The elusive role of store depletion in the control of intracellular calcium release

The contractile cycle of striated muscles, skeletal and cardiac, is controlled by a cytosolic [Ca²⁺] transient that requires rapid movements of the ion through channels in the sarcoplasmic reticulum (SR). A functional signature of these channels is their closure after a stereotyped time lapse of Ca²⁺ release. In cardiac muscle there is abundant evidence that termination of release is mediated by depletion of the Ca²⁺ store, even if the linkage mechanism remains unknown. By contrast, in skeletal muscle the mechanisms of release termination are not understood. This article reviews measurements of store depletion, the experimental evidence for dependence of Ca²⁺ release on the [Ca²⁺] level inside the SR, as well as tests of the molecular nature of putative intra-store Ca²⁺ sensors. Because Ca²⁺ sparks exhibit the basic release termination mechanism, much attention is dedicated to the studies of store depletion caused by sparks and its relationship with termination of sparks. The review notes the striking differences in volume, content and buffering power of the stores in cardiac vs. skeletal muscle, differences that explain why functional depletion is much greater for cardiac than skeletal muscle stores. Because in skeletal muscle store depletion is minimal and reduction in store [Ca²⁺] does not appear to greatly inhibit Ca²⁺ release, it is concluded that decrease in free SR [Ca²⁺] does not mediate physiological termination of Ca²⁺ release in this type of muscle. In spite of the apparent absence of store depletion and its putative channel closing effect, termination of Ca²⁺ sparks is faster and more robust in skeletal than cardiac muscle. A gating role of a hypothetical “proximate store” constituted by polymers of calsequestrin and associated proteins is invoked in an attempt to preserve a role for store depletion and unify mechanisms in both types of striated muscle.     

Ca2+ store determines gating of store operated calcium entry in mammalian skeletal muscle

This work describes the gating of the store operated calcium entry (SOCE) in adult mammalian skeletal muscle. Flexor digitorum brevis fibers (FDB) were isolated from adult mice and exposed to conditions to deplete the sarcoplasmic reticulum (SR). A transient SR depletion caused either by repetitive depolarizations, chlorocresol (CMC) or, cyclopiazonic acid (CPA) induced a bell shaped calcium entry that raised the [Ca²⁺]_i to a maximum of 27.09 ± 4.35 nM from the resting value. The activation time to reach 10–90% of the maximum amplitude was 112 ± 10 s (n = 22). On the other hand, any mechanism that caused a permanent SR depletion (like thapsigargin, continuous CPA, or continuous CMC) triggered a calcium entry pathway that lasted 325 ± 23 s and raised the [Ca²⁺]_i to 129.50 ± 13.05 nM from the resting level (n = 28). Then, a prolonged depletion triggered an increase in [Ca²⁺]_i to higher values and for a longer time than when the SR is transiently depleted (p < 0.001). Our results, in skeletal muscle, showed that calcium store depletion was the signal for SOCE activation and how the SR got depleted was not relevant. Also, we found that SOCE deactivation was not caused by [Ca²⁺]_i but by the SR content. Our results suggest that the SR calcium content plays an important role in SOCE gating in mammalian skeletal muscle and a calcium sensor is located inside the SR.     

