A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. I. Effects of caffeine on intracellular Ca2+ stores

Summary The effects of agents known to interfere with Ca²⁺ release processes of endoplasmic reticulum were investigated in bradykinin (BK)-stimulated bovine aortic endothelial cells (BAE cells), via the activation of Ca²⁺-activated potassium channels [K(Ca²⁺) channels]. In cell-attached patch experiments, the external application of caffeine (1 mm) caused a brief activation of K(Ca²⁺) channels in Ca²⁺-free and Ca²⁺-containing external solutions. The application of BK (10 nm) during cell stimulation by caffeine (1–20 mm) invariably led to a drastic channel activation which was maintained during a recording period longer than that observed in caffeine-free conditions. In addition, the cell exposure to caffeine (20 mm) during the BK stimulation enhanced systematically the channel activation process. Since a rapid inhibition of BK-evoked channel activity was also produced by removing caffeine from the bath medium, it is proposed that the sustained single-channel response recorded in the concomittant presence of both agents was due to their synergic action on internal stores and/or the external Ca²⁺ entry pathway resulting in an increased [Ca²⁺]_i. In addition, the local anesthetic, procaine, depressed the initial BK-induced K(Ca²⁺) channel activity and completely blocked the secondary phase of the channel activation process related to the external Ca²⁺ influx into stimulated cells. In contrast, this blocking effect of procaine was not observed on the initial caffeine-elicited channel activity and could not suppress the external Ca²⁺-dependent phase of this channel activation process. Our results confirm the existence of at least two pharmacologically distinct types of Ca²⁺-release from internal stores in BAE cells: an inositol 1,4,5-triphosphate (InsP₃)-dependent and a caffeine-induced Ca²⁺-release process.The authors would like to thank Dr. A. Diarra for his contribution to the fluorescence measurements and Diane Vallerand for preparing cell cultures. These data were presented in part at the 14th Scientific Meeting of the International Society of Hypertension (Madrid, Spain, June 14–18, 1992), and have been published in abstract form in the Journal of Hypertension (1992). Dominique Thuringer is a fellow of the Heart and Stroke Foundation of Canada. Rémy Sauvé is a senior fellow from the Fonds de la Recherche en Santé du Québec. This work was supported by a grant from the Medical Research Council of Canada.     

Norepinephrine activates store-operated Ca2+ entry coupled to large-conductance Ca2+-activated K+ channels in rat pinealocytes

Norepinephrine (NE) is one of the major neurotransmitters that determine melatonin production in the pineal gland. Although a substantial amount of Ca²⁺ influx is triggered by NE, the Ca²⁺ entry pathway and its physiological relevance have not been elucidated adequately. Herein we report that the Ca²⁺ influx triggered by NE significantly regulates the protein level of serotonin N-acetyltransferase, or arylalkylamine N-acetyltransferase (AANAT), a critical enzyme in melatonin production, and is responsible for maintaining the Ca²⁺ response after repetitive stimulation. Ca²⁺ entry evoked by NE was dependent on PLC activation. NE evoked a substantial amount of Ca²⁺ entry even after cells were treated with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an analog of diacylglycerol. To the contrary, further OAG treatment after cells had been exposed to OAG did not evoke additional Ca²⁺ entry. Moreover, NE failed to induce further Ca²⁺ entry after the development of Ca²⁺ entry induced by thapsigargin (Tg), suggesting that the pathway of Ca²⁺ entry induced by NE might be identical to that of Tg. Interestingly, Ca²⁺ entry evoked by NE or Tg induced membrane hyperpolarization that was reversed by iberiotoxin (IBTX), a specific inhibitor of large-conductance Ca²⁺-activated K⁺ (BK) channels. Moreover, IBTX-sensitive BK current was observed during application of NE, suggesting that activation of the BK channels was responsible for the hyperpolarization. Furthermore, the activation of BK channels triggered by NE contributed to regulation of the protein level of AANAT. Collectively, these results suggest that NE triggers Ca²⁺ entry coupled to BK channels and that NE-induced Ca²⁺ entry is important in the regulation of AANAT. serotonin N-acetyltransferase; pineal gland     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Ca2+ Mobilization

