Channels In Chromaffin Cells Research Articles

Lead exerts a depression of neurotransmitter release through a blockade of voltage dependent calcium channels in chromaffin cells

The present work, using chromaffin cells of bovine adrenal medullae (BCCs), aims to describe what type of ionic current alterations induced by lead (Pb2+) underlies its effects reported on synaptic transmission. We observed that the acute application of Pb2+ lead to a drastic depression of neurotransmitters release in a concentration-dependent manner when the cells were stimulated with both K+ or acetylcholine, with an IC50 of 119,57 μM and of 5,19 μM, respectively. This effect was fully recovered after washout. Pb2+ also blocked calcium channels of BCCs in a time- and concentration-dependent manner with an IC50 of 6,87 μM. This blockade was partially reversed upon washout. This compound inhibited the calcium current at all test potentials and shows a shift of the I-V curve to more negative values of about 8 mV. The sodium current was not blocked by acute application of high Pb2+ concentrations. Voltage-dependent potassium current was also shortly affected by high Pb2+. Nevertheless, the calcium- and voltage-dependent potassium current was drastically depressed in a dose-dependent manner, with an IC50 of 24,49 μM. This blockade was related to the prevention of Ca2+ influx through voltage-dependent calcium channels coupled to Ca2+-activated K+-channels (BK) instead a direct linking to these channels. Under current-clamp conditions, BCCs exhibit a resting potential of −52.7 mV, firing spontaneous APs (1–2 spikes/s) generated by the opening of Na+ and Ca2+-channels, and terminated by the activation of K+ channels. In spite of the effect on ionic channels exerted by Pb2+, we found that Pb2+ didn’t alter cellular excitability, no modification of the membrane potential, and no effect on action potential firing. Taken together, these results point to a neurotoxic action evoked by Pb2+ that is associated with changes in neurotransmitter release by blocking the ionic currents responsible for the calcium influx.

Nav1.3 and FGF14 are primary determinants of the TTX-sensitive sodium current in mouse adrenal chromaffin cells.

Adrenal chromaffin cells (CCs) in rodents express rapidly inactivating, tetrodotoxin (TTX)-sensitive sodium channels. The resulting current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate via two independent pathways which differ in their time course of recovery. One subpopulation recovers with time constants similar to traditional fast inactivation and the other ∼10-fold slower, but both pathways can act within a single homogenous population of channels. Here, we use Nav1.3 KO mice to probe the properties and molecular components of Nav current in CCs. We find that the absence of Nav1.3 abolishes all Nav current in about half of CCs examined, while a small, fast inactivating Nav current is still observed in the rest. To probe possible molecular components underlying slow recovery from inactivation, we used mice null for fibroblast growth factor homology factor 14 (FGF14). In these cells, the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. The use dependence of Nav current reduction during trains of stimuli in WT cells is completely abolished in FGF14 KO mice, directly demonstrating a role for slow recovery from inactivation in determining Nav current availability. Our results indicate that FGF14-mediated inactivation is the major determinant defining use-dependent changes in Nav availability in CCs. These results establish that Nav1.3, like other Nav isoforms, can also partner with FGF subunits, strongly regulating Nav channel function.

Human native Cav1 channels in chromaffin cells: contribution to exocytosis and firing of spontaneous action potentials

The present study was performed to evaluate the Cav1 channel subtypes expressed in human chromaffin cells and the role that these channels play in exocytosis and cell excitability. Here we show that human chromaffin cells obtained from organ donors express Cav1.2 and Cav1.3 subtypes using molecular and pharmacological techniques. Immunocytochemical data demonstrated the presence of Cav1.2 and Cav1.3 subtypes, but not Cav1.1 or Cav1.4. Electrophysiological experiments were conducted to investigate the contribution of Cav1 channels to the exocytotic process and cell excitability. Cav1 channels contribute to the exocytosis of secretory vesicles, evidenced by the block of 3μM nifedipine (36.5±2%) of membrane capacitance increment elicited by 200ms depolarizing pulses. These channels show a minor contribution to the initiation of spontaneous action potential firing, as shown by the 2.5 pA of current at the threshold potential (−34mV), which elicits 10.4mV of potential increment. In addition, we found that only 8% of human chromaffin cells exhibit spontaneous action potentials. These data offer novel information regarding human chromaffin cells and the role of human native Cav1 channels in exocytosis and cell excitability.

