Effects of 2,3-Butanedione 2-Monoxime on Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Single channel and [³H]ryanodine binding measurements were performed to test for a direct functional interaction between 2,3-butanedione 2-monoxime (BDM) and the skeletal and cardiac muscle sarcoplasmic reticulum Ca²⁺ release channels (ryanodine receptors). Single channel measurements were carried out in symmetric 0.25 m KCl media using the planar lipid bilayer method. BDM (1–10 mm) activated suboptimally Ca²⁺-activated (0.5–1 μm free Ca²⁺) single, purified and native cardiac and skeletal release channels in a concentration-dependent manner by increasing the number of channel events without a change of single channel conductances. BDM activated the two channel isoforms when added to either side of the bilayer. At a maximally activating cytosolic Ca²⁺ concentration of 20 μm, BDM was without effect on the cardiac channel, whereas it inhibited skeletal channel activities with IC₅₀≈ 2.5 mm. In agreement with single channel measurements, high-affinity [³H]ryanodine binding to the two channel isoforms was increased in a concentration-dependent manner at ≤1 μm Ca²⁺. BDM was without a noticeable effect at low (≤0.01 μm) Ca²⁺ concentrations. At 20 μm Ca²⁺, BDM inhibited the skeletal but not cardiac channel. These results suggest that BDM regulates the Ca²⁺ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle in a concentration, Ca²⁺ and tissue-dependent manner. Received: 31 December 1998/Revised: 9 March 1999     

Skeletal Muscle Ryanodine Receptor Channels Are Activated by the Fungal Metabolite, Gliotoxin

Interactions between the reactive disulfide fungal metabolite, gliotoxin (GTX), and rabbit skeletal ryanodine receptor (RyR) calcium release channels have been examined. RyRs in terminal cisternae vesicles formed a covalent complex with 100 μm ³⁵S-GTX, which was reversed by 1 mm dithiothreitol (DTT) or 1 mm glutathione. GTX (80–240 μm), added to either cytoplasmic (cis) or luminal (trans) solutions, increased the rate of Ca²⁺ release from SR vesicles and the frequency of opening of single RyR channels in lipid bilayers. Channel activation was reversed upon addition of 2 mm DTT to the cis solution, showing that the activation was due to an oxidation reaction (2 mm DTT added to the cis solution in the absence of GTX did not affect RyR activity). Furthermore, RyRs were not activated by trans GTX if the cis chamber contained DTT, suggesting that GTX oxidized a site in or near the membrane. In contrast to cis DTT, 2 mm DTT in the trans solution increased RyR activity when added either alone or with 200 μm trans GTX. The results suggest that (i) GTX increases RyR channel activity by oxidizing cysteine residues that are close to the membrane and located on RyR, or associated proteins, and (ii) a disulfide bridge or nitrosothiol, accessible only from the luminal solution, normally suppresses RyR channel activity. Some of the actions of GTX in altering Ca²⁺ homeostatsis might depend on its modification of RyR calcium channels. Received: 12 November 1999/Revised: 14 March 2000     

Sarcoplasmic Reticulum Ca2+ Release in Rat Slow- and Fast-Twitch Muscles

The same isoform of ryanodine receptor (RYR1) is expressed in both fast and slow mammalian skeletal muscles. However, differences in contractile activation and calcium release kinetics in intact and skinned fibers have been reported. In this work, intracellular Ca²⁺ transients were measured in soleus and extensor digitorum longus (EDL) single muscle fibers using mag-fura-2 (K _D for Ca²⁺= 49 μm) as Ca²⁺ fluorescent indicator. Fibers were voltage-clamped at V _h =−90 mV and sarcoplasmic reticulum calcium release was measured at the peak (a) and at the end (b) of 200 msec pulses at +10 mV. Values of a-b and b were assumed to correspond to Ca²⁺-gated and voltage-gated Ca²⁺ release, respectively. Ratios (b/a-b) in soleus and EDL fibers were 0.41 ± 0.05 and 1.01 ± 0.13 (n= 12), respectively. This result suggested that the proportion of dihydropyridine receptor (DHPR)-linked and unlinked RYRs is different in soleus and EDL muscle. The number of DHPR and RYR were determined by measuring high-affinity [³H]PN200-110 and [³H]ryanodine binding in soleus and EDL rat muscle homogenates. The B _max values corresponded to a PN200-110/ryanodine binding ratio of 0.34 ± 0.05 and 0.92 ± 0.11 for soleus and EDL muscles (n= 4–8), respectively. These data suggest that soleus muscle has a larger calcium-gated calcium release component and a larger proportion of DHPR-unlinked RYRs. Received: 31 August 1995/Revised: 25 January 1996     