Many physiological processes are controlled by a great diversity of Ca^{2 +} signals that depend on Ca^{2 +} entry into the cell and/or Ca^{2 +} release from internal Ca^{2 +} stores. Ca^{2 +} mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP₃), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca^{2 +} release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP₃ and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca^{2 +} stores. Activation of the NAADP-sensitive Ca^{2 +} channels evokes complex changes in cytoplasmic Ca^{2 +} levels by means of channel chatter with other intracellular Ca^{2 +} channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca^{2 +} signaling.     

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP₃)-mediated Ca²⁺ release in renal epithelial LLC-PK₁ cells. AVP-induced changes in the intracellular free calcium concentration ([Ca²⁺]_i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca²⁺ mobilization was also observed in the absence of extracellular Ca²⁺, but was completely abolished after depletion of the intracellular Ca²⁺ stores by 2 μM thapsigargin. Using ⁴⁵Ca²⁺ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP₃ affected the Ca²⁺ content of nonmitochondrial Ca²⁺ pools in different loading and release conditions. Less than 10% of the Ca²⁺ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca²⁺. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca²⁺, but Ca²⁺ sequestered in this pool could not be released by InsP₃. The thapsigargin-sensitive Ca²⁺ pool, in contrast, was nearly completely InsP₃ sensitive. A submaximal [InsP₃], however, released only a fraction of the sequestered Ca²⁺. This fraction was dependent on the cytosolic as well as on the luminal [Ca²⁺]. The cytosolic free [Ca²⁺] affected the InsP₃-induced Ca²⁺ release in a biphasic way. Maximal sensitivity toward InsP₃ was found at a free cytosolic [Ca²⁺] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca²⁺] decreased the InsP₃ sensitivity. Other divalent cations or La³⁺ did not provoke similar inhibitory effects on InsP₃-induced Ca²⁺ release. The luminal free [Ca²⁺] was manipulated by varying the time of incubation of Ca²⁺ -loaded cells in an EGTA-containing medium. Reduction of the Ca²⁺ content to one-third of its initial value resulted in a fivefold decrease in the InsP₃ sensitivity of the Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

Mitochondrial Calcium Uniporter MCU Supports Cytoplasmic Ca2+ Oscillations,Store-Operated Ca2+ Entry and Ca2+-Dependent Gene Expression in Response to Receptor Stimulation

Ca²⁺ flux into mitochondria is an important regulator of cytoplasmic Ca²⁺ signals, energy production and cell death pathways. Ca²⁺ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU) but whether the MCU is involved in shaping Ca²⁺ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC₄ evokes a series of cytoplasmic Ca²⁺ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca²⁺ entry into the matrix, prevents the mitochondrial Ca²⁺ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca²⁺ oscillations appeared to be a consequence of enhanced Ca²⁺-dependent inactivation of InsP₃ receptors, which arose from the loss of mitochondrial Ca²⁺ buffering. Ca²⁺ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca²⁺ release, mitochondria also sequestrated Ca²⁺ entry through store-operated Ca²⁺ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca²⁺.     

Impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels in the coronary artery smooth muscle cells of Zucker Diabetic Fatty rats

