Voltage- and Ca2+-activated potassium channels in Ca2+ store control Ca2+ release Masayuki Yamashita, Miho Sugioka and Yoichi Ogawa Department of Physiology I, Nara Medical University, Kashihara, Japan Keywords Ca2+ oscillation; Ca2+ release; endoplasmic reticulum membrane potential; voltagesensitive dye; retina Correspondence M Yamashita, Department of Physiology I, Nara Medical University, Shijo-cho 840, Kashihara 634–8521, Japan Fax: +81 744 29 0306 Tel: +81 744 29 8827 E-mail: yama@naramed-u.ac.jp (Received 14 April 2006, revised June 2006, accepted June 2006) doi:10.1111/j.1742-4658.2006.05365.x Ca2+ release from Ca2+ stores is a ‘quantal’ process; it terminates after a rapid release of stored Ca2+ To explain the quantal nature, it has been supposed that a decrease in luminal Ca2+ acts as a ‘brake’ on store release However, the mechanism for the attenuation of Ca2+ efflux remains unknown We show that Ca2+ release is controlled by voltage- and Ca2+activated potassium channels in the Ca2+ store The potassium channel was identified as the big or maxi-K (BK)-type, and was activated by positive shifts in luminal potential and luminal Ca2+ increases, as revealed by patch-clamp recordings from an exposed nuclear envelope The blockage or closure of the store BK channel due to Ca2+ efflux developed lumennegative potentials, as revealed with an organelle-specific voltage-sensitive dye [DiOC5(3); 3,3’-dipentyloxacarbocyanine iodide], and suppressed Ca2+ release The store BK channels are reactivated by Ca2+ uptake by Ca2+ pumps regeneratively with K+ entry to allow repetitive Ca2+ release Indeed, the luminal potential oscillated bistably by 45 mV in amplitude Our study suggests that Ca2+ efflux-induced store BK channel closures attenuate Ca2+ release with decreases in counter-influx of K+ The release of Ca2+ from intracellular Ca2+ stores is a pivotal event in Ca2+ signaling, which regulates a variety of cellular activities [1] There are at least two types of Ca2+ releasing channels that allow Ca2+ efflux from Ca2+ stores: inositol 1,4,5-trisphosphate (InsP3) receptor channels and ryanodine receptor channels These channels are involved in the highly versatile Ca2+ signal systems with various spatial and temporal dynamics [1] However, the openings of these channels alone cannot account for the complex patterns of Ca2+ signals It has been known that the Ca2+ release is a ‘quantal’ rather than a continuous process; it consists of a rapid release of a fraction of stored Ca2+ followed by no or a much slower efflux of Ca2+ [2] This transient and partial release behavior requires a mechanism for the attenuation of Ca2+ efflux, e.g inactivation of InsP3 receptor channels However, many studies have provided evidence for the lack of inactivation or desensitization of InsP3 receptor channels [3–9] The quantal Ca2+ release also occurs following the activation of ryanodine receptors [10,11], and appears to be a rather general phenomenon [12,13] To explain the quantal nature of this phenomenon, it was first proposed that Ca2+ is released in an ‘allor-none’ fashion from multiple stores with different sensitivities to InsP3 (‘all-or-none’ model) [2,14] Irvine [15] has proposed another model, in which the Ca2+ efflux through InsP3 receptor channels is regulated from the luminal side by the concentration of Ca2+ in the Ca2+ store (‘steady-state’ model) Since this model was proposed, many studies have demonstrated that a reduction in the luminal [Ca2+] concentration attenuates Ca2+ efflux [8,9,16,17] Koizumi et al [11] have shown that the Ca2+ release from ryanodine-sensitive Abbreviations BK channel, big or maxi-K channel; CICR, Ca2+-induced Ca2+ release; DiOC5(3), 3,3’-dipentyloxacarbocyanine iodide; ECa, equilibrium potential for Ca2+ ions; E3, embryonic day 3; ER, endoplasmic reticulum; IK channel, intermediate conductance Ca2+-activated K channel; InsP3, inositol 1,4,5-trisphosphate; NBS, normal bath solution; SK