Exocytosis from chromaffin cells: hydrostatic pressure slows vesicle fusion.

Pressure affects reaction kinetics because chemical transitions involve changes in volume, and therefore pressure is a standard thermodynamic parameter to measure these volume changes. Many organisms live in environments at external pressures other than one atmosphere (0.1 MPa). Marine animals have adapted to live at depths of over 7000 m (at pressures over 70 MPa), and microorganisms living in trenches at over 110 MPa have been retrieved. Here, kinetic changes in secretion from chromaffin cells, measured as capacitance changes using the patch-clamp technique at pressures of up to 20 MPa are presented. It is known that these high pressures drastically slow down physiological functions. High hydrostatic pressure also affects the kinetics of ion channel gating and the amount of current carried by them, and it drastically slows down synaptic transmission. The results presented here indicate a similar change in volume (activation volume) of 390 ± 57 Å(3) for large dense-core vesicles undergoing fusion in chromaffin cells and for degranulation of mast cells. It is significantly larger than activation volumes of voltage-gated ion channels in chromaffin cells. This information will be useful in finding possible protein conformational changes during the reactions involved in vesicle fusion and in testing possible molecular dynamic models of secretory processes.

Lower density of L-type and higher density of P/Q-type of calcium channels in chromaffin cells of hypertensive, compared with normotensive rats

Enhanced activity of the sympatho-adrenal axis and augmented circulating catecholamines has been implicated in the development of hypertension. Release of catecholamine from stimulated adrenal medulla chromaffin cells has been shown to be higher and longer in spontaneously hypertensive rats (SHRs), compared with normotensive Wistar rats (NWRs). Whether differences in the functional expression of voltage-dependent calcium channels (VDCCs) of the L-, N-, or P/Q subtypes may contribute to such distinct secretory behaviour, is unknown. We therefore approached here this study in voltage-clamped NWR and SHR chromaffin cells, using 10mM Ba2+ as charge carrier (IBa) and selective blockers of each channel type. We found that compared with NWR cells, SHR chromaffin cells exhibited the following differences: (1) 30% diminution of the IBa fraction carried by L channels; (2) a doubling of the IBa fraction carried by P/Q channels; (3) more visible current modulation by ATP that could be linked to a 10-fold higher mRNA levels for purinergic receptors of the P2Y2 subtype; and (3) a higher contribution of PQ channels to the transients of the cytosolic calcium concentrations ([Ca2+]c) generated by K+, compared with L channels. These results may contribute to the better understanding of the greater calcium signalling and exocytotic responses of SHR compared with NWR chromaffin cells, found in three previous reports from our laboratories.

Are Cav1.3 pacemaker channels in chromaffin cells? Possible bias from resting cell conditions and DHP blockers usage

Mouse and rat chromaffin cells (MCCs, RCCs) fire spontaneously at rest and their activity is mainly supported by the two L-type Ca2+ channels expressed in these cells (Cav1.2 and Cav1.3). Using Cav1.3-/- KO MCCs we have shown that Cav1.3 possess all the prerequisites for carrying subthreshold currents that sustain low frequency cell firing near resting (0.5 to 2 Hz at -50 mV)1: low-threshold and steep voltage dependence of activation, slow and incomplete inactivation during pulses of several hundreds of milliseconds. Cav1.2 contributes also to pacemaking MCCs and possibly even Na+ channels may participate in the firing of a small percentage of cells. We now show that at potentials near resting (–50 mV), Cav1.3 carries equal amounts of Ca2+ current to Cav1.2 but activates at 9 mV more negative potentials. MCCs express only TTX-sensitive Nav1 channels that activate at 24 mV more positive potentials than Cav1.3 and are fully inactivating. Their blockade prevents the firing only in a small percentage of cells (13%). This suggests that the order of importance with regard to pacemaking MCCs is: Cav1.3, Cav1.2 and Nav1. The above conclusions, however, rely on the proper use of DHPs, whose blocking potency is strongly holding potential dependent. We also show that small increases of KCl concentration steadily depolarize the MCCs causing abnormally increased firing frequencies, lowered and broadened AP waveforms and an increased facility of switching “non-firing” into “firing” cells that may lead to erroneous conclusions about the role of Cav1.3 and Cav1.2 as pacemaker channels in MCCs.2