The large-conductance Ca²⁺-activated K⁺ (BK) channels play an important role in the regulation of cellular excitability in response to changes in intracellular metabolic state and Ca²⁺ homeostasis. In vascular smooth muscle, BK channels are key determinants of vasoreactivity and vital-organ perfusion. Vascular BK channel functions are impaired in diabetes mellitus, but the mechanisms underlying such changes have not been examined in detail. We examined and compared the activities and kinetics of BK channels in coronary arterial smooth muscle cells from Lean control and Zucker Diabetic Fatty (ZDF) rats, using single-channel recording techniques. We found that BK channels in ZDF rats have impaired Ca²⁺ sensitivity, including an increased free Ca²⁺ concentration at half-maximal effect on channel activation, a reduced steepness of Ca²⁺ dose-dependent curve, altered Ca²⁺-dependent gating properties with decreased maximal open probability, and a shortened mean open-time and prolonged mean closed-time durations. In addition, the BK channel β-subunit-mediated activation by dehydrosoyasaponin-1 (DHS-1) was lost in cells from ZDF rats. Immunoblotting analysis confirmed a 2.1-fold decrease in BK channel β₁-subunit expression in ZDF rats, compared with that of Lean rats. These abnormalities in BK channel gating lead to an increase in the energy barrier for channel activation, and may contribute to the development of vascular dysfunction and complications in type 2 diabetes mellitus.     

Methylglyoxal evokes acute Ca2+ transients in distinct cell types and increases agonist-evoked Ca2+ entry in endothelial cells via CRAC channels

Methylglyoxal (MG) is a by-product of glucose metabolism and its accumulation has been linked to the development of diabetic complications such as retinopathy and nephropathy by affecting multiple signalling pathways. However, its influence on the intracellular Ca²⁺ homeostasis and particularly Ca²⁺ entry, which has been reported to be mediated via TRPA1 channels in DRG neurons, has not been studied in much detail in other cell types. In this study, we report the consequences of acute and long-term MG application on intracellular Ca²⁺ levels in endothelial cells. We showed that acute MG application doesn’t evoke any instantaneous changes in the intracellular Ca²⁺ concentration in immortalized mouse cardiac endothelial cells (MCECs) and murine microvascular endothelial cells (muMECs). In contrast, an MG-induced rise in intracellular Ca²⁺ level was observed in primary mouse mesangial cells within 30 s, indicating that the modulation of Ca²⁺ homeostasis by MG is strictly cell type specific. The formation of the MG-derived advanced glycation end product (AGE) MG-H1 was found to be time and concentration-dependent in MCECs. Likewise, MG pre-incubation for 6 h increased the angiotensin II-evoked Ca²⁺ entry in MCECs and muMECs which was abrogated by inhibition of Calcium release activated calcium (CRAC) channels with GSK-7975A, but unaffected by an inhibitor specific to TRPA1 channels. Quantitative PCR analysis revealed that MG pre-treatment did not affect expression of the genes encoding the angiotensin receptors AT1R (Agtr 1a & Agtr 1b), Trpa1 nor Orai1, Orai2, Orai3, Stim1, Stim2 and Saraf which operate as constituents or regulators of CRAC channels and store-operated Ca²⁺ entry (SOCE) in other cell types. Together, our results show that long-term MG stimulation leads to the formation of glycation end products, which facilitates the agonist-evoked Ca²⁺ entry in endothelial cells, and this could be a new pathway that might lead to MG-evoked vasoregression observed in diabetic vasculopathies.     

Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

The presence and physiological role of Ca²⁺-induced Ca²⁺ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca²⁺] ([Ca²⁺]_c). Using targeted aequorin, we have directly monitored [Ca²⁺] changes inside the ER ([Ca²⁺]_ER) in bovine adrenal chromaffin cells. Ca²⁺ entry induced by cell depolarization triggered a transient Ca²⁺ release from the ER that was highly dependent on [Ca²⁺]_ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca²⁺ release was quantal in nature due to modulation by [Ca²⁺]_ER. Whereas caffeine released essentially all the Ca²⁺ from the ER, inositol 1,4,5-trisphosphate (InsP₃)- producing agonists released only 60–80%. Both InsP₃ and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca²⁺ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca²⁺]_c measurements showed that the wave of [Ca²⁺]_c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca²⁺ pool that can release Ca²⁺ both via InsP₃ receptors or CICR.     