channel, small conductance Ca2+-activated channel FEBS Journal 273 (2006) 3585–3597 ª 2006 The Authors Journal compilation ª 2006 FEBS 3585 BK channels in Ca2+ store control Ca2+ release M Yamashita et al stores is also regulated by the luminal [Ca2+] concentration Caroppo et al [18] reevaluated this issue by introducing a membrane-permeant, low affinity Ca2+ chelator [TPEN; N,N,N¢,N¢-tetrakis(2-pyridylmethyl)ethylenediamine] to intact BHK-21 cells They have shown that a reduction in the luminal [Ca2+] does indeed attenuate the Ca2+ release However, it remains unclear whether the regulation by luminal Ca2+ occurs either directly at the InsP3 receptor channel itself or indirectly [19,20] Recently, a Ca2+-dependent protein has been reported to regulate InsP3 receptor channels from the luminal side [21] However, this protein affects InsP3 receptor type alone At present, the underlying mechanism for the attenuation of Ca2+ efflux still remains unknown The release of Ca2+ from a Ca2+ store should cause ancillary movements of other ions, such as influx of K+, to compensate for electrical charge movements across the store membrane [22,23] Otherwise, a lumen-negative potential is rapidly built up by Ca2+ efflux to such a degree that the negative shift can reach the equilibrium potential for Ca2+ ions, and the Ca2+ efflux is likely to cease with the loss of the electrochemical driving force for Ca2+ efflux The counter-movement of K+ has been proposed to compensate for charge movements across the membrane of sarcoplasmic reticulum and thereby to support rapid Ca2+ release [24] It may be supposed that the membrane potential of Ca2+ store is regulated not only by Ca2+ releasing channels but also by other channels in the Ca2+ store such as big or maxi-K (BK)-type potassium channels [25] At present, however, despite its functional importance, there is very little direct experimental information about the membrane potential of endoplasmic reticulum (ER) or sarcoplasmic reticulum, as Burdakov et al [26] have pointed out in a recent review article The aims of the present study are to reveal dynamic changes in the membrane potential of the Ca2+ store (luminal potential) and to estimate the effect of counter-movements of K+ on Ca2+ release In order to detect the changes in luminal potential, we applied 3,3’-dipentyloxacarbocyanine iodide [DiOC5(3)], a voltage-sensitive fluorescent probe for organelle membrane [27,28], to the neuroepithelium of embryonic chick retina, where the activation of G protein-coupled purinoceptor by ATP causes a robust Ca2+ release and Ca2+ oscillations occur [29–31] The ATP-induced Ca2+ mobilization is largest at embryonic day (E3) [29], when almost all cells are undergoing interkinetic nuclear migration along the vertical (outer–inner) axis of the retina during the cell cycle [32,33] We have already revealed the distribution of ER and nuclear envelope in the E3 chick retina with the DiOC5(3) staining [31] In the present study, we studied the changes in the DiOC5(3) fluorescence intensity in the E3 chick retina to gain an insight into the dynamics of the luminal potential of Ca2+ store in intact cells Results Luminal potential changes measured with DiOC5(3) The voltage sensitivity of DiOC5(3) was evaluated by voltage clamping of an excised membrane patch stained with DiOC5(3) The bath solution (outside the membrane patch) mimicked intracellular solution and the pipette solution (inside the membrane patch) mimicked the lumen of Ca2+ store in ionic composition (see Experimental procedures) The DiOC5(3) fluorescence intensity increased with a negative change in the pipette potential and decreased with a positive change (Fig 1A) The rate of change in DiOC5(3) fluorescence intensity against voltage change was )1.3 ± 0.3% ⁄ mV (mean ± SD, n ¼ patches, Fig 1B) Figure 1C illustrates the vertical plane of the neuroepithelium of E3 chick retina and Fig 1D shows the horizontal plane of the inner