Spontaneous tension oscillation in skinned bovine cardiac muscle

 Skinned fibres from bovine ventricles exhibited spontaneous tension oscillations when MgADP and inorganic phosphate (Pi) were added to the solution bathing fibres in the relaxed state (ADP-SPOC). A similar type of oscillation was observed at intermediate concentrations of free Ca²⁺ in the absence of MgADP and Pi (Ca-SPOC). To investigate the correlation between ADP-SPOC and Ca-SPOC, we constructed two-dimensional state diagrams of cardiac muscle using different concentrations of Pi (0–20 mM) and free Ca²⁺ [pCa=around 5 (+Ca²⁺), pCa=5.15–6.9 and +EGTA (–Ca²⁺)], with varying concentrations of MgADP (0–10 mM), with 2 mM MgATP and 2 mM free Mg²⁺ maintaining ionic strength at 0.15±0.01 M, pH 7.0, 25 °C. The three-dimensional (pCa-Pi-MgADP) state diagram thus obtained was divided into three regions, i.e. the contraction region in which tension oscillation was undetectable, the spontaneous tension oscillation (SPOC) region and the relaxation region. We found that the regions of ADP-SPOC and Ca-SPOC were continuously connected by a single oscillation region sandwiched between the contraction and relaxation regions. The state diagram, which encompasses physiological conditions, shows that the probability of SPOC is higher in cardiac muscle than in skeletal muscle. From these results, we suggest that, despite distinct ionic conditions, the molecular state of cross-bridges during SPOC is common to both ADP-SPOC and Ca-SPOC. Received 19 February 1996 / Received after revision: 16 July 1996 / Accepted: 14 August 1996     

Early effects of denervation on sarcoplasmic reticulum properties of slow-twitch rat muscle fibres

 The Ca²⁺ release activity of the sarcoplasmic reticulum (SR) in chemically skinned single slow-twitch fibres from control, 2-day and 7-day denervated rat soleus muscle was studied. Histochemical fibre type composition of the whole muscle, electrophysiological properties and the Ca²⁺ sensitivity of tension development by single muscle fibres were also studied. All the data were correlated with contractile properties of the in vitro muscle. In the 2-day denervated muscle the SR Ca²⁺ capacity and the rate of Ca²⁺ uptake decreased from the control values of 0.384 ± 0.030 μmol (mg fibre protein)^–1 and 19.8 ± 1.9 nmol min^–1 (mg fibre protein)^–1, respectively, to 0.210 ± 0.016 μmol (mg fibre protein)^–1 and 13.5 ± 0.9 nmol min^–1 (mg fibre protein)^–1; the calculated amount of Ca²⁺ released upon stimulation by caffeine decreased from the control value of 0.148 to 0.078 μmol (mg fibre protein)^–1. In the 7-day denervated muscle, the SR Ca²⁺ capacity and the rate of Ca²⁺ uptake increased to 0.517 ± 0.06 μmol (mg fibre protein)^–1 and 21.6 ± 2.3 nmol min^–1 (mg fibre protein)^–1, respectively; the calculated amount of Ca²⁺ released increased to 0.217 μmol (mg fibre protein)^–1. Both contraction time and tension of the isometric twitch decreased in 2-day denervated and increased in 7-day denervated muscles. Electrophysiological and histochemical changes, as well as changes in the Ca²⁺ sensitivity of the muscle fibres did not show any apparent correlation with mechanical changes. It is therefore concluded that the SR plays a prominent role in the early changes of contraction time and tension following denervation. Received: 15 October 1996 / Received after revision: 28 March 1997 / Accepted: 8 April 1997     

Role for protein kinase in Ca2+-dependent feedback modulation of divalent cation influx in internal-Ca2+-store-depleted rat parotid gland cells

 Divalent cation (Ca²⁺ and Mn²⁺) influx, stimulated by internal Ca²⁺ store depletion, into rat parotid acinar cells is inhibited by conditions which increase protein phosphorylation [T. Sakai and I.S. Ambudkar (1996) Am J Physiol 271:C284–C294]. The present study examines the involvement of this protein phosphorylation and Ca²⁺ in the store-dependent inactivation of divalent cation entry. Internal Ca²⁺ store depletion, achieved by incubation (30 min) of cells in nominally Ca²⁺-free medium containing either carbachol or thapsigargin, stimulated Ca²⁺, and Mn²⁺, influx into cells. In either case, inclusion of 1.5 mM Ca²⁺ for the last 5 min of incubation resulted in a decrease in Ca²⁺ (33–41%) and Mn²⁺ (50%) influx, which could not be accounted for by internal Ca²⁺ store refill. The inhibition was prevented when internal-store-depleted cells were treated (prior to incubation with Ca²⁺) with either staurosporine or K-252a, but not with H-7 or KN-93. Refilling of internal Ca²⁺ store(s) in carbachol-treated cells (incubation with Ca²⁺+atropine) induced complete inhibition of divalent cation influx, which was not prevented by treatment with protein kinase inhibitors. These data suggest the staurosporine-sensitive (and K-252a-sensitive) protein phosphorylation is not involved in Ca²⁺-store-refilling-dependent inactivation of Ca²⁺ influx but mediates a Ca²⁺-dependent feedback modulation of divalent cation influx in rat parotid gland acinar cells. Received: 24 July 1996 / Received after revision: 26 September 1996 / Accepted: 2 October 1996     