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP₃)-mediated increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). Local Ca²⁺ release from an InsP₃ receptor (InsP₃R) cluster present on the sarcoplasmic reticulum is termed a Ca²⁺ puff. Ca²⁺ released via InsP₃R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca²⁺ rise. In smooth muscle, mitochondrial Ca²⁺ uptake maintains global InsP₃-mediated Ca²⁺ release by preventing a negative feedback effect of high [Ca²⁺] on InsP₃R. Mitochondria may regulate InsP₃-mediated Ca²⁺ signals by operating between or within InsP₃R clusters. In the former mitochondria could regulate only global Ca²⁺ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP₃-mediated Ca²⁺ release by operating within (local) or between (global) InsP₃R clusters has been addressed. Ca²⁺ puffs evoked by localized photolysis of InsP₃ in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5–4.0 F/F₀, durations of ∼112 ms at half-maximum amplitude, and were abolished by the InsP₃R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized ΔΨ_M to prevent mitochondrial Ca²⁺ uptake and attenuated Ca²⁺ puffs by ∼66 or ∼60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca²⁺ puffs by ∼62%. The “fast” Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acted like mitochondria to prolong InsP₃-mediated Ca²⁺ release suggesting that mitochondrial influence is via their Ca²⁺ uptake facility. These results indicate Ca²⁺ uptake occurs quickly enough to influence InsP₃R communication at the intra-cluster level and that mitochondria regulate both local and global InsP₃-mediated Ca²⁺ signals.     

Ca2+ release and Ca2+ influx in Chinese hamster ovary cells expressing the cloned mouse B2 bradykinin receptor: tyrosine kinase inhibitor-sensitive and -insensitive processes

A cDNA encoding a mouse B₂ bradykinin (BK) receptor was stably transfected in Chinese hamster ovary (CHO) cells. In two resulting transformants, mouse B₂ BK receptor was found to induce a twofold elevation in the inositol-1,4,5-trisphosphate level. In a pertussis toxin-insensitive manner, BK also produced a biphasic increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). The initial elevation in [Ca²⁺]_i was abolished by thapsigargin pretreatment in Ca²⁺-free medium. The second phase was dependent on external Ca²⁺. The BK/inositol trisphosphate- and thapsigargin-sensitive Ca²⁺ stores required extracellular Ca²⁺ for refilling. Ca²⁺ influx induced by BK and thapsigargin was confirmed by Mn²⁺ entry through Ca²⁺ influx pathways producing Mn²⁺ quenching. Genistein, a tyrosine kinase inhibitor, partially decreased the BK-induced [Ca²⁺]_i increase during the sustained phase and the rate of Mn²⁺ entry. BK had essentially no effect on the intracellular cyclic AMP level. The results suggest that the mouse B₂ BK receptor couples to phospholipase C in CHO cells and that its activation results in biphasic [Ca²⁺]_i increases, by mobilization of intracellular Ca²⁺ and store-depletion-mediated Ca²⁺ influx, the latter of which is tyrosine phosphorylation-dependent.     

Divergence of Ca2+ selectivity and equilibrium Ca2+ blockade in a Ca2+ release-activated Ca2+ channel

Prevailing models postulate that high Ca²⁺ selectivity of Ca²⁺ release-activated Ca²⁺ (CRAC) channels arises from tight Ca²⁺ binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca²⁺ binding for Ca²⁺ selectivity in recombinant Orai3 channels, which function as highly Ca²⁺-selective channels when gated by the endoplasmic reticulum Ca²⁺ sensor STIM1 or as poorly Ca²⁺-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca²⁺ blocked Na⁺ currents in both gating modes with a similar inhibition constant (K_i; ∼25 µM). Thus, equilibrium binding as set by the K_i of Ca²⁺ blockade cannot explain the differing Ca²⁺ selectivity of the two gating modes. Unlike STIM1-gated channels, Ca²⁺ blockade in 2-APB–gated channels depended on the extracellular Na⁺ concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na⁺ pore occupancy. Moreover, the second-order rate constants of Ca²⁺ blockade were eightfold faster in 2-APB–gated channels than in STIM1-gated channels. A four-barrier, three–binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca²⁺ and Na⁺ to simulate the faster rate constants of 2-APB–gated channels qualitatively reproduces their low Ca²⁺ selectivity, suggesting that ion entry and exit rates strongly affect Ca²⁺ selectivity. Noise analysis indicated that the unitary Na⁺ conductance of 2-APB–gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (P_o; ∼0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high P_o state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca²⁺ binding and kinetic factors contribute to high Ca²⁺ selectivity in CRAC channels.     

