor nerve terminals, was investigated by means of perineurial recordings. The effects of Ca²⁺ chelators with different binding kinetics on the activation of I _K(Ca) were also examined. The calcium channel blockers of the P/Q family, ω-agatoxin IVA (ω-Aga-IVA) and funnel-web spider toxin (FTX), have been shown to exert a strong blocking effect on I _K(Ca). In contrast, nitrendipine and ω-conotoxin GVIA (ω-CgTx) did not affect the Ca²⁺-activated K⁺ currents. The intracellular action of the fast Ca²⁺ buffers BAPTA and DM-BAPTA prevented the activation of the I _K(Ca), while the slow Ca²⁺ buffer EGTA was ineffective at blocking it. These data indicate that P/Q-type VDCC mediate the Ca²⁺ influx which activates I _K(Ca). The spatial association between Ca²⁺ and Ca²⁺-gated K⁺ channels is discussed, on the basis of the differential effects of the fast and slow Ca²⁺ chelators. Received: 20 December 1996 / Received after revision: 21 March 1997 / Accepted: 2 April 1997     

Localized L-type calcium channels control exocytosis in cat chromaffin cells

Depolarizing 1-s pulses to 0 mV from a holding potential of −70 mV, induced whole-cell currents through Ca²⁺ channels (I _Ca) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 μM) reduced the peak current by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) reduced the peak I _Ca by 42% and the late I _Ca by 55%. Pulses (10 s duration) with 70 mM K⁺/2.5 mM Ca²⁺ solution (70 K⁺/2.5 Ca²⁺), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i from 0.1 to 2.21 μM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K⁺/2.5 Ca²⁺ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca²⁺]_o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca²⁺ channels in cat chromaffin cells. It seems, howevever, that though extracellular Ca²⁺ entry through both channel types leads to similar increments of averaged [Ca²⁺]_i, the control of catecholamine release is dominated only by Ca²⁺ entering through L-type Ca²⁺ channels. This supports the idea of a preferential segregation of L-type Ca²⁺ channels to localized “hot spots” in the plasmalemma of chromaffin cells where exocytosis occurs.     

Voltage-gated calcium channels may be involved in the regulation of the mechanosensitivity of slowly conducting knee joint afferents in rat

Voltage-gated Ca²⁺ channels play an important role in the central processing of nociceptive information. Recently, it has been shown that L- and N-type voltage-gated Ca²⁺ channels are also present on peptidergic, fine afferent nerve fibers in the knee joint capsule. Therefore, the influence of specific blockers for L-type (verapamil) or N-type (ω-conotoxin GVIA) Ca²⁺ channels on the mechanosensitivity of slowly conducting afferents was tested in the rat knee joint. Topical application of 100 μM verapamil onto the receptive field reduced the mean response to knee joint rotation to 67±8% (SEM, n=12), obtained by outward rotations with a torque of 10 mNm above the mechanical threshold and compared with control movements. In the presence of 50 μM ω-conotoxin GVIA, the mean response decreased to 44±5% (n=12), a reduction that was also observed during rotations of other intensities. Simultaneous application of both substances further reduced the response to 25±11% (n=6). In additional experiments it was shown that L- and N-type voltage-gated Ca²⁺ channels do not influence activity-dependent changes of the mechanical excitability. In conclusion, the data of the present study indicate that voltage-gated Ca²⁺ channels may also be involved in the regulation of the mechanosensitivity of nociceptive nerve fiber endings. Electronic Publication     

Cholinergic agonists decrease quantal output at the frog neuromuscular junction by targeting a calcium channel blocked by ω-conotoxin