layer, where the somata of S-phase cells are located The S-phase cell soma Fig Measurement of the membrane potential of Ca2+ store with DiOC5(3) (A,B) Voltage sensitivity of DiOC5(3) An excised membrane patch was voltage-clamped at )10 mV (negative inside the pipette) and stained with DiOC5(3) (A) The command voltage (pipette potential) was changed from )10 mV to +30 mV and )70 mV (B) The change in DiOC5(3) fluorescence intensity [DF ⁄ F0 DiOC5(3)] is plotted against the pipette potential (Membrane potential) for the same DiOC5(3)-stained membrane patch as shown in (A) (C) A schematic drawing of the vertical plane of E3 chick retinal neuroepithelium (total thickness,  40 lm) (D) Nomarski optics (DIC) view of the horizontal plane of the inner layer (5 lm inside of the inner surface) (E) A schematic drawing of a cell with the soma in the inner layer, which is occupied with a nucleus as revealed by DNA staining with SYTO 24 [31] N, nucleus; NE, nuclear envelope (F) A DiOC5(3)-stained retinal cell enlarged with a low-Ca2+ hypotonic solution Its DiOC5(3) fluorescence image (upper) and DIC image (lower) (G) Fluo-4 and DiOC5(3) fluorescence responses to ATP recorded from the inner layer of E3 chick retinae ATP was bath-applied during the bar (500 lM, maximal dose for Ca2+ response [29]) The measurement area of DiOC5(3) fluorescence was 17 · 17 lm The fluo-4 trace ([Ca2+]i) is an averaged recordings from seven cells in a different retina D[Ca2+]i is the derivative of the fluo-4 trace (H) Possible movements of Ca2+ and K+ during Ca2+ release and uptake 3586 FEBS Journal 273 (2006) 3585–3597 ª 2006 The Authors Journal compilation ª 2006 FEBS BK channels in Ca2+ store control Ca2+ release M Yamashita et al A B C D E F G H FEBS Journal 273 (2006) 3585–3597 ª 2006 The Authors Journal compilation ª 2006 FEBS 3587 BK channels in Ca2+ store control Ca2+ release M Yamashita et al is occupied with a nucleus and hence the nuclear envelope forms a continuous circular structure in close apposition to the plasma membrane in the horizontal plane (Fig 1E) With this structural characteristic, DiOC5(3) staining shows continuous circular structures in the inner layer [31] (see also Fig 3E in the Discussion) The circular structure is also labeled with fluorescent thapsigargin [31], an ER-specific dye in living cells [34] As the criterion of the labeling of ER or nuclear envelope with DiOC5(3) is a continuous structure [28], it seems likely that the circular structure is either perinuclear ER or a nuclear envelope In fact, enlargement of the DiOC5(3)-stained cell by applying a lowCa2+ hypotonic solution showed the labeling of nuclear envelope (Fig 1F) The bath-application of ATP causes a robust Ca2+ release in the E3 retina [29,31] In response to the bath-applied ATP, a biphasic change occurred in the DiOC5(3) fluorescence intensity; it increased and then decreased (Fig 1G, measured in the inner layer) From the fluorescence–voltage relationship of DiOC5(3) (Figs 1A,B), it is suggested that the biphasic change indicates a negative-positive change in the luminal potential As it is supposed that the luminal potential changes in a negative direction with Ca2+ release, and in a positive direction with Ca2+ uptake by Ca2+ pumps [22,35], it seems likely that the initial increase reflects the negative potential change due to Ca2+ release and the subsequent decrease reflects the positive change by Ca2+ uptake The DiOC5(3) fluorescence increase appears rather earlier than the increase in the intracellular Ca2+ concentration ([Ca2+]i) measured with fluo-4 However, it should be noted here that Ca2+ fluxes (rates of charge movements by Ca2+, i.e Ca2+ currents) generate the voltage drops across the membrane of Ca2+ store A rise in [Ca2+]i is the integration of Ca2+ effluxes from the store and hence develops later than the Ca2+ efflux The Ca2+ flux can be estimated by the derivative of fluo-4 trace (D[Ca2+]i), and the efflux of Ca2+ precedes