Role ofDrosophila TRP in inositide-mediated Ca2+ entry

Inositol lipid signaling relies on an InsP₃-induced Ca²⁺ release from intracellular stores and on extracellular Ca²⁺ entry, which takes place when the Ca²⁺ stores become depleted of Ca²⁺. This interplay between Ca²⁺ release and Ca²⁺ entry has been termed capacitative Ca²⁺ entry and the inward current calcium release activated current (CRAC) to indicate gating of Ca²⁺ entry by Ca²⁺-store depletion. The signaling pathway and the gating mechanism of capacitative Ca²⁺ entry, however, are largely unknown and the molecular participants in this process have not been identified. In this article we review genetic, molecular, and functional studies of wild-type and mutantDrosophila photoreceptors, suggesting that thetransient receptor potential mutant (trp) is the first putative capacitative Ca²⁺ entry mutant. Furthermore, several lines of evidence suggest that thetrp gene product TRP is a candidate subunit of the plasma membrane channel that is activated by Ca²⁺ store depletion.     

A re-evaluation of the apparent effects of luminal Ca on inositol 1,4,5-trisphosphate-induced Ca release

It has been suggested that the release of Ca²⁺ from intracellular stores by inositol 1,4,5-trisphosphate (InsP₃) is modulated by the luminal Ca²⁺ content of the stores and that such an effect could underlie the apparent ‘quantal’ nature of InsP3-induced release. Although initial studies failed to find evidence in support of such a modulation, several subsequent reports have indicated luminal Ca²⁺ effects that become apparent only after a greater than 70–75% depletion of Ca²⁺ stores. In these studies, Ca²⁺ release was expressed as a percentage of an A23187-releasable pool which comprised both InsP₃-sensi-tive and InsP₃-insensitive components. In model calculations we have found that the presence of even a minor InsP₃-insensitive component in the total Ca²⁺ pool significantly distorts interpretation of the data. We show that the published results can be accurately duplicated without any requirement for a shift in the true InsP3 sensitivity of Ca²⁺ release if either: (a) the InsP₃-insensitive component does not remain a constant proportion of the total pool during depletion (i.e. depletion disproportionally affects the InsP₃-sensitive component); or (b) during generation of InsP₃-response curves, additional Cal ²⁺ is released from the InsP₃-insensitive component as the InsP₃-sensitive component is progressively emptied. Examination indicates that either, or both, of these conditions apply in the published reports and we conclude that the demonstrated effects of luminal Ca²⁺ may be artifacts.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca²⁺ release by photolysis of caged InsP₃ have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca²⁺-activated Cl⁻ and K⁺ currents. Photolytic release of InsP₃ from caged InsP₃ at 100 Joules caused transient inward (V_H = 60 mV) and outward (V_H = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl⁻ concentration was decreased, and the outward current was suppressed by K⁺ channel blockers, indicating that they were carried by Cl⁻ and K⁺, respectively. Intracellular pre-loading of the InsP₃ receptor antagonist heparin or the Ca²⁺ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca²⁺-dependent Cl⁻ and K⁺ currents underlies the inward and the outward currents. At low flash intensities, InsP₃ caused Ca²⁺ release which normally activated the K⁺ and Cl⁻ currents in a mono-transient manner. At higher intensities, however, InsP₃ induced an additional delayed outward K⁺ current (I_K(delay)). I_K(delay) was independent of the initial K⁺ current, independent of extracellular Ca²⁺, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca²⁺ from caged Ca²⁺ did not mimic the I_K(delay). It is suggested that Ca²⁺ releases from the InsP₃-sensitive pools in an InsP₃ concentration-dependent manner. Low concentrations of InsP₃ induce the transient Ca²⁺-dependent Cl⁻ and K⁺ currents, which reflects the local Ca²⁺ release, whereas high concentrations of InsP₃ induce a delayed Ca²⁺-dependent K⁺ current, which may reflect the Ca²⁺ wave propagation. J. Cell. Physiol. 174:387–397, 1998. © 1998 Wiley-Liss, Inc.     