Novel regulators of RyR Ca2+ release channels: insight into molecular changes in genetically-linked myopathies

There are many mutations in the ryanodine receptor (RyR) Ca²⁺ release channel that are implicated in skeletal muscle disorders and cardiac arrhythmias. More than 80 mutations in the skeletal RyR1 have been identified and linked to malignant hyperthermia, central core disease or multi-minicore disease, while more than 40 mutations in the cardiac RyR2 lead to ventricular arrhythmias and sudden cardiac death in patients with structurally normal hearts. These RyR mutations cause diverse changes in RyR activity which either excessively activate or block the channel in a manner that disrupts Ca²⁺ signalling in the muscle fibres. In a different myopathy, myotonic dystrophy (DM), a juvenile isoform of the skeletal RyR is preferentially expressed in adults. There are two regions of RyR1 that are variably spiced and developmentally regulated (ASI and ASII). The juvenile isoform (ASI (−)) is less active than the adult isoform (ASI(+)) and its over-expression in adults with DM may contribute to functional changes. Finally, mutations in an important regulator of the RyR, the Ca²⁺ binding protein calsequestrin (CSQ), have been linked to a disruption of Ca²⁺ homeostasis in cardiac myocytes that results in arrhythmias. We discuss evidence supporting the hypothesis that mutations in each of these situations alter protein/protein interactions within the RyR complex or between the RyR and its associated proteins. The disruption of these protein–protein interactions can lead either to excess Ca²⁺ release or reduced Ca²⁺ release and thus to abnormal Ca²⁺ homeostasis. Much of the evidence for disruption of protein–protein interactions has been provided by the actions of a group of novel RyR regulators, domain peptides with sequences that correspond to sequences within the RyR and which compete with the endogenous residues for their interaction sites.     

Activation of non-selective cation conductance by carbachol in freshly isolated bovine ciliary muscle cells

 In smooth muscle cells freshly isolated from the bovine ciliary body, effects of carbachol (CCh) on the membrane potential and current were examined by the whole-cell clamp method. The resting membrane potential of the muscle cells used was –60 ± 1 mV (n=111). Extracellular application of CCh (2 μM) depolarized the cells to –15 ± 5 mV (n=50) with an apparent increase in membrane conductance. Under voltage-clamp conditions, CCh (2 μM) evoked an inward current which exhibited inward-going rectification and reversed the polarity at about 0 mV. Removal of Na⁺ from the external solution caused a reduction of the amplitude of the current and a shift of the reversal potential to the negative direction. CCh was able to elicit an inward current even under a condition where Ca²⁺ was the only cation producing an inwardly directed electrochemical gradient. The current was not affected by verapamil or by tetrodotoxin. The CCh-induced current was inhibited by antimuscarinic agents with the affinity sequence: atropine ≈4–DAMP >> pirenzepine > AF-DX116, indicating that the response is mediated by a muscarinic cholinoceptor that belongs to the M₃-subtype. Unlike the non-selective cation channel current in intestinal smooth muscles, which is activated by elevation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i), the current of the ciliary muscle was inactivated when the [Ca²⁺]_i was increased. The conductance, which admits Ca²⁺, may serve as a pathway for Ca²⁺ entry required for contraction. Received: 2 December 1996 / Received after revision: 7 January 1997 / Accepted: 8 January 1997     

Residual sarcoplasmic reticulum Ca<Superscript>2+</Superscript> concentration after Ca<Superscript>2+</Superscript> release in skeletal myofibers from young adult and old mice