Inhibition of Ca2+ Channels and Adrenal Catecholamine Release by G Protein Coupled Receptors

Catecholamines and other transmitters released from adrenal chromaffin cells play central roles in the "fight-or-flight" response and exert profound effects on cardiovascular, endocrine, immune, and nervous system function. As such, precise regulation of chromaffin cell exocytosis is key to maintaining normal physiological function and appropriate responsiveness to acute stress. Chromaffin cells express a number of different G protein coupled receptors (GPCRs) that sense the local environment and orchestrate this precise control of transmitter release. The primary trigger for catecholamine release is Ca2+ entry through voltage-gated Ca2+ channels, so it makes sense that these channels are subject to complex regulation by GPCRs. In particular G protein βγ heterodimers (Gbc) bind to and inhibit Ca2+ channels. Here I review the mechanisms by which GPCRs inhibit Ca2+ channels in chromaffin cells and how this might be altered by cellular context. This is related to the potent autocrine inhibition of Ca2+ entry and transmitter release seen in chromaffin cells. Recent data that implicate an additional inhibitory target of Gβγ on the exocytotic machinery and how this might fine tune neuroendocrine secretion are also discussed.

Loose coupling between calcium channels and sites of exocytosis in chromaffin cells

Calcium microdomains generated by tight clusters of calcium channels regulate fusion of small vesicles at the synaptic terminal and have also been suggested to trigger exocytosis of large dense-core vesicles from neuroendocrine cells. To test this idea, we have compared sites of exocytosis and the spatial distribution of calcium channels in chromaffin cells. Fusion of individual vesicles was visualized using interference reflection microscopy and the submembranous calcium signal was assessed using total internal reflection fluorescence microscopy. Depolarization triggered a burst of exocytosis from up to seven sites in a membrane area of 11 microm(2), but these sites did not colocalize with calcium microdomains. Instead, calcium influx occurred in large patches (averaging 34 microm(2)) containing a mixture of P/Q- and N-type channels. About 20% of fusion events occurred outside calcium channel patches. Further, the delay between the onset of stimulation and a burst of exocytosis was prolonged for several seconds by increasing the concentration of the slow calcium chelator EGTA from 1.5 to 5 mM. These results demonstrate that while calcium channels and release sites tend to congregate in specialized regions of the surface membrane, these have dimensions of several micrometres. The dominant calcium signal regulating release in chromaffin cells is generated by the cooperative action of many channels operating over distances of many micrometres rather than discrete clusters of calcium channels generating localized microdomains.

Inhibition of N and PQ calcium channels by calcium entry through L channels in chromaffin cells

Why adrenal chromaffin cells express various subtypes of voltage-dependent Ca(2+) channels and whether a given channel is specialized to perform a specific function are puzzling and unanswered questions. In this study, we have used the L Ca(2+) channel activator FPL64176 (FPL) to test the hypothesis that enhanced Ca(2+) entry through this channel favors the inhibition of N and PQ channels in voltage-clamped bovine adrenal chromaffin cells. Using 2 mM Ca(2+) as charge carrier and under the perforated-patch configuration (PPC) of the patch-clamp technique, FPL caused a paradoxical inhibition of the whole-cell inward Ca(2+) current (I (Ca)). Such inhibition turned on into an augmentation upon cell loading with EGTA-AM. Also, under the whole-cell configuration (WCC) of the patch-clamp technique, FPL decreased I (Ca) in the absence of EGTA from the pipette solution and increased the current in its presence. Using 2 mM Ba(2+) as charge carrier, FPL augmented the Ba(2+) current under both recording conditions, WCC and PPC. FPL augmented the residual current remaining after blockade of N and PQ channels with omega-conotoxin MVIIC or by holding the membrane potential at -50 mV. The data support the view that Ca(2+) entering the cell through the lesser inactivating L channels serves to modulate the more inactivating N and PQ channels. They also suggest a close colocalization of L and N/PQ Ca(2+) channels. This kind of L channel specialization may be relevant to cell excitability, exocytosis, and cell survival mechanisms.