Inositol 1,4,5-Trisphosphate But Not Ryanodine-Receptor Agonists Induces Calcium Release from Rat Liver Golgi Apparatus Membrane Vesicles

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptor agonists on Ca²⁺ release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with ⁴⁵Ca²⁺. Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP₃ at concentrations ranging from 100 nm to 100 μm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases, promoted a rapid decrease in the Ca²⁺ content of GA vesicles. The amount of Ca²⁺ released from the vesicles was a function of IP₃ concentration, reaching about 60% in both GA and ER fractions at 100 μm IP₃. Calcium release was inhibited by heparin, an antagonist of IP₃ receptors. Calcium exhibited a bell-shaped effect on IP₃-dependent Ca²⁺ released from GA vesicles: it activated Ca²⁺ release at concentrations up to 1 μm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca²⁺ release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP₃-sensitive Ca²⁺ release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca²⁺ release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane. Received: 13 March 2000/Revised: 13 July 2000     

Evidence for Negative Charge in the Conduction Pathway of the Cardiac Ryanodine Receptor Channel Provided by the Interaction of K+ Channel N-type Inactivation Peptides

We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH₂-terminal domain of the Shaker K⁺ channel with purified, ryanodine-modified, cardiac Ca²⁺-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec⁻¹μm ⁻¹ for ShB and 2000 ± 200 sec⁻¹μm ⁻¹ for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K_off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K⁺ channels, this is mediated by a large increase in K_on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel. Received: 15 December 1997/Revised: 13 March 1998     

Regulatory Volume Decrease by SV40-transformed Rabbit Corneal Epithelial Cells Requires Ryanodine-sensitive Ca2+-induced Ca2+ Release

The relationship between relative cell volume and time-dependent changes in intracellular Ca²⁺ concentration ([Ca²⁺] _i ) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (τ _vr ) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca²⁺] _i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca²⁺] _i transients were inhibited by either: (a) Ca²⁺-free medium; (b) 5 mm Ni²⁺ (inhibitor of plasmalemma Ca²⁺ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca²⁺ content); or (d) 100 μm ryanodine (inhibitor of Ca²⁺ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca²⁺ influx and the emptying of intracellular Ca²⁺ stores during the [Ca²⁺] _i transients, Mn²⁺ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn²⁺ permeability 6-fold. However, Mn²⁺ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca²⁺] _i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca²⁺ release from an intracellular store leads to a subsequent increase in plasmalemma Ca²⁺ influx, and that both are required for cells to undergo RVD. Received: 7 November 1996/Revised: 6 January 1997     

Modulation of the Calmodulin-induced Inhibition of Sarcoplasmic Reticulum Calcium Release Channel (Ryanodine Receptor) by Sulfhydryl Oxidation in Single Channel Current Recordings and [3H]Ryanodine Binding

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [³H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P _o ) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P _o (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [³H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P _o > 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P _o = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[¹²⁵I]calmodulin binding to HSR. 1 mm MgCl₂ reduced P _o from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P _o > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel. Received: 22 July 1999/Revised: 15 November 1999     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998     

Characterization of Ryanodine-sensitive Ca2+ Release from Microsomal Vesicles of Rat Parotid Acinar Cells: Regulation by Cyclic ADP-ribose