Contrasting information suggests either almost complete depletion of sarcoplasmic reticulum (SR) Ca²⁺ or significant residual Ca²⁺ concentration after prolonged depolarization of the skeletal muscle fiber. The primary obstacle to resolving this controversy is the lack of genetically encoded Ca²⁺ indicators targeted to the SR that exhibit low-Ca²⁺ affinity, a fast biosensor: Ca²⁺ off-rate reaction, and can be expressed in myofibers from adult and older adult mammalian species. This work used the recently designed low-affinity Ca²⁺ sensor (Kd = 1.66 mM in the myofiber) CatchER (calcium sensor for detecting high concentrations in the ER) targeted to the SR, to investigate whether prolonged skeletal muscle fiber depolarization significantly alters residual SR Ca²⁺ with aging. We found CatchER a proper tool to investigate SR Ca²⁺ depletion in young adult and older adult mice, consistently tracking SR luminal Ca²⁺ release in response to brief and repetitive stimulation. We evoked SR Ca²⁺ release in whole-cell voltage-clamped flexor digitorum brevis muscle fibers from young and old FVB mice and tested the maximal SR Ca²⁺ release by directly activating the ryanodine receptor (RyR1) with 4-chloro-m-cresol in the same myofibers. Here, we report for the first time that the Ca²⁺ remaining in the SR after prolonged depolarization (2 s) in myofibers from aging (~220 μM) was larger than young (~132 μM) mice. These experiments indicate that SR Ca²⁺ is far from fully depleted under physiological conditions throughout life, and support the concept of excitation–contraction uncoupling in functional senescent myofibers.     

Alterations in calcium homeostasis reduce membrane excitability in amphibian skeletal muscle

The effects of alterations in intracellular calcium homeostasis on surface membrane excitability were investigated in resting Rana temporaria sartorius muscle. This was prompted by initial results from a fatiguing stimulation protocol study that demonstrated a fibre subpopulation in which action potential generation in response to a standard 1.5 V electrical stimulus failed despite mean membrane potentials [E _m, −69±2.3 mV (n=14)] compatible with spike firing in a control set of quiescent muscle fibres. Intracellular micro-electrode recordings showed a similar reversible loss of excitability, attributable to an increased threshold, despite only small (7.1±1.8 mV) positive changes in E _m after approximately 60-min exposures to nominally 0 Ca²⁺ Ringer solutions in which Ca²⁺ was replaced by Mg²⁺. This effect was not reproduced by addition of Mg²⁺ to the Ringer solution and persisted under conditions of Cl⁻ deprivation. The effects of three pharmacological agents, cyclopiazonic acid (CPA), caffeine and 4-chloro-m-cresol (4-CmC), each known to deplete store Ca²⁺ and increase cytosolic Ca²⁺ through contrasting mechanisms without influencing E _m, were then investigated. All three agents produced a more rapid, but nevertheless still reversible, loss of membrane excitability than in 0 Ca²⁺ Ringer solution alone. This reduction in membrane excitability persisted in fibres studied in solutions containing a normal [Ca²⁺] following prior depletion of store Ca²⁺ using CPA- and 4-CmC-containing solutions. These novel findings suggest that sarcoplasmic reticulum Ca²⁺ content profoundly influences surface membrane excitability, thereby providing a potential mechanism by which spike firing fails in well-polarised fibres during fatigue.The authors Usher-Smith and Xu were equal contributors to this paper.     

Bridging the myoplasmic gap: recent developments in skeletal muscle excitation–contraction coupling

Conformational coupling between the L-type voltage-gated Ca²⁺ channel (or 1,4–dihydropyridine receptor; DHPR) and the ryanodine-sensitive Ca²⁺ release channel of the sarcoplasmic reticulum (RyR1) is the mechanistic basis for excitation–contraction (EC) coupling in skeletal muscle. In this article, recent findings regarding the roles of the individual cytoplasmic domains (the amino- and carboxyl-termini, cytoplasmic loops I–II, II–III, and III–IV) of the DHPR α_1S subunit in bi-directional communication with RyR1 will be discussed.     

