`Quantal' Ca2+ release at the cytoplasmic aspect of the Ins(1,4,5)P3R channel in smooth muscle

McCarron, John G.; Chalmers, Susan; Muir, Thomas C.

doi:10.1242/jcs.017541

Smooth muscle responds to activation of the inositol (1,4,5)-trisphosphate receptor [Ins(1,4,5)P₃R] with a graded concentration-dependent (`quantal') Ca²⁺ release from the sarcoplasmic reticulum (SR) store. Graded release seems incompatible both with the finite capacity of the store and the Ca²⁺-induced Ca²⁺ release (CICR)-like facility, at Ins(1,4,5)P₃Rs, that, once activated, should release the entire content of SR Ca²⁺. The structural organization of the SR and the regulation of Ins(1,4,5)P₃R activity by inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] and Ca²⁺ have each been proposed to explain `quantal' Ca²⁺ release. Here, we propose that regulation of Ins(1,4,5)P₃R activity by lumenal Ca²⁺ acting at the cytoplasmic aspect of the receptor might explain `quantal' Ca²⁺ release in smooth muscle. The entire SR store was found to be lumenally continuous and Ca²⁺ could diffuse freely throughout: peculiarities of SR structure are unlikely to account for `quantal' release. While Ca²⁺ release was regulated by [Ca²⁺] within the SR, the velocity of release increased (accelerated) during the release process. The extent of acceleration of release determined the peak cytoplasmic [Ca²⁺] and was attenuated by a reduction in SR [Ca²⁺] or an increase in cytoplasmic Ca²⁺ buffering. Positive feedback by released Ca²⁺ acting at the cytoplasmic aspect of Ins(1,4,5)P₃Rs (i.e. CICR-like) might (a) account for the acceleration, (b) provide the regulation of release by SR [Ca²⁺] and (c) explain the `quantal' release process itself. During Ca²⁺ release, SR [Ca²⁺] and thus unitary Ins(1,4,5)P₃R currents decline, CICR reduces and stops. With increasing [Ins(1,4,5)P₃], coincidental activation of several neighbouring Ins(1,4,5)P₃Rs offsets the reduced Ins(1,4,5)P₃R current to renew CICR and Ca²⁺ release.

Introduction

The second-messenger inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃ or IP₃] binds to its receptor [Ins(1,4,5)P₃R] on the internal Ca²⁺ store – the sarcoplasmic reticulum (SR) of muscle or endoplasmic reticulum (ER) of non-muscle cells – to release stored Ca²⁺ into the cell. The subsequent changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) are essential for many receptor-mediated signalling pathways in several cell types, including smooth muscle. For example, cell contraction, division and death are each regulated by changes in [Ca²⁺]_c evoked by Ins(1,4,5)P₃ (Berridge et al., 2000; McCarron et al., 2006; Straub et al., 2006). Ins(1,4,5)P₃Rs on the SR exist in clusters of ∼25-35 receptors (Shuai et al., 2006), each cluster separated by a few micrometers (Bootman et al., 1997a; Shuai et al., 2006; Swillens et al., 1999). The receptor arrangement provides spatially discrete release sites (Bootman et al., 1997a; Horne and Meyer, 1995) and enables Ca²⁺ signals arising from Ins(1,4,5)P₃Rs to exist in a variety of spatial forms, including `blips', arising from the opening of a single Ins(1,4,5)P₃R in a cluster, or `puffs', when several Ins(1,4,5)P₃Rs in a cluster open (Bootman et al., 1997a; Parker et al., 1996). Larger [Ca²⁺]_c increases occur when `puffs' coalesce to form transient Ca²⁺ increases (spikes) or propagate through the cell as waves (Bootman et al., 1997a; McCarron et al., 2004). For signals to progress from single to multiple Ins(1,4,5)P₃R activations, the initial Ca²⁺ release event must be amplified by positive-feedback processes. One such process operates on the receptor itself. Ca²⁺ released through the Ins(1,4,5)P₃R can evoke further release from adjacent Ins(1,4,5)P₃-occupied receptors in a Ca²⁺-induced Ca²⁺ release (CICR)-like process (e.g. Bootman et al., 1997b; Dargan and Parker, 2003; Iino et al., 1994) (reviewed in Bootman et al., 1997a; McCarron et al., 2004). This process can determine the very characteristics (e.g. amplitude) of several Ins(1,4,5)P₃-mediated Ca²⁺ signals, such as puffs, waves and spikes. For example, the transition of a signal from a `blip' to a `puff' or from a `puff' to a wave might each require local feedback in a CICR-like process; in the former case to recruit within individual clustered Ins(1,4,5)P₃Rs (Bootman et al., 1997a; Rose et al., 2006) and, in the latter, to recruit among clusters (Bootman et al., 1997a; MacMillan et al., 2005; McCarron et al., 2004).