We have measured ryanodine (caffeine)-sensitive ⁴⁵Ca²⁺ release from isolated microsomal vesicles of endoplasmic reticulum prepared from rat parotid acinar cells. After a steady state of ATP-dependent ⁴⁵Ca²⁺ uptake, the addition of caffeine (40 mm), ryanodine (10∼500 μm) or an NAD⁺ metabolite, cyclic ADP-ribose (cADPR, 4 μm) released about 10% of the ⁴⁵Ca²⁺ that had been taken up. The ⁴⁵Ca²⁺ release was not inhibited by heparin, an antagonist of IP₃ receptor. The effects of caffeine, ryanodine and cADPR on ⁴⁵Ca²⁺ release were also tested in the presence of thapsigargin (TG), an inhibitor of microsomal Ca²⁺-ATPase. When caffeine (10∼40 mm), ryanodine (10 μm) or cADPR (1∼10 μm) was added in the medium with 100 nm TG, a significant ⁴⁵Ca²⁺ release was seen, while higher concentrations of ryanodine (>100 μm) did not cause any ⁴⁵Ca²⁺ release in the presence of TG. The initial rate of caffeine (40 mm)-induced ⁴⁵Ca²⁺ release was increased by a pretreatment with 10 μm ryanodine, whereas the caffeine-induced ⁴⁵Ca²⁺ release was strongly inhibited by the presence of a higher concentration (500 μm) of ryanodine. cADPR-induced ⁴⁵Ca²⁺ release was also inhibited by 500 μm ryanodine. Caffeine (40 mm)- or cADPR (4 μm)-induced ⁴⁵Ca²⁺ release was abolished by a presence of ruthenium red (50∼100 μm). The presence of a low concentration (0.5 μm) of cADPR shifted the dose-response curve of caffeine-induced ⁴⁵Ca²⁺ release to the left. These results indicate the presence of a ryanodine sensitive Ca²⁺ release mechanism in the endoplasmic reticulum of rat parotid acinar cells that is distinct from the IP₃-sensitive Ca²⁺ channel and is activated by caffeine, cADPR and a low concentration (10 μm) of ryanodine, but is inhibited by higher concentrations (>100 μm) of ryanodine and ruthenium red. The properties of the ryanodine-sensitive mechanism are similar to that of the ryanodine receptor as described in muscle cells. Received: 11 June 1996/Revised: 14 November 1996     

Identification, Characterization and Partial Purification of a Thiol-protease Which Cleaves Specifically the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel

A 94 kDa large subunit thiol-protease, as identified by anti-calpain antibodies, has been isolated from skeletal muscle junctional sarcoplasmic reticulum (SR). This protease cleaves specifically the skeletal muscle ryanodine receptor (RyR)/Ca²⁺ release channel at one site resulting in the 375 kDa and 150 kDa fragments. The 94 kDa thiol-protease degrades neither other SR proteins nor the ryanodine receptor of cardiac nor brain membranes. The partially purified 94 kDa protease, like the SR associated protease, had an optimal pH of about 7.0, was absolutely dependent on the presence of thiol reducing reagents, and was completely inhibited by HgCl₂, leupeptin and the specific calpain I inhibitor. However, while the SR membrane-associated protease requires Ca²⁺ at a submicromolar concentration, the isolated thiol-protease has lost the Ca²⁺ requirement. The 94 kDa thiol-protease had no effect on ryanodine binding but modified the channel activity of RyR reconstituted into planar lipid bilayer: in a time-dependent manner, the channel activity decreases and within several minutes the channel is converted into a subconducting state. The protease-modified channel activity is still Ca²⁺-dependent and ryanodine sensitive. This 94 kDa thiol-protease cross react with anti-calpain antibodies thus, may represent the novel large subunit of the skeletal muscle specific calpain p94. Received: 10 December 1996/Revised: 11 August 1997     

Magnesium Inhibition of Ryanodine-Receptor Calcium Channels: Evidence for Two Independent Mechanisms