Cross-bridge movement in rat slow skeletal muscle as a function of calcium concentration

A single fibre bundle from rat soleus muscle was chemically skinned with saponin and the transfer of myosin heads from the thick filaments to the thin filaments at a sarcomere length of 2.4 μm was measured as a function of Ca²⁺ concentration using an x-ray diffraction method at 4–7 °C. In the relaxed state, the 1,0 spacing was 42.08 nm. The spacing showed no significant decrease when the Ca²⁺ concentration was below the threshold (−log₁₀ [Ca²⁺] or pCa 5.8). No significant transfer of the myosin heads occurred when the Ca²⁺concentration was below the threshold (pCa 5.8). When the muscle was maximally activated at pCa 4.4, the spacing decreased to 40.35 nm. During the maximum isometric contraction at pCa 4.4, 54.9 ± 6.5% (±SE of the mean) of the myosin heads were transferred to the thin filaments. The transfer of the myosin heads was approximately proportional to relative tension. These results suggest that myosin heads of both fast-twitch and slow-twitch skeletal muscles transferred on the common movement as a function of Ca²⁺ concentration. Received: 1 December 1995/Received after revision and accepted: 20 May 1996     

Changes of the biophysical properties of calcium-activated potassium channels of rat skeletal muscle fibres during aging

 In the present work, we have investigated the effects of the aging process on Ca²⁺-activated K⁺ channels (K_Ca2+) of rat skeletal muscle fibres. K_Ca2+ channels of adult (5–7 months old) and aged (24–26 months old) rats were surveyed by the patch-clamp technique. In aged rats, K_Ca2+ channels were routinely detected on the surface membrane of the fibres in both cell-attached and inside-out configurations. Conversely, in adult rat fibres, K_Ca2+ channels were rarely detected. In the cell-attached configuration, the open probability of the aged rat K_Ca2+ channel, measured in the range of potentials from –60 mV to +20 mV, was about 1.5–2 times higher than that of the adult one. The number of functional channels was abnormally increased by aging. An average of three channels per patch/area was counted in the inside-out patches of aged rat fibres, whereas no more than one open channel per patch/area was detected in the adult rat fibres. The frequency of finding channels in the patches also increased with aging, i.e. 11.5% and 30.1% in the adult and in the aged rat fibres, respectively. However, no significant change in the single-channel conductance has been observed with aging: it was 227 pS and 231 pS for adult and aged rat channels, respectively. In detached patches, both the adult and aged rat channels showed a similar voltage dependence of open probability and a similar sensitivity to Ca²⁺ ions. The aging process did not alter the response of the single channel to charybdotoxin, or its modulation by nucleotides, MgATP and adenosine 5’-O-(3-thiotriphosphate) (ATP[γ-S]). On the other hand, charybdotoxin reduced the abnormally high resting macroscopic K⁺ conductance of the aged rat fibres, recorded using the two-intracellular-microelectrode technique. These findings indicate that, in skeletal muscle, the activity of K_Ca2+ channels increases with advancing age. Received: 10 April 1997 / Received after revision and accepted: 4 June 1997     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