Ca2+-activated K channels in parotid acinar cells: The functional basis for the hyperpolarized activation of BK channels

Fluid secretion relies on a close interplay between Ca²⁺-activated Cl and K channels. Salivary acinar cells contain both large conductance, BK, and intermediate conductance, IK1, K channels. Physiological fluid secretion occurs with only modest (<500 nM) increases in intracellular Ca²⁺ levels but BK channels in many cell types and in heterologous expression systems require very high concentrations for significant activation. We report here our efforts to understand this apparent contradiction. We determined the Ca²⁺ dependence of IK1 and BK channels in mouse parotid acinar cells. IK1 channels activated with an apparent Ca²⁺ affinity of about 350 nM and a hill coefficient near 3. Native parotid BK channels activated at similar Ca²⁺ levels unlike the BK channels in other cell types. Since the parotid BK channel is encoded by an uncommon splice variant, we examined this clone in a heterologous expression system. In contrast to the native parotid channel, activation of this expressed “parslo” channel required very high levels of Ca²⁺. In order to understand the functional basis for the special properties of the native channels, we analyzed the parotid BK channel in the context of the horrigan-Aldrich model of BK channel gating. We found that the shifted activation of parotid BK channels resulted from a hyperpolarizing shift of the voltage dependence of voltage sensor activation and channel opening and included a large change in the coupling of these two processes.Key words: ion channels, Ca²⁺-activated K channels, maxi-K channels, IK1 channels     

Synaptic NMDA Receptor-Dependent Ca2+ Entry Drives Membrane Potential and Ca2+ Oscillations in Spinal Ventral Horn Neurons

During vertebrate locomotion, spinal neurons act as oscillators when initiated by glutamate release from descending systems. Activation of NMDA receptors initiates Ca²⁺-mediated intrinsic membrane potential oscillations in central pattern generator (CPG) neurons. NMDA receptor-dependent intrinsic oscillations require Ca²⁺-dependent K⁺ (K_Ca2) channels for burst termination. However, the location of Ca²⁺ entry mediating K_Ca2 channel activation, and type of Ca²⁺ channel – which includes NMDA receptors and voltage-gated Ca²⁺ channels (VGCCs) – remains elusive. NMDA receptor-dependent Ca²⁺ entry necessitates presynaptic release of glutamate, implying a location at active synapses within dendrites, whereas VGCC-dependent Ca²⁺ entry is not similarly constrained. Where Ca²⁺ enters relative to K_Ca2 channels is crucial to information processing of synaptic inputs necessary to coordinate locomotion. We demonstrate that Ca²⁺ permeating NMDA receptors is the dominant source of Ca²⁺ during NMDA-dependent oscillations in lamprey spinal neurons. This Ca²⁺ entry is synaptically located, NMDA receptor-dependent, and sufficient to activate K_Ca2 channels at excitatory interneuron synapses onto other CPG neurons. Selective blockade of VGCCs reduces whole-cell Ca²⁺ entry but leaves membrane potential and Ca²⁺ oscillations unaffected. Furthermore, repetitive oscillations are prevented by fast, but not slow, Ca²⁺ chelation. Taken together, these results demonstrate that K_Ca2 channels are closely located to NMDA receptor-dependent Ca²⁺ entry. The close spatial relationship between NMDA receptors and K_Ca2 channels provides an intrinsic mechanism whereby synaptic excitation both excites and subsequently inhibits ventral horn neurons of the spinal motor system. This places the components necessary for oscillation generation, and hence locomotion, at glutamatergic synapses.     