The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca²⁺ and Mg²⁺ concentrations. RyRs from skeletal and cardiac muscle are activated by μm Ca²⁺ and inhibited by mm Ca²⁺ and Mg²⁺. ⁴⁵Ca²⁺ release from skeletal SR vesicles suggests two mechanisms for Mg²⁺-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236–244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg²⁺- and Ca²⁺-dependent gating kinetics confirm that there are two mechanisms for Mg²⁺ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg²⁺ reduces P _o by competing with Ca²⁺ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca²⁺ and Mg²⁺ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg²⁺ effect depend on cytoplasmic [Ca²⁺] in such a way that Mg²⁺ inhibition has the properties of Types-I and II inhibition at low and high [Ca²⁺] respectively. Both mechanisms are equally important when [Ca²⁺] = 10 μm in cardiac RyRs or 1 μm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg²⁺ inhibition, as is often assumed, and we discuss the physiological implications of this finding. Received: 1 January 1996/Revised: 14 November 1996     

The Essential Role of Cytosolic Cl− in Ca2+ Regulation of an Amiloride-Sensitive Channel in Fetal Rat Pneumocyte

An amiloride-sensitive, Ca²⁺-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na⁺ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca²⁺ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P _o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca²⁺. The P _o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca²⁺ to 0.26 ± 0.05 (n= 8). The cytosolic Ca²⁺ concentration ([Ca²⁺] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl⁻ concentration ([Cl⁻] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca²⁺] _c from 100 to 20 nm itself had no significant effects on the P _o if the [Cl⁻] _c was reduced to 20 mm; the P _o was 0.58 ± 0.10 at 100 nm [Ca²⁺] _c and 0.55 ± 0.09 at 20 nm [Ca²⁺] _c (n= 8) with 20 mm [Cl⁻] _c in inside-out patches. On the other hand, the P _o (0.28 ± 0.10) at 20 nm [Ca²⁺] _c with 40 mm [Cl⁻] _c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca²⁺] _c with 20 mm [Cl⁻] _c , suggesting that reduction of [Cl⁻] _c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca²⁺ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl⁻] _c . Received: 11 August 2000/Revised: 4 December 2000     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

ADH Action on Whole-Cell Currents by Cytosolic Ca2+-dependent Pathways in Aldosterone-treated A6 Cells

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca²⁺ concentrations ([Ca²⁺] _c , 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca²⁺] _c , basal conductances mainly consisted of Cl⁻ conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca²⁺] _c to 2 nm abolished the basal Cl⁻ conductance. AVT evoked Cl⁻ conductances at 2 as well as 30 nm [Ca²⁺] _c . In addition to Cl⁻ conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca²⁺] _c but not at 2 nm [Ca²⁺] _c . Thus, cytosolic Ca²⁺ regulates NSC and Cl⁻ conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca²⁺] _c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells. Received: 6 May 1996/Revised: 28 June 1996     

``Frustrated Exocytosis' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca²⁺] _i transient and exocytosis of dense core vesicles (``trichocysts') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>] <sub>o</sub> = 50 μm). When [Ca<sup>2+</sup>] <sub>o</sub> is kept at 30 nm (<[Ca<sup>2+</sup>]<sup>rest</sup> <sub>i</sub> ), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca<sup>2+</sup> <sub>o</sub> . Upon readdition of Ca<sup>2+</sup> <sub>o</sub> or some other appropriate Me<sup>2+</sup> <sub>o</sub> at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca<sup>2+</sup>, Sr<sup>2+</sup> or Mn<sup>2+</sup> are vesicular, but when formed in presence of Mg<sup>2+</sup>, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg<sup>2+</sup>] <sub>o</sub> = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg²⁺] _o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me²⁺] to maintain docking sites in a functional state, (ii) requirement of Ca²⁺ _o or of some other Me²⁺ _o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis'. Received: 20 January 2000/Revised: 5 May 2000     

Cations and Anions as Modifiers of Ryanodine Binding to the Skeletal Muscle Calcium Release Channel