Calcium channels in chromaffin cells: focus on L and T types

Voltage-gated Ca2+ channels (Cav) are highly expressed in the adrenal chromaffin cells of mammalian species. Besides shaping action potential waveforms, they are directly involved in the excitation-secretion coupling underlying catecholamine release and, possibly, control other Ca2+-dependent events that originate near the membrane. These functions are shared by a number of Cav channel types (L, N, P/Q, R and T) which have different structure-function characteristics and whose degree of expression changes remarkably among mammalian species. Understanding precisely the functioning of each voltage-gated Ca2+ channels is a crucial task that helps clarifying the Ca2+-dependent mechanisms controlling exocytosis during physiological and pathological conditions. In this paper, we focus on classical and new roles that L- and T-type channels play in the control of chromaffin cell excitability and neurotransmitter release. Interestingly, L-type channels are shown to be implicated in the spontaneous autorhythmicity of chromaffin cells, while T-type channels, which are absent in adult chromaffin cells, are coupled with secretion and can be recruited following long-term beta-adrenergic stimulation or chronic hypoxia. This suggests that like other cells, adrenal chromaffin cells undergo effective remodelling of membrane ion channels and cell functioning during prolonged stress conditions.

Fast exocytosis mediated by T- and L-type channels in chromaffin cells: distinct voltage-dependence but similar Ca2+-dependence

Expression, spatial distribution and specific roles of different Ca(2+) channels in stimulus-secretion coupling of chromaffin cells are intriguing issues still open to discussion. Most of the evidence supports a role of high-voltage activated (HVA) Ca(2+) channels (L-, N-, P/Q- and R-types) in the control of exocytosis: some suggesting a preferential coupling of specific Ca(2+) channel subunits with the secretory apparatus, others favoring the idea of a contribution to secretion proportional to the expression density and gating properties of Ca(2+) channels. In this work we review recent findings and bring new evidence in favor of the hypothesis that also the LVA (low-voltage-activated, T-type) Ca(2+) channels effectively control fast exocytosis near resting potential in adrenal chromaffin cells of adult rats. T-type channels recruited after long-term treatments with pCPT-cAMP (or chronic hypoxia) are shown to control exocytosis with the same efficacy of L-type channels, which are the dominant Ca(2+) channel types expressed in rodent chromaffin cells. A rigorous comparison of T- and L-type channel properties shows that, although operating at different potentials and with different voltage-sensitivity, the two channels possess otherwise similar Ca(2+)-dependence of exocytosis, size and kinetics of depletion of the immediately releasable pool and mobilize vesicles of the same quantal size. Thus, T- and L-type channels are coupled with the same Ca(2+)-efficiency to the secretory apparatus and deplete the same number of vesicles ready for release. The major difference of the secretory signals controlled by the two channels appear to be the voltage range of operation, suggesting the idea that stressful conditions (hypoxia and persistent beta-adrenergic stimulation) can lower the threshold of cell excitability by recruiting new Ca(2+) channels and activate an additional source of catecholamine secretion.