the main part of Ca2+ rise (Fig 1G) It is also noted that the DiOC5(3) fluorescence increase appears broader than the efflux of Ca2+ and that the DiOC5(3) fluorescence decrease returns to the initial level later than the recovery of [Ca2+]i These temporal characteristics may be due to the slow time constant of DiOC5(3) fluorescence response (s  s, Fig 1A) and the fact that [Ca2+]i is reduced by plasma membrane Ca2+ pumps and intracellular Ca2+-binding proteins as well as store Ca2+ pumps, which will continue to work until the store is replenished Figure 1H illustrates possible movements of Ca2+ and K+ during Ca2+ release and uptake, although other ionic movements cannot be excluded 3588 The DiOC5(3) fluorescence change might have reflected contributions from mitochondria However, the application of FCCP [carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone] (10 lm, for 60 s), a mitochondrial protonophore, did not change the DiOC5(3) fluorescence (data not shown) Potassium channel in store membrane If the potassium conductance of the store membrane regulates the luminal potential, it should depend on the difference in [K+] between the lumen and cytosol To test this idea, a mm-K+ solution containing nystatin (an ionophore for monovalent cations and Cl–) was bath-applied to a DiOC5(3)-stained retina It caused an increase in the DiOC5(3) fluorescence (Fig 2A), suggesting that the lowering of cytosolic [K+] shifts the luminal potential in a negative direction The subsequent fluorescence decrease might reflect a positive change due to influx of Na+ or efflux of Cl–; NaCl was replaced with sodium gluconate in the nystatincontaining solution In a steady state without changes in [K+] or [Ca2+], the membrane potential of Ca2+ store will be determined by the equilibrium potential for Ca2+ ions (ECa), which is lumen-negative, and by the proportion of calcium conductance to potassium conductance (see also Discussion) To test this idea, we applied quinidine (200 lm), a membrane-permeant BK channel blocker [36], to a DiOC5(3)-stained retina It caused an increase in the DiOC5(3) fluorescence (Fig 2B), indicating that a negative shift is caused in the luminal potential The negative shift may be due to a decrease in the potassium conductance by the blockage of store BK channels, and thus the luminal potential shifts towards the ECa The negative shift could exclude the possibility that quinidine primarily blocks Ca2+-releasing channels, because a reduction in calcium conductance should cause a positive shift The closing of store BK channels may decrease the counter-influx of K+, and the negative shift in the luminal potential may decrease the driving force for Ca2+ efflux In accordance with this idea, the ATPinduced [Ca2+]i rise was significantly inhibited by quinidine (Student’s t-test, P < 0.1%); changes in the ratio of fluo-4 fluorescence intensities (DF ⁄ F0) were 2.04 ± 0.60 (mean ± sd, n ¼ 33 cells) without quinidine and 0.54 ± 0.39 (n ¼ 32) with quinidine (Fig 2C) Quinidine itself caused no change in the fluo4 fluorescence (data not shown) Next, we examined iberiotoxin, a specific peptide blocker for BK channels [37] When iberiotoxin (100 nm) was bath-applied, there was no inhibitory effect on the ATP-induced [Ca2+]i rise (DF ⁄ F0: 2.26 ± 0.57, n ¼ 32) In contrast, the FEBS Journal 273 (2006) 3585–3597 ª 2006 The Authors Journal compilation ª 2006 FEBS BK channels in Ca2+ store control Ca2+ release M Yamashita et al A C Fig K channel in Ca2+ store (A) DiOC5(3) response to bath-application of mM-K+ solution containing nystatin (200 lgỈmL)1) (B) DiOC5(3) response to quinidine (200 lM) (C,D) Effects of quinidine (200 lM, C) and iberiotoxin (IbTx, introduced by electroporation, D) on Ca2+ responses to ATP (500 lM) recorded from somata in the inner layer (E) Single channel currents recorded from an exposed nuclear envelope in the ‘nucleusattached’ mode [25] at pipette potentials indicated Negative changes