Rate and equilibrium measurements of ryanodine binding to terminal cysternae fractions of heavy sarcoplasmic reticulum vesicles demonstrate that its activation by high concentrations of monovalent salts is based on neither elevated osmolarity nor ionic strength. The effect of the ions specifically depends on their chemical nature following the Hofmeister ion series for cations (Li⁺ < NH⁺ ₄ < K⁻∼ Cs⁺≤ Na⁺) and anions (gluconate⁻ < Cl⁻ < NO₃ ⁻∼ ClO₄ ⁻∼ SCN⁻) respectively, indicating that both are involved in the formation of the salt-protein complex that can react with ryanodine. Activation by rising salt concentrations exhibits saturation kinetics with different dissociation constants (25–11 m) and different degrees of cooperativity (n= 1.5–4.0) for the respective salts. Maximal second order binding rates between 40,000 and 80,000 (m ⁻¹· sec⁻¹) were obtained for chlorides and nitrates of 1a group alkali ions with the exception of lithium supporting only rates of maximally 10,000 (M⁻¹· sec⁻¹). The nitrogen bases, NH⁺ ₄ and Tris⁺, in combination with chloride or nitrate, behave divergently. High maximal binding rates were achieved only with NH₄NO₃. The dissociation constants for the ryanodine–protein complexes obtained by measurements at equilibrium proved to depend differently on salt concentration, yet, converging to 1–3 nm for the applied salts at saturating concentrations. The salts do not affect dissociation of the ryanodine protein complex proving that the effect of salts on the protein's affinity for ryanodine is determined by their effect on the on-rate of ryanodine binding. ATP and its analogues modify salt action resulting in elevated maximal binding rates and reduction or abolition of binding cooperativity. Linear relations have been obtained by comparing the rates of ryanodine binding at different salt concentrations with the rates or the initial amplitudes (15 sec) of salt induced calcium release from actively loaded heavy vesicles indicating that the various salts promote specifically and concentration dependently channel opening and its reaction with ryanodine. Received: 9 February 1998/Revised: 24 April 1998     

Veratridine-Mediated Ca2+ Dynamics and Exocytosis in Paramecium Cells

We analyzed [Ca²⁺] _i transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben & Plattner, 1994. J. Cell Biol. 127:935–945; Plattner et al., 1994. J. Membrane Biol. 158:197–208). Wild-type cells (7S), nondischarge strain nd9–28°C and trichocyst-free strain ``trichless' (tl), respectively, displayed similar, though somewhat diverging time course and plateau values of [Ca<sup>2+</sup>] <sub>i</sub> transients with moderate [Ca<sup>2+</sup>] <sub>o</sub> in the culture/assay fluid (50 μm or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca<sup>2+</sup>] <sub>o</sub> = 30 nm (c.f. [Ca<sup>2+</sup>] <sup>rest</sup> <sub>i</sub> =∼50 to 100 nm), veratridine produced only a small cortical [Ca<sup>2+</sup>] <sub>i</sub> transient. This increased in size and spatial distribution at [Ca<sup>2+</sup>] <sub>o</sub> = 50 μm of 1 mm. Interestingly with unusually high yet nontoxic [Ca<sup>2+</sup>] <sub>o</sub> = 10 mm, [Ca<sup>2+</sup>] <sub>i</sub> transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With [Ca<sup>2+</sup>] <sub>o</sub> = 30 nm, the restricted residual response observed is due to Ca<sup>2+</sup> mobilization from subplasmalemmal stores. (ii) With moderate [Ca<sup>2+</sup>] <sub>o</sub> = 50 μm to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca<sup>2+</sup> <sub>o</sub> influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl cells, where free docking sites allow for rapid Ca<sup>2+</sup> spread, and least in 7S cells, whose perfectly assembled docking sites may ``consume' a large part of the [Ca²⁺] _i increase. (iii) With unusually high [Ca²⁺] _o , mobilization of cortical stores and/or Ca²⁺ _o influx may be impeded by the known membrane stabilizing effect of Ca²⁺ _o counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and, hence, not to be toxic side-effects, as confirmed by retention of injected calcein. (v) Finally, Mn²⁺ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific Me²⁺ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine is of particular and continous interest. Received: 8 December 1998/Revised: 2 March 1999     

Differential desensitization of Ca2+ mobilization and vasoconstriction by ET(A) receptors in the gerbil spiral modiolar artery