Calcium Signaling and Exocytosis in Adrenal Chromaffin Cells

At a given cytosolic domain of a chromaffin cell, the rate and amplitude of the Ca2+ concentration ([Ca2+]c) depends on at least four efficient regulatory systems: 1) plasmalemmal calcium channels, 2) endoplasmic reticulum, 3) mitochondria, and 4) chromaffin vesicles. Different mammalian species express different levels of the L, N, P/Q, and R subtypes of high-voltage-activated calcium channels; in bovine and humans, P/Q channels predominate, whereas in felines and murine species, L-type channels predominate. The calcium channels in chromaffin cells are regulated by G proteins coupled to purinergic and opiate receptors, as well as by voltage and the local changes of [Ca2+]c. Chromaffin cells have been particularly useful in studying calcium channel current autoregulation by materials coreleased with catecholamines, such as ATP and opiates. Depending on the preparation (cultured cells, adrenal slices) and the stimulation pattern (action potentials, depolarizing pulses, high K+, acetylcholine), the role of each calcium channel in controlling catecholamine release can change drastically. Targeted aequorin and confocal microscopy shows that Ca2+ entry through calcium channels can refill the endoplasmic reticulum (ER) to nearly millimolar concentrations, and causes the release of Ca2+ (CICR). Depending on its degree of filling, the ER may act as a sink or source of Ca2+ that modulates catecholamine release. Targeted aequorins with different Ca2+ affinities show that mitochondria undergo surprisingly rapid millimolar Ca2+ transients, upon stimulation of chromaffin cells with ACh, high K+, or caffeine. Physiological stimuli generate [Ca2+]c microdomains in which the local subplasmalemmal [Ca2+]c rises abruptly from 0.1 to approximately 50 microM, triggering CICR, mitochondrial Ca2+ uptake, and exocytosis at nearby secretory active sites. The fact that protonophores abolish mitochondrial Ca2+ uptake, and increase catecholamine release three- to fivefold, support the earlier observation. This increase is probably due to acceleration of vesicle transport from a reserve pool to a ready-release vesicle pool; this transport might be controlled by Ca2+ redistribution to the cytoskeleton, through CICR, and/or mitochondrial Ca2+ release. We propose that chromaffin cells have developed functional triads that are formed by calcium channels, the ER, and the mitochondria and locally control the [Ca2+]c that regulate the early and late steps of exocytosis.

An activity-dependent increased role for L-type calcium channels in exocytosis is regulated by adrenergic signaling in chromaffin cells

Chromaffin cells of the adrenal medulla represent a primary output of the sympathetic nervous system. Their electrical stimulation evokes the fusion of large dense core granules with the cell membrane and the exocytic release of multiple transmitter molecules into the circulation. There the transmitters contribute to the regulation of basic metabolism of the organism. Under physiological activity, granule fusion and transmitter release are limited by activity-dependent Ca2+ influx, entering through multiple isoforms of voltage-gated calcium channels. In this study we utilize perforated-patch voltage-clamp recordings and depolarize mouse chromaffin cells in situ with action potential-like waveforms to mimic physiological firing. We measure calcium influx through specific isoforms and measure cell capacitance as an index of granule fusion. Combining these approaches we calculate specific stimulus-secretion efficiencies for L-type, N-type, P/Q-type and R-type calcium channels under varied physiological activity levels. Current influx through all channel subtypes exhibited an activity-dependent depression. As expected P/Q-type channels, while responsible for modest Ca2+ influx, are tightly coupled to catecholamine secretion under all conditions. We further find that stimulation designed to match sympathetic input under the acute stress response recruits L-type channels to a state of enhanced stimulus-secretion efficiency. N- and R-type channels do not undergo activity-dependent recruitment and remain loosely coupled to the secretion. Thus, only L-type channels exhibit activity-dependent changes in their stimulus-secretion function under physiological stimulation. Lastly, we show that treatment with the β-adrenergic agonist, isoproterenol, specifically blocks the increase in the stimulus-secretion function of L-type channels. Thus, increased cell firing specifically enhances stimulus-secretion coupling of L-type Ca2+ channels in chromaffin cells in situ. This mechanism is regulated by an adrenergic signaling pathway.

Intracellular Ca 2+ microdomain-triggered exocytosis in neuroendocrine cells

Colocalization of voltage-gated Ca 2+ channels and exocytotic sites at the active zones of nerve terminals underlies ‘synchronous’ action potential discharge and synaptic vesicle exocytosis, thus allowing fast interneuronal signalling. Such a demand for a rapid release is not expected in neuroendocrine cells whose secretory products act throughout the entire organism. Nevertheless, by using evanescent field imaging of near-membrane Ca 2+ concentrations and fluorescently labelled vesicles, Becherer et al. have recently reported exocytosis of individual large dense-core vesicles triggered by Ca 2+ microdomains formed around clusters of open L-type Ca 2+ channels in chromaffin cells from the adrenal medulla. This finding, besides illustrating the power of new microscopy imaging techniques, directly demonstrates in neuroendocrine cells a functional interaction between Ca 2+ channels and secretory vesicles very much reminiscent of that in neurons.