 Nicotinic cholinergic agonists are known to decrease synchronous evoked quantal output at the frog neuromuscular junction [Van der Kloot 1993, J Physiol (Lond) 468:567–589]. Here we also show that carbachol decreases the frequency of miniature endplate potentials (F _MEPP) in solutions containing elevated levels of K⁺ and Ca²⁺. Carbachol did not decrease F _MEPP in hypertonic solutions or in solutions containing the Ca²⁺ ionophore ionomycin and Ca²⁺. We conclude that the nicotinic agonists decrease Ca²⁺ influx through voltage-gated Ca²⁺ channels. Carbachol did not alter two-pulse facilitation. A blocker of N-type Ca²⁺ channels, ω-conotoxin GVIA, antagonized the nicotinic agonist-induced decrease in evoked quantal output. The effect of carbachol was not altered by ω-conotoxin MVIIC, a blocker of P-type and certain other Ca²⁺channels. The Ca²⁺ channel targeted by the nicotinic agonists appears to be of the N-type. Received: 6 March 1997 / Received after revision: 6 May 1997 / Accepted: 4 June 1997     

Functional expression of rat brain cloned α1E calcium channels in COS-7 cells

 The properties of the rat brain α1E Ca²⁺ channel subunit and its modulation by accessory rat brain α2-δ and β1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether α1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca²⁺ channel and to examine its pharmacology in detail. α1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric α1E/α2-δ/β1b channels were largely dictated by the presence of the β1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. α1E/α2-δ/β1b channels did not appear to be regulated by Ca²⁺-induced inactivation. α1E was shown to exhibit a unique pharmacological profile. ω-Agatoxin IVA blocked the current in a dose-dependent manner with an IC₅₀ of approximately 50 nM and a maximum inhibition of about 80%, whilst ω-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 μM) produced an inhibition of 51 ± 7%, whereas the DHP agonist S-(–)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the α1E Ca²⁺ channel subunit; we propose that rat brain α1E forms a novel Ca²⁺ channel with properties more similar to a subtype of LVA than HVA Ca²⁺ current. Received: 30 August 1996 / Received after revision and accepted: 28 October 1996     

Autocrine/paracrine modulation of calcium channels in bovine chromaffin cells

 Applying 10-s pulses of 10 mM Ba²⁺ to resting or K⁺-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca²⁺ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr²⁺ or Ca²⁺ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca²⁺, Sr²⁺ and Ba²⁺. Ca²⁺ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I _Ca by 19%, I _Sr by 31%, and I _Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I _Ca, I _Sr and I _Ba similarly. On the other hand, the use of Na⁺ as a charge carrier through Ca²⁺ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating secretion from unpatched cells, by either 10 mM Ba²⁺, 100 μM acetylcholine or 70 mM K⁺, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca²⁺ channels through autocrine/paracrine mechanisms. Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998     

Opioid potentiation of N-type Ca2+ channel currents via pertussis-toxin-sensitive G proteins in NG108-15 cells

Opioids have both inhibitory and stimulatory effects on neurotransmitter release. While the inhibitory effect has been ascribed to presynaptic inhibition of Ca²⁺ channels, the cellular mechanism underlying the stimulatory effect is not clear. In order to address this issue, we analyzed the effects of [d-Ala², d-Leu⁵]-enkephalin (DADLE) on whole-cell Ba²⁺ currents (I _Ba) through voltage-gated Ca²⁺ channels in NG108–15 neuroblastoma × glioma hybrid cells. Application of DADLE inhibited and washout of DADLE transiently potentiated I _Ba. Furthermore, potentiation of I _Ba was elicited even in the presence of DADLE, when inhibition was relieved by a large depolarizing prepulse. DADLE-induced potentiation, as well as inhibition, had both voltage-sensitive and -insensitive components and was abolished by treatment with ICI174864, a δ-opioid antagonist, pertussis toxin (PTX) and ω-conotoxin GVIA. Potentiation developed over @3 min and took 5–20 min to recover, whereas inhibition was complete within 30 s and recovered within 1 min. Although this potentiation should contribute to DADLE-induced desensitization of Ca²⁺ channel inhibition, it was not the sole mechanism for desensitization. We conclude that the δ-opioid receptor exerts a dual action on N-type Ca²⁺ channels via PTX-sensitive G proteins, i.e., rapid inhibition followed by slowly developing potentiation. Received: 31 March 1999 / Received after revision: 27 April 1999 / Accepted: 28 April 1999     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

Neomycin inhibits K+-induced force and Ca2+ channel current in rat arterial smooth muscle