The action of perchlorate on malignant-hyperthermia-susceptible muscle

 To better understand the altered skeletal muscle excitation-contraction (E-C) coupling that occurs in malignant hyperthermia, we have examined the potentiating actions of perchlorate in intact muscle fiber bundles, isolated sarcoplasmic reticulum (SR) vesicles, and the purified ryanodine receptor/Ca²⁺ release channel (RyR) isolated from malignant-hyperthermia-susceptible (MHS) and normal porcine muscle. The concentration of perchlorate that half-maximally potentiated twitch tension (2.5–3.5 mM) was not significantly different for MHS and normal muscles. The effect of perchlorate on fractional twitch force was significantly greater for normal than for MHS muscle, although the absolute twitch potentiation was similar for both muscle types. The K-contracture threshold of MHS muscle bundles is significantly lower than that of normal bundles; perchlorate shifted the K-contraction activation curves of both MHS and normal muscle bundles to lower K⁺ concentrations. Perchlorate both increased ryanodine binding to MHS and normal SR vesicles and increased single-channel open probability of the purified MHS and normal RyR. In both cases, the percentage increase was greater for normal than for MHS preparations; however, the absolute increase in activity was not different for MHS and normal RyR indicating that there is no difference in the perchlorate sensitivity of MHS and normal SR Ca²⁺ release channels. Thus, the greater absolute responses of the MHS Ca²⁺ release channel in the presence of perchlorate is likely to be due to the greater basal activity of the MHS release channel and does not reflect an underlying defect in the site of action of perchlorate on the MHS skeletal muscle Ca²⁺ release channel. Received: 17 April 1997 / Accepted: 9 July 1997     

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres

 The effects of the sarcoplasmic reticulum (SR) Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) were studied in saponin-permeabilized frog skeletal muscle fibres. Release of Ca²⁺ from the SR was triggered by brief (2 s) applications of 40 mM caffeine at 2-min intervals. Changes in [Ca²⁺] within the fibre were monitored continuously using Fura-2 fluorescence. At a bathing [Ca²⁺] of 100 nM, introduction of 20 μM CPA induced a slow release of Ca²⁺ from the SR. The following one to two caffeine-induced Ca²⁺ transients were markedly increased in amplitude and duration. Thereafter, the caffeine-induced Ca²⁺ transients decreased progressively and were barely detectable 6–7 min after introduction of CPA. However, increasing the bathing [Ca²⁺] or increasing the Ca²⁺ loading period resulted in a partial recovery of the caffeine-induced Ca²⁺ transients, suggesting that pump inhibition is incomplete, even in the presence of 100 μM CPA. The slow Ca²⁺ efflux induced by CPA was insensitive to ryanodine, but absent following abolition of SR Ca²⁺ pump activity by ATP withdrawal. These results suggest that the caffeine-induced Ca²⁺ transient reflects a balance between efflux via the SR Ca²⁺ channel and reuptake by the Ca pump. Ca²⁺ release upon addition of CPA may result from inhibition of SR Ca²⁺ uptake, which reveals a tonic Ca²⁺ efflux that is independent of the Ca²⁺ release channels. Received: 26 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998     

Regulation of smooth muscle delayed rectifier K+ channels by protein kinase A

We identified voltage-activated K⁺ channels in freshly dispersed smooth muscle cells from the circular layer of the canine colon in patch-clamp experiments using 200 nM charybdotoxin to suppress 270-pS Ca²⁺-activated K⁺ channels (BK channels). Three channel types were distinguished in symmetrical 140 mM KCl solutions: 19.5 ± 1.7 pS channels (K_DR1), 90.6 ± 5.4 pS channels (K_DR2) and 149 ± 4 pS intermediate-conductance Ca²⁺-activated K⁺ channels (IK channels). All three types showed an increase in open probability with membrane depolarization. Ensemble average current from K_DR1 channels inactivated with a time constant of 1.7 ± 0.1 s at +60 mV test potential, while K_DR2 and IK channels did not show inactivation. IK channels were activated by free cytoplasmic [Ca²⁺] (10⁻⁶M) but were insensitive to 4-aminopyridine (4-AP, 10 mM) and intracellular tetraethylammonium (TEA, 1 mM). K_DR1 channels were sensitive to 4-AP (10 mM) and intracellular TEA (1–10 mM) but not to Ca²⁺. K_DR2 channels did not have a consistent pharmacological profile, suggesting that this class may be comprised of several subtypes. At +40 mV membrane potential, the catalytic subunit of protein kinase A (PKA) increased the open probability of K_DR1 channels 3.4-fold and of K_DR2 channels 3.9-fold, but had no effect on IK channels. In the absence of Mg-ATP, PKA did not affect channel open probabilities. At physiological membrane potentials (−60 mV) only openings of K_DR1 channels could be induced by PKA, suggesting that these 4-AP-sensitive 20-pS K⁺ channels are primarily responsible for the cAMP-mediated hyperpolarization of colonic smooth muscle cells. Received: 20 June 1995/Received after revision: 25 January 1996/Accepted: 7 February 1996     