mean lumenpositive changes (F) Immunolabeling of exposed nuclei with an antibody against BK channels (upper, fluoro-labeled with Alexa Fluor 488) and negative control (lower) The cells were enlarged with a low-Ca2+ hypotonic solution and plasma membranes were removed by perfusion with 0.2% Triton X-100 (G) DiOC5(3) response to Ca2+-free solution containing thapsigargin (500 nM) (H) DiOC5(3) response to 100 mM-K+ solution Measurement area, 13 · 13 lm in the inner layer (A,B,G,H) B D E F G H introduction of iberiotoxin into the cells by electroporation significantly inhibited it; DF ⁄ F0 was decreased from 1.98 ± 0.46 (n ¼ 87) in the control response (electrical pulses plus vehicle alone) to 1.27 ± 0.46 (n ¼ 85, electrical pulses with iberiotoxin, Fig 2D) We made patch-clamp recordings from an exposed nuclear envelope in the ‘nucleus-attached’ mode [25] after removing the plasma membrane (see Experimen- tal procedures) Recordings in the low-Ca2+ hypotonic solution for cell enlargement showed no channel activity (n ¼ patches), whereas channel activities were found after perfusion with a Ca2+-increasing solution containing 10 lm ionomycin and 1.1 mm CaCl2 (Supplementary Fig S1) Single channel outward currents were activated by positive shifts in the luminal potential (Fig 2E) Because the recordings FEBS Journal 273 (2006) 3585–3597 ª 2006 The Authors Journal compilation ª 2006 FEBS 3589 BK channels in Ca2+ store control Ca2+ release M Yamashita et al were made with a patch pipette containing a highNa+, low-Cl– solution, the outward current would be carried by K+ ions, although the luminal [K+] seemed to be considerably lowered by the hypotonic solution for cell enlargement The exposed nucleus was immunolabeled with an antibody against BK channels (Fig 2F) The above result of Ca2+ dependence suggests that the store BK channel will be closed by a decrease in luminal [Ca2+] The luminal [Ca2+] can be lowered by blocking store Ca2+ pumps with thapsigargin in the E3 retina [38] When thapsigargin (500 nm) was applied to a DiOC5(3)-stained retina, the DiOC5(3) fluorescence was remarkably increased (Fig 2G) This large negative shift in the luminal potential may be due to a leak of Ca2+ from the store and also an increase in the membrane resistance by closure of store BK channels with the decrease in luminal [Ca2+] The DiOC5(3) fluorescence was not changed by the depolarization of plasma membrane with a 100 mm-K+ solution (Fig 2H), which could suggest that Ca2+ influx through voltage-dependent calcium channels [31,39] does not induce Ca2+ release Ca2+ oscillation and luminal potential oscillation We observed Ca2+ oscillations ( 6–30 min)1 in frequency), which occurred spontaneously or after ATPinduced Ca2+ release at rather high temperature (‡ 28 °C) (Fig 3A,B) When ATP was applied to the retina that was showing spontaneous Ca2+ oscillation, excess increases in [Ca2+]i were caused with different onsets (Fig 3C,D) Nevertheless, the Ca2+ oscillation persisted and the frequency of Ca2+ oscillation was raised at the peak level of the agonist-induced [Ca2+]i increase (Fig 3D), where the lower level of Ca2+ oscillation was distinctly higher than the upper level of the Ca2+ oscillation before agonist application Thus it seems difficult to explain the generation of Ca2+ oscillation by [Ca2+]idependent negative feedback models, which predicts that the Ca2+ oscillation should be arrested by the excess increase in [Ca2+]i Alternatively, the Ca2+ oscillation might have been caused by spontaneous action potentials independently of the agonist-induced Ca2+ release [40] However, the cells of E3 retina not fire action potentials [39], even with current injections (M Yamashita, unpublished data) Therefore, we suppose that the Ca2+ oscillation is caused by the repetition of quantal Ca2+ release that depends on luminal [Ca2+] [41] or by luminal potential oscillation, as described below Bistable oscillations of DiOC5(3) fluorescence