Adrenal medulla calcium channel population is not conserved in bovine chromaffin cells in culture

During the stress response adrenal medullary chromaffin cells release catecholamines to the bloodstream. Voltage-activated calcium channels present in the cell membrane play a crucial role in this process. Although the electrophysiological and pharmacological properties of chromaffin cell calcium channels have been studied in detail, the molecular composition of these channels has not been defined yet. Another aspect that needs to be explored is the extent to which chromaffin cells in culture reflect the adrenal medulla calcium channel characteristics. In this sense, it has been described that catecholamine release in the intact adrenal gland recruits different calcium channels than those recruited during secretion from cultured chromaffin cells. Additionally, recent electrophysiological studies show that chromaffin cells in culture differ from those located in the intact adrenal medulla in the contribution of several calcium channel types to the whole cell current. However there is not yet any study that compares the population of calcium channels in chromaffin cells with that one present in the adrenal medulla. In order to gain some insight into the roles that calcium channels might play in the adrenal medullary cells we have analyzed the α 1 subunit mRNA expression profile. We demonstrate that the expression pattern of voltage-dependent calcium channels in cultured bovine chromaffin cells markedly differs from that found in the native adrenal medulla and that glucocorticoids are only partially involved in those differences. Additionally, we show, for the first time, that the cardiac isoform of L-type calcium channel is present in both bovine adrenal medulla and cultured chromaffin cells and that its levels of expression do not vary during culture.

SNX482 selectively blocks P/Q Ca 2+ channels and delays the inactivation of Na + channels of chromaffin cells

The effects of the toxin SXN482 on Ca 2+ channel currents ( I Ca), Na + currents ( I Na), and K + currents ( I K) have been studied in bovine adrenal medullary chromaffin cells voltage-clamped at −80 mV. Currents were elicited by depolarising pulses to 0–10 mV ( I Ca and I Na) or to +60 mV ( I K). SNX482 blocked I Ca in a concentration-dependent manner. The inhibition curve exhibited two phases. The first high-affinity phase comprised 28% of the whole-cell current and exhibited an IC 50 of 30.2 nM. The second low-affinity phase comprised over 70% of I Ca and had an IC 50 of 758.6 nM. Blockade was rapid and fully reversible upon washout of the toxin. Occlusion experiments showed additivity of blockade exerted by nifedipine plus SNX482 (0.3 μM) and by ω-conotoxin GVIA plus SNX482. In contrast, blockade exerted by combined ω-agatoxin IVA plus SNX482 (about 50% of the whole cell) did not show additivity. At 0.3 μM and higher concentrations, SNX482 delayed the inactivation of I Na. The time constant ( τ) for inactivation of I Na in control conditions doubled in the presence of 0.5 μM SNX482. At 0.3 μM, SNX482 did not affect I K. Our data demonstrate that: (i) SNX482 selectively blocks P/Q Ca 2+ channels at submicromolar concentrations; (ii) the toxin partially blocks Na + channels; (iii) SNX482 delays the inactivation of Na + channels. These results reveal novel properties of SNX482 and cast doubts on the claimed selectivity and specificity of the toxin to block the R-type Ca 2+ channel.

Opposite Action of β1- and β2-Adrenergic Receptors on CaV1 L-Channel Current in Rat Adrenal Chromaffin Cells