Endothelins are known to be among the most potent endogenous vasoconstrictors. Vasoconstriction of the spiral modiolar artery, which supplies the cochlea, may be implicated in hearing loss and tinnitus. The purpose of the present study was to determine whether the spiral modiolar artery responds to endothelin, whether a change in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) mediates the response and which endothelin receptors are present. The vascular diameter and [Ca²⁺]_i were measured simultaneously by videomicroscopy and microfluorometry in the isolated spiral modiolar artery from the gerbil. ET-1 induced a transient [Ca²⁺]_i increase and a strong and long-lasting vasoconstriction. The transient [Ca²⁺]_i increase underwent rapid desensitization, was independent of extracellular Ca²⁺ and inhibited by the IP₃-receptor blocker (75 μm) 2-aminoethoxydiphenyl borate (2-APB) and by depletion of Ca²⁺ stores with 10⁻⁶ m thapsigargin. In contrast, the vasoconstriction displayed no comparable desensitization. The initial vasoconstriction was independent of extracellular Ca²⁺ but maintenance of the constriction depended on the presence of extracellular Ca²⁺. The half-maximal concentration values (EC ₅₀) for the agonists ET-1, ET-3 and sarafotoxin S6c were 0.8 nm, >10 nm and >100 nm, respectively. Affinity constants for the antagonists BQ-123 and BQ-788 were 24 nm and 77 nm, respectively. These observations demonstrate that ET-1 mediates a vasoconstriction of the gerbil spiral modiolar artery via ET_A receptors and an IP₃ receptor-mediated release of Ca²⁺ from thapsigargin-sensitive Ca²⁺ stores. The marked difference in desensitization between Ca²⁺ mobilization and vasoconstriction suggests that Ca²⁺ mobilization is not solely responsible for the vasoconstriction and that other signaling mechanisms must be present. Received: 4 January 2001/Revised: 23 April 2001     

Cardiac Sarcalumenin: Phosphorylation, Comparison with the Skeletal Muscle Sarcalumenin and Modulation of Ryanodine Receptor

Cardiac sarcoplasmic reticulum (SR) contains an endogenous phosphorylation system that under specific conditions phosphorylates two proteins with apparent molecular masses of 150 and 130 kDa. The conditions for their phosphorylation are as for the skeletal muscle sarcalumenin and the histidine-rich Ca²⁺ binding protein (HCP) with respect to: (i) Ca²⁺ and high concentrations of NaF are required; (ii) phosphorylation is obtained with no added Mg²⁺ and shows a similar time course and ATP concentration dependence; (iii) inhibition by similar concentrations of La³⁺; (iv) phosphorylation is obtained with [γ-³²P]GTP; (v) ryanodine binding is inhibited parallel to the phosphorylation of the two proteins. The endogenous kinase is identified as casein kinase II (CK II) based on its ability to use GTP as effectively as ATP, and its inhibition by La³⁺. The association of CK II with the cardiac SR, even after EGTA extraction at alkaline pH, is demonstrated using antibodies against CK II. The cardiac 130 kDa protein is identified as sarcalumenin based on its partial amino acid sequence and its blue staining with Stains-All. Cardiac sarcalumenin is different from the skeletal muscle protein based on electrophoretic mobilities, immunological analysis, peptide and phosphopeptide maps, as well as amino acid sequencing. Preincubation of SR with NaF and ATP, but not with NaF and AMP-PNP caused strong inhibition of ryanodine binding. This is due to decrease in Ca²⁺- and ryanodine-binding affinities of the ryanodine receptor (RyR) by about 6.6 and 18-fold, respectively. These results suggest that cardiac sarcalumenin is an isoform of the skeletal muscle protein. An endogenous CK II can phosphorylate sarcalumenin, and in parallel to its phosphorylation the properties of the ryanodine receptor are modified. Received: 15 December 1998/Revised: 25 March 1999     

Calcium-Sensitive Nonselective Cation Channel Identified in the Epithelial Cells Isolated from the Endolymphatic Sac of Guinea Pigs

We identified a Ca²⁺-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K⁺= Na⁺ > Ca²⁺≫ Cl⁻. This channel was weakly voltage-dependent but was strongly activated by Ca²⁺ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca²⁺ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), activated the channel at an estimated [Ca²⁺]_i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca²⁺ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na⁺ under certain pathological conditions that will increase [Ca²⁺]_i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells. Received: 24 October 2000/Revised: 10 April 2001