were observed in the inner layer (Fig 3E,F) and the vertical 3590 plane (Fig 3G,H) The magnitude of potential oscillation was estimated to be up to 45 mV (Fig 3F,H) It was noted that the falling phase was more rapid than the rising phase Quinidine lowered the frequency of DiOC5(3) fluorescence oscillation (Fig 4A,B), and then the oscillation ceased (Fig 4C) After washout of quinidine, the DiOC5(3) fluorescence oscillation turned out to be irregular high frequency flickering (Fig 4D) Paxilline (10 lm), another membrane-permeant BK channel blocker [42], also lowered the frequency of DiOC5(3) fluorescence oscillation in a reversible manner (data not shown) Discussion Changes in the membrane potential across the ER are likely to impact upon both the rate and extent of Ca2+ release from the store, and hence influence the spatial and temporal dynamics of intracellular Ca2+ signals Despite its potential importance, we know very little about how the ER potential can change, or whether this can affect the profile of intracellular Ca2+ signals The major hurdle to our understanding of this area is the technical difficulty in measuring membrane potential changes specific to the ER without contamination from other organelles within living cells In the present study, it was attempted to tackle this issue directly, using a voltage-sensitive fluorescent dye [DiOC5(3)] We applied DiOC5(3) to the intact cells of embryonic chick retina to measure changes in the membrane potential of Ca2+ store The DiOC5(3) fluorescence signal may include contributions not only from the membrane of the Ca2+ store (nuclear envelope and ER), but also from the plasma membrane or mitochondrial membranes To address this issue, the DiOC5(3)-stained cell was enlarged by applying a low-Ca2+ hypotonic solution Figure 1F clearly shows that the dye is located in the nuclear envelope or perinuclear ER, and not in the plasma membrane The DiOC5(3) fluorescence was not changed by the depolarization of plasma membrane (Fig 2H) This result may also exclude the contribution from the plasma membrane There was also a concern that the DiOC5(3) fluorescence signal might have reflected contributions from mitochondria To address this issue, we tested FCCP (a mitochondria-specific protonophore), which did not change the DiOC5(3) fluorescence This result may exclude the contribution from mitochondria We examined the distribution of mitochondria with rhodamine 123 (a specific fluorescent probe for mitochondria) The fluorescence image showed discrete granular structures, which were quite different from the circular structures (unpublished observation) Thus it is supposed that the DiOC5(3) FEBS Journal 273 (2006) 3585–3597 ª 2006 The Authors Journal compilation ª 2006 FEBS BK channels in Ca2+ store control Ca2+ release M Yamashita et al A B C D G E F H Fig Ca2+ oscillation and DiOC5(3) fluorescence oscillation (A) Fluo-4 images of the inner layer at t1 and t2 in (B) (B) Fluo-4 fluorescence changes of the three cells marked in (A) (C,D) Same as (A,B) ATP (500 lM) was applied during the bar (E–H) DiOC5(3) fluorescence oscillations in the inner layer (E,F) and the vertical plane (G,H) fluorescence signal mainly reflects the changes in the membrane potential of the nuclear envelope or ER Patch-clamp recordings from an exposed nuclear envelope showed that channel activities appeared after perfusion with a Ca2+-increasing solution containing ionomycin and Ca2+, and that the channel activity was voltage-dependent Ca2+-activated potassium channels are classified into three groups [BK-, intermediate-conductance Ca2+-activated (IK)- and smallconductance Ca2+-activated (SK)-types] according to voltage dependence and single-channel conductance [36] Among the three groups, BK-type alone shows voltage dependence Our patch-clamp recording shows clear voltage dependence We tried to excise a membrane patch from the exposed nuclear envelope to estimate single-channel