Voltage-gated Ca(2+) channels of chromaffin cells are modulated by locally released neurotransmitters through autoreceptor-activated G-proteins. Clear evidence exists in favor of a Ca(2+) channel gating inhibition mediated by purinergic, opioidergic, and alpha-adrenergic autoreceptors. Few and contradictory data suggest also a role of beta-adrenergic autoreceptors (beta-ARs), the action of which, however, remains obscure. Here, using patch-perforated recordings, we show that rat chromaffin cells respond to the beta-AR agonist isoprenaline (ISO) by either upmodulating or downmodulating the amplitude of Ca(2+) currents through two distinct modulatory pathways. ISO (1 microm) could cause either fast inhibition (approximately 25%) or slow potentiation (approximately 25%), or a combination of the two actions. Both effects were completely prevented by propranolol. Slow potentiation was more evident in cells pretreated with pertussis toxin (PTX) or when beta(1)-ARs were selectively stimulated with ISO + ICI118,551. Potentiation was absent when the beta(2)-AR-selective agonist zinterol (1 microm), the protein kinase A (PKA) inhibitor H89, or nifedipine was applied, suggesting that potentiation is associated with a PKA-mediated phosphorylation of L-channels (approximately 40% L-current increase) through beta(1)-ARs. The ISO-induced inhibition was fast and reversible, preserved in cell treated with H89, and mimicked by zinterol. The action of zinterol was mostly on L-channels (38% inhibition). Zinterol action preserved the channel activation kinetics, the voltage-dependence of the I-V characteristic, and was removed by PTX, suggesting that beta(2)AR-mediated channel inhibition was mainly voltage independent and coupled to G(i)/G(o)-proteins. Sequential application of zinterol and ISO mimicked the dual action (inhibition/potentiation) of ISO alone. The two kinetically and pharmacologically distinct beta-ARs signaling uncover alternative pathways, which may serve the autocrine control of Ca(2+)-dependent exocytosis and other related functions of rat chromaffin cells.

Steady-State and Closed-State Inactivation Properties of Inactivating BK Channels

Calcium-dependent potassium (BK-type) Ca 2+ and voltage-dependent K + channels in chromaffin cells exhibit an inactivation that probably arises from coassembly of Slo1 α subunits with auxiliary β subunits. One goal of this work was to determine whether the Ca 2+ dependence of inactivation arises from any mechanism other than coupling of inactivation to the Ca 2+ dependence of activation. Steady-state inactivation and the onset of inactivation were studied in inside-out patches and whole-cell recordings from rat adrenal chromaffin cells with parallel experiments on inactivating BK channels resulting from cloned α + β2 subunits. In both cases, steady-state inactivation was shifted to more negative potentials by increases in submembrane [Ca 2+] from 1 to 60 μM. At 10 and 60 μM Ca 2+, the maximal channel availability at negative potentials was similar despite a shift in the voltage of half availability, suggesting there is no strictly Ca 2+-dependent inactivation. In contrast, in the absence of Ca 2+, depolarization to potentials positive to +20 mV induces channel inactivation. Thus, voltage-dependent, but not solely Ca 2+-dependent, kinetic steps are required for inactivation to occur. Finally, under some conditions, BK channels are shown to inactivate as readily from closed states as from open states, indicative that a key conformational change required for inactivation precedes channel opening.

Cause for excite-M-ent in adrenal chromaffin cells

Cause for excite-M-ent in adrenal chromaffin cells

Neuronal Ca2+ Sensor-1/Frequenin Functions in an Autocrine Pathway Regulating Ca2+ Channels in Bovine Adrenal Chromaffin Cells

NCS-1/frequenin belongs to a family of EF-hand-containing Ca(2+) sensors expressed mainly in neurons. Overexpression of NCS-1/frequenin has been shown to stimulate neurotransmitter release but little else is known of its cellular roles. We have constructed an EF-hand mutant, NCS-1(E120Q), as a likely dominant inhibitor of cellular NCS-1 function. Recombinant NCS-1(E120Q) showed an impaired Ca(2+)-dependent conformational change but could still bind to cellular proteins. Transient expression of this mutant, but not NCS-1, in bovine adrenal chromaffin cells increased non-L-type Ca(2+) channel currents. Cells expressing NCS-1(E120Q) no longer responded effectively to the removal of autocrine purinergic/opioid inhibition of Ca(2+) currents but still showed voltage-dependent facilitation. These data are consistent with the existence of both voltage-dependent and voltage-independent pathways for Ca(2+) channel inhibition in chromaffin cells. Our results suggest a novel function for NCS-1 specific for the voltage-independent autocrine pathway that negatively regulates non-L-type Ca(2+) channels in chromaffin cells.