Ins(1,4,5)P₃-mediated Ca²⁺ release is a graded concentration-dependent process in which low concentrations of Ins(1,4,5)P₃ release only part of the available Ca²⁺ content of the SR store (Bootman et al., 1994; Meyer and Stryer, 1990; Muallem et al., 1989; Oldershaw et al., 1991; Shin et al., 2001) (reviewed in Parys et al., 1996). The Ca²⁺ store is not fully depleted even after prolonged stimulation. Such a graded release seems, at first sight, incompatible both with the finite capacity of the store and the positive feedback CICR-like facility at Ins(1,4,5)P₃Rs (Iino, 1990) that, once activated, would be anticipated to release the entire SR Ca²⁺ content. Yet Ca²⁺ release from the SR by means of Ins(1,4,5)P₃Rs is both controlled and graded by the concentration of Ins(1,4,5)P₃ (Meyer and Stryer, 1990). This process of graded Ca²⁺ release through Ins(1,4,5)P₃Rs has been termed `quantal' (Muallem et al., 1989), initially to imply an all-or-none release of discrete Ca²⁺ pools, analogous to events involving acetylcholine at the skeletal neuromuscular junction (Fatt and Katz, 1952). The term `quantal' release has now been applied more generally to denote the graded release of Ca²⁺ from the store. So called `quantal' release has been observed in cells depleted of ATP, exposed to inhibitors of the SR Ca²⁺ pump or following the use of poorly metabolizable forms of Ins(1,4,5)P₃ (Combettes et al., 1996; Meyer and Stryer, 1990; Parys et al., 1996; Shuttleworth, 1992). These results suggest that neither rapid activation of the SR Ca²⁺ pump, a phosphorylation-mediated event nor breakdown of Ins(1,4,5)P₃ accounts for the `quantal' release process (Combettes et al., 1996; Meyer and Stryer, 1990; Parys et al., 1996; Shuttleworth, 1992).

Several proposals exist to account for `quantal' release, but none has gained widespread acceptance. In one proposal (Hirose and Iino, 1994; Muallem et al., 1989; Parker and Ivorra, 1990; Shin et al., 2001), cells might contain a series of Ca²⁺ stores, each with different sensitivities to Ins(1,4,5)P₃, so that, at any given concentration of the inositide, only some stores will be activated to release their Ca²⁺ content. At low [Ins(1,4,5)P₃], only those stores most sensitive to the inositide will release their contents, whereas others will be activated only at higher [Ins(1,4,5)P₃]. This proposal not only requires there to be numerous stores but also a sensitivity gradient to Ins(1,4,5)P₃ that will permit a small range of [Ins(1,4,5)P₃] to empty some stores, while leaving others unaffected. The sensitivity gradient might exist, at least theoretically. Computer simulations of Ins(1,4,5)P₃-mediated Ca²⁺ release versus [Ins(1,4,5)P₃] suggest that the EC₅₀ for Ins(1,4,5)P₃ depends critically on the relative Ins(1,4,5)P₃R density ([Ins(1,4,5)P₃R]) of the store. Lower [Ins(1,4,5)P₃R] caused a rightward shift in the Ins(1,4,5)P₃ dose-response relationship. For example, at a constant [Ins(1,4,5)P₃] [3 μM (Dawson et al., 2003)], a store with an Ins(1,4,5)P₃R density of 0.02 μM discharged its entire Ca²⁺ content, while one with a receptor density of 0.005 μM was largely unaffected (Dawson et al., 2003).