Stochastic aspects of oscillatory Ca2+ dynamics in hepatocytes

Signal-induced Ca²⁺ oscillations have been observed in many cell types and play a primary role in cell physiology. Although it is the regular character of these oscillations that first catches the attention, a closer look at time series of Ca²⁺ increases reveals that the fluctuations on the period during individual spike trains are far from negligible. Here, we perform a statistical analysis of the regularity of Ca²⁺ oscillations in norepinephrine-stimulated hepatocytes and find that the coefficient of variation lies between 10% and 15%. Stochastic simulations based on Gillespie's algorithm and considering realistic numbers of Ca²⁺ ions and inositol trisphosphate (InsP₃) receptors account for this variability if the receptors are assumed to be grouped in clusters of a few tens of channels. Given the relatively small number of clusters (∼200), the model predicts the existence of repetitive spikes induced by fluctuations (stochastic resonance). Oscillations of this type are found in hepatocytes at subthreshold concentrations of norepinephrine. We next predict with the model that the isoforms of the InsP₃ receptor can affect the variability of the oscillations. In contrast, possible accompanying InsP₃ oscillations have no impact on the robustness of signal-induced repetitive Ca²⁺ spikes.     

Effects of mechanical stretch on the functions of BK and L-type Ca2+ channels in vascular smooth muscle cells

It is well recognized that pathologically increased mechanical stretch plays a critical role in vascular remodeling during hypertension. However, how the stretch modulates the functions of ion channels of vascular smooth muscle cells (VSMCs) remains to be elucidated. Here, we demonstrated the effects of mechanical stretch on the activity of large conductance calcium, voltage-activated potassium (BK) and L-type Ca²⁺ channels. In comparison with 5% stretch (physiological), 15% stretch (pathological) upregulated the current density of L-type Ca²⁺ and BK channels as well as the frequency and amplitude of calcium oscillation in VSMCs. 15% stretch also increased the open probability and mean open time of the BK channel compared with 5% stretch. BK and L-type Ca²⁺ channels participated in the mechanical stretch-modulated calcium oscillation. Our results suggested that during hypertension, pathological stretch altered the activity of BK and L-type Ca²⁺ channels and manipulated the calcium oscillation of VSMCs.     

Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca²⁺] ([Ca²⁺]_i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP₃-evoked Ca²⁺ release. The kinetics of both processes were studied with flash photolytic release of InsP₃ and time resolved measurements of [Ca²⁺]_i in single cells. InsP₃ evoked Ca²⁺ flux into the cytosol was measured as d[Ca²⁺]_i/dt, and the kinetics of Ca²⁺ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP₃ concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP₃ suggests that metabolism of InsP₃ is important in determining the minimal concentration needed to produce Ca²⁺ release. A distinct latency or delay of several hundred milliseconds after release of low InsP₃ concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca²⁺]_i, suggesting a cooperative action of Ca²⁺ in InsP₃ receptor activation. InsP₃-evoked flux and peak [Ca²⁺]_i increased with InsP₃ concentration up to 5–10 μM, with large variation from cell to cell at each InsP₃ concentration. The duration of InsP₃-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca²⁺]_i/dt and much less cell to cell variation than the dependence of flux on InsP₃ concentration, suggesting that the rate of termination of the Ca²⁺ flux depends on the free Ca²⁺ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca²⁺ flux, up to 50 μM · s⁻¹ and in Purkinje neurons at high flux up to 1,400 μM · s⁻¹. Experiments in which [Ca²⁺]_i was controlled at resting or elevated levels support a mechanism in which InsP₃-evoked Ca²⁺ flux is inhibited by Ca²⁺ inactivation of closed receptor/channels due to Ca²⁺ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP₃-evoked Ca²⁺ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP₃ receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP₃ receptor density promotes fast rising, rapidly inactivating InsP₃-evoked [Ca²⁺]_i transients.