Involvement of the IP3 cascade in the damage to guinea-pig ventricular myocytes induced by cytotoxic T lymphocytes

 We have shown previously that the interaction between cytotoxic T lymphocytes (CTL) and ventricular myocytes, an in vitro model for heart transplant rejection, results in electrophysiological and morphological alterations indicative of overload of the intracellular [Ca²⁺] ([Ca²⁺]_i). Since these deleterious effects cannot be accounted for by increased L-type Ca²⁺ current (I _Ca,L), we hypothesize that [Ca²⁺]_i overload due to Ca²⁺ release from intracellular stores, e.g. sarcoplasmic reticulum (SR), is initiated by CTL-induced activation of the inositol trisphosphate (IP₃) cascade. Patch-clamp and fura-2-fluorescence techniques were utilized to record transmembrane potentials and [Ca²⁺]_i from ventricular myocytes bound to peritoneal exudate CTL (PEL). In ventricular myocyte-PEL conjugates (after 60 min), resting potential was reduced (compared with the nonconjugated state) from –80.9 ± 0.7 to –59.9 ± 2.5 mV, action potential amplitude from 139.5 ± 1.4 to 80.6 ± 1.7 mV and action potential duration to 50% repolarization (APD₅₀) from 797 ± 97 to 52 ± 12 ms. The ratio of fluorescence at 340 and 380 nm (R _340/380) increased from a control value (in nonconjugated myocytes) of 0.71 ± 0.02 to 2.07 ± 0.03, 30 min after conjugate formation, and exceeded 4.0 at 60 min, before myocyte destruction. Heparin (50 μg/ml), an antagonist of IP₃-induced Ca²⁺ release from SR channels, or U-73122 (2 μM), a phospholipase C (PLC) inhibitor (drugs were included in the pipette solution), prevented PEL-induced morphological and electrophysiological alterations. Accordingly, heparin attenuated the PEL-induced increase in [Ca²⁺]_i; after 60 min of PEL-myocyte interaction, R _340/380 was 1.15 ± 0.09 (compared with approximately 4.0 in the absence of heparin). The results indicate that CTL-mediated damage to ventricular myocytes is, at least partially, mediated by PLC activation and IP₃-induced Ca²⁺ release from intracellular stores. Pharmacological targeting of IP₃ in heart transplant rejection is thus suggested. Received: 3 July 1996 / Received after revision: 21 October 1996 / Accepted: 3 December 1996     

Reciprocal dihydropyridine and ryanodine receptor interactions in skeletal muscle activation

Dihydropyridine (DHPR) and ryanodine receptors (RyRs) are central to transduction of transverse (T) tubular membrane depolarisation initiated by surface action potentials into release of sarcoplasmic reticular (SR) Ca²⁺ in skeletal muscle excitation–contraction coupling. Electronmicroscopic methods demonstrate an orderly positioning of such tubular DHPRs relative to RyRs in the SR at triad junctions where their membranes come into close proximity. Biochemical and genetic studies associated expression of specific, DHPR and RyR, isoforms with the particular excitation–contraction coupling processes and related elementary Ca²⁺ release events found respectively in skeletal and cardiac muscle. Physiological studies of intramembrane charge movements potentially related to voltage triggering of Ca²⁺ release demonstrated a particular q_γ charging species identifiable with DHPRs through its T-tubular localization, pharmacological properties, and steep voltage-dependence paralleling Ca²⁺ release. Its nonlinear kinetics implicated highly co-operative conformational events in its transitions in response to voltage change. The effects of DHPR and RyR agonists and antagonists upon this intramembrane charge in turn implicated reciprocal rather than merely unidirectional DHPR–RyR interactions in these complex reactions. Thus, following membrane potential depolarization, an orthograde q_γ-DHPR–RyR signaling likely initiates conformational alterations in the RyR with which it makes contact. The latter changes could then retrogradely promote further q_γ-DHPR transitions through reciprocal co-operative allosteric interactions between receptors. These would relieve the resting constraints on both further, delayed, nonlinear q_γ-DHPR charge transfers and on RyR-mediated Ca²⁺ release. They would also explain the more rapid charging and recovery q_γ transients following larger depolarizations and membrane potential repolarization to the resting level.     