conductance with defined K+ concentrations However, it was very difficult to excise a membrane patch in the low-Ca2+ hypotonic solution, or to maintain a seal at GW-values while changing bath solutions to a high-K+ solution Thus we could not estimate the single-channel conductance in a correct manner Nevertheless, the Ca2+- and voltagedependence strongly suggests that BK channels are functioning there Our result is in accordance with the FEBS Journal 273 (2006) 3585–3597 ª 2006 The Authors Journal compilation ª 2006 FEBS 3591 BK channels in Ca2+ store control Ca2+ release M Yamashita et al A B C D E Fig Bistable change in the membrane potential of Ca2+ store (A–D) Inhibition by quinidine (200 lM) of DiOC5(3) fluorescence oscillation (measurement area, 13 · 13 lm in the inner layer) Records before (A) and after beginning of quinidine application (B), after application for 11 (C) and after washout for 100 (D) (E) Two states of Ca2+ store Left, openings of store BK channels maintain the driving force for Ca2+ efflux with counter-influx of K+ CICR, Ca2+-induced Ca2+ release Right, Ca2+ release declines due to closings of store BK channels study of Maruyama et al [25], i.e that the BK channel in nuclear envelope is activated by positive shifts in the luminal potential and luminal Ca2+ increases Furthermore, the immunolabeling of the exposed nuclei with an antibody against BK channels indicates that BK channels are present there Openings of store BK channels tend to nullify the luminal potential unless a [K+] gradient is formed across the store membrane As the ECa is lumen-negative, the openings of store BK channels would maintain the driving force for Ca2+ efflux When Ca2+ is released, the Ca2+ efflux may be amplified by Ca2+induced Ca2+ release (CICR) at the Ca2+ releasing site (Fig 4E, left) A decrease in luminal [Ca2+] and a negative potential shift due to the Ca2+ efflux could close the store BK channels, which decreases the counter-influx of K+ The increase in store membrane resistance should enhance the negative potential shift 3592 towards the ECa and the Ca2+ release would decline with a decrease in the driving force for Ca2+ efflux (Fig 4E, right) It should also be considered that the lumen-negative potential could induce blockage of Ca2+ releasing channels by binding of Mg2+ to sites in the conduction pathway [43] The rate of Ca2+ uptake by Ca2+ pumps is accelerated by a lumen-negative potential [44] and a decrease in luminal [Ca2+] [45] When the Ca2+-pumping activity is thus raised, the store BK channel would be reactivated by an increase in luminal [Ca2+] and a positive shift in the luminal potential due to the uptake of Ca2+ Capacitative Ca2+ entry [38,46,47] also caused a positive shift in the luminal potential, even when the Ca2+ pump was inhibited (unpublished observation) The efflux of Cl– may also contribute to the depolarization (Fig 4E, right) The reactivation process of store BK channels should be regenerative, because an FEBS Journal 273 (2006) 3585–3597 ª 2006 The Authors Journal compilation ª 2006 FEBS BK channels in Ca2+ store control Ca2+ release M Yamashita et al influx of K+ causes a positive shift in the luminal potential and this positive shift further activates the store BK channels Such regenerative activation may account for the rapid decrease in DiOC5(3) fluorescence (i.e positive shift) during oscillation The DiOC5(3) fluorescence oscillation appears synchronous in the vertical plane (Fig 3G,H), which could suggest that the regenerative depolarization rapidly propagates throughout the Ca2+ store The rapid depolarization may also give a clue to the explanation for the synchronicity of Ca2+ releases across the oscillating cells (see Supplementary Doc S1) The oscillatory change in luminal potential of 45 mV in amplitude could regulate Ca2+ release, because ECa would be  )50 mV if the luminal [Ca2+] falls below 50 lm [48] and the peak [Ca2+]i exceeds lm At luminal [Ca2+]