 The techniques of small vessel isometric myography and patch clamp were used to investigate the action of neomycin on K⁺-induced isometric force and voltage-gated Ca²⁺ channel currents in rat arterial smooth muscle. Neomycin and the dihydropyridine (DHP) Ca²⁺ channel antagonist (–)202–791 concentration-dependently and reversibly inhibited 40 mM K⁺-induced isometric force in rings of rat mesenteric and basilar arteries (IC₅₀ values of 70 μM and 1.2 nM, respectively, n = 10 and 4). Elevation of [Ca²⁺]_o by a factor of 2 significantly reduced the IC₅₀ values for inhibition of K⁺-induced force for both neomycin and (–)202–791 (192 μM and 3.7 nM, respectively, n = 6 and 4), but did not affect the Hill coefficient of the concentration/effect relationships. In patch-clamp experiments using freshly isolated basilar arterial myocytes, the voltage-gated inward current carried by Ba²⁺ was reversibly and concentration-dependently inhibited by neomycin (IC₅₀ 32 μM, n = 3). The concentration/effect curve for inhibition of the inward Ba²⁺ current by neomycin was significantly shifted to the right when [Ba²⁺]_o was raised from 1.8 mM to 10 mM (IC₅₀ 144 μM, n = 8). Our findings suggest that neomycin relaxes high-K⁺-induced force in rat isolated mesenteric and basilar arteries largely by inhibition of voltage-dependent and DHP-sensitive Ca²⁺ channels. Received: 1 August 1996 / Received after revision and accepted: 11 September 1996     

Tyrosine kinase inhibitors suppress N-type and T-type Ca2+ channel currents in NG108–15 cells

 Modulation of Ca²⁺ channel activity by protein kinases constitutes one of the major mechanisms regulating neuronal functions. Here, we explored the possible modulation of neuronal Ca²⁺ channels by protein tyrosine kinases (PTKs). To this end, the effects of PTK inhibitors on whole-cell Ba²⁺ currents (I _Ba) through voltage-gated Ca²⁺ channels were analysed in differentiated NG108–15 neuroblastoma × glioma hybrid cells. Genistein suppressed I _Ba in a concentration-dependent fashion (IC₅₀ = 22 μM). Although daidzein, an analogue of genistein that is devoid of PTK inhibitory activity, also suppressed I _Ba, we estimated that specific PTK inhibition by genistein reduced I _Ba amplitude by 30%. In addition, lavendustin A (20 μM) and herbimycin A (20 μM), two other distinct PTK inhibitors, depressed I _Ba by 22% and 20%, respectively. Genistein suppressed N-type and T-type currents, sparing L-type current, and its effect was independent of G protein activation. The results suggest that the activity of neuronal Ca²⁺ channels can be modulated by PTKs, opening the possibility that some of the functions of PTKs in the nervous system are mediated by Ca²⁺ channel modulation. Received: 21 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998     

Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre-and postsynaptic calcium fluxes in rat hippocampal slices

The contribution of T-, L-, N-, P-, and Q-type Ca²⁺ channels to pre-and postsynaptic Ca²⁺ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca²⁺ concentration ([Ca²⁺]₀). [Ca²⁺]₀ was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca²⁺ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca²⁺]₀ decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca²⁺ entry (Ca_pre), while [Ca²⁺]⁰ decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca²⁺ entry (Ca_post). Ethosuximide and trimethadione were ineffective m concentrations up to 1 mM. At 10 mM, they reduced Ca_post and, much less, also Ca_pre Nimodipine (25 M) reduced Ca_post and, to a minor extent, Ca_pre. -Agatoxin IVA (0.4–1 M) and -conotoxin MVIIC (1 M) also reduced both Ca_pre and Ca_post, but with a stronger action on Ca_pre. -Conotoxin GVIA (3–8 M) reduced Ca_post without effect on Ca_pre. We conclude that during stimulus-induced, high-frequency neuronal activity Ca_post is carried by P/Q-, N-, and L-type channels and probably a further channel type different from these channels. Ca_pre includes at least P/Q-and possibly L-type channels. N-type channels did not contribute to Ca_pre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca²⁺ entry in this situation.