Binding property of avian skeletal muscle ryanodine receptor isoforms with dihydropyridine receptor and calmodulin

Ca²⁺ release during excitation–contraction coupling in avian skeletal muscle is controlled by two ryanodine receptor isoforms, αRYR and βRYR. Two other proteins, dihydropyridine receptor (DHPR) and calmodulin (CaM), have been shown to play important roles in regulating the RYR channel activity. In the current study, we measured the protein contents of DHPR and RYR in turkey skeletal muscle and obtained a ratio of 1:1 between DHPR and αRYR which suggests that only a subpopulation of αRYR is interacting with DHPR. Two CaM derivatives, the photoactivable crosslinking probe [¹²⁵I]-Bz-CaM and metabolically labeled probe [³⁵S]CaM, were used to study the interaction between CaM and RYR isoforms in turkey skeletal muscle. The αRYR and βRYR displayed a marked difference in their CaM binding behavior. At a Ca²⁺ concentration of 200 μM, CaM bound to both isoforms at a ratio of one CaM molecule per one RYR subunit. At a Ca²⁺ concentration of <10 nM, CaM bound primarily to αRYR and the binding affinity was significantly lower than that at micromolar level of Ca²⁺ concentration. Cloning and sequencing of putative CaM binding sites in αRYR and βRYR suggests that differences in primary structures of the CaM binding sites of each RYR isoform may contribute to the differential CaM binding behavior of αRYR and βRYR.     

Effects of calciseptine on unitary barium channel currents in guinea-pig portal vein

 Effects of synthesized calciseptine (CaS), found naturally in the venom of the black mamba, on voltage-dependent Ca²⁺ channels in smooth muscle cells of the guinea-pig portal vein were investigated. In the whole-cell voltage-clamp configuration, extracellular application of CaS (≥ 10 nM) inhibited the inward current in a concentration- and voltage-dependent manner at a holding potential of –90 mV. The Ca²⁺ current recorded at a high holding potential (–50 mV) was approximately 8 times more sensitive to CaS than that at a more negative holding potential (–90 mV). CaS (50 nM) shifted to the left the steady-state inactivation curve obtained by using single 8-s conditioning pulses of various amplitudes. When CaS (≥ 200 nM) was present in the pipette, the Ca²⁺ current remained for the duration of the experiments (more than 60 min) in the whole-cell configuration. Two different Ca²⁺ channel conductances are present in this tissue (25-pS and 12-pS channels). Both channels are blocked by dihydropyridine (DHP) derivatives, but have different sensitivities. In the cell-attached condition, CaS hardly changed the activity of either unitary Ca²⁺ channel current. To prevent the ”run down” of the Ca²⁺ channels in cell-free conditions, we added cardiac cytosol, a supernatant from homogenized cardiac cells and an endogenous Ca²⁺ channel activating factor, in the pipette. The unitary Ca²⁺ channel currents were then recorded using the outside-out membrane patch configuration. Application of CaS (1 μM) in the bath completely blocked the open events of the 25-pS Ca²⁺ channel. CaS (10 nM) in the bath reduced the mean open time and channel availability, resulting in a decrease in the open probability of the 25-pS channel currents without affecting the amplitude of the single-channel conductance. CaS also reduced the open probability (though less potently) and channel availability of the 12-pS Ca²⁺ channel without a change in its amplitude. From these results, we conclude that CaS has inhibitory effects on the voltage-dependent Ca²⁺ current that are similar to those of DHP derivatives and that it acts from the outside of the membrane. Received: 4 October 1995 / Received after revision: 20 February 1996 / Accepted: 1 March 1996