Indeed, there is evidence for numerous stores with different sensitivities to Ins(1,4,5)P₃ in intact cells (Parker and Ivorra, 1990; Parker and Yao, 1991). Ins(1,4,5)P₃-mediated Ca²⁺ release in localized (micrometre scale) regions of Xenopus oocytes varied in an approximately all-or-none manner with increasing [Ins(1,4,5)P₃] (Parker and Ivorra, 1990; Shin et al., 2001), but, when measured in larger areas, grew progressively as [Ins(1,4,5)P₃] increased above a threshold value (Parker and Ivorra, 1990). These results were interpreted as indicating the presence of an Ins(1,4,5)P₃-sensitive Ca²⁺ store comprising multiple compartments that each released Ca²⁺ in an all-or-none manner (Parker and Ivorra, 1990). An all-or-none emptying of stores (Bootman et al., 1992; Oldershaw et al., 1991; Shin et al., 2001) and regional differences in store sensitivity to [Ins(1,4,5)P₃] (Bootman et al., 1992; Kasai et al., 1993) have also been observed in other cell types (e.g. hepatocytes, HeLa and pancreatic acinar cells). This interesting proposal has been examined in the present study.

Other proposed `quantal' release mechanisms do not require the presence of numerous stores (Moran and Turner, 1996) but suggest that Ins(1,4,5)P₃R activity declines (`adapts') to a particular [Ins(1,4,5)P₃]. At any given [Ins(1,4,5)P₃], the entire Ca²⁺ store(s) is activated and releases some of its content to become partially depleted. Partial depletion deactivates Ca²⁺ release (Missiaen et al., 1992; Tanimura and Turner, 1996). Raising the [Ins(1,4,5)P₃] reactivates Ins(1,4,5)P₃R to renew the Ca²⁺ release process. This proposal requires rather more-complex adaptive changes in Ins(1,4,5)P₃R activity that might involve negative-feedback processes either at the cytoplasmic or the lumenal aspects of Ins(1,4,5)P₃Rs. For example, Ins(1,4,5)P₃ binding might initially activate, then partially inactivate, Ins(1,4,5)P₃Rs in a concentration-dependent way to produce `quantal' Ca²⁺ release (Hajnoczky and Thomas, 1994; Marchant and Taylor, 1998; Wilcox et al., 1996). Alternatively, the rise in cytoplasmic [Ca²⁺]_c, which derives from the activity of Ins(1,4,5)P₃R, might itself inactivate the receptor (Adkins and Taylor, 1999; Iino and Tsukioka, 1994; Oancea and Meyer, 1996). In another proposal, the sensitivity of Ins(1,4,5)P₃Rs to Ins(1,4,5)P₃ is controlled by the lumenal [Ca²⁺] such that, as the concentration of the ion within the SR lumen falls, so does Ins(1,4,5)P₃R activity (e.g. Irvine, 1990; Tanimura and Turner, 1996). Indeed, the control of Ins(1,4,5)P₃Rs by lumenal Ca²⁺, although disputed, is currently the most commonly proposed mechanism of `quantal' release. It is particularly unclear whether the control of Ins(1,4,5)P₃R activity by lumenal Ca²⁺ operates over the physiological Ca²⁺ concentration range. For example, the threshold for lumenal regulation of Ins(1,4,5)P₃Rs to begin is a depletion of the store by in excess of ∼70% of the steady-state lumenal Ca²⁺ concentration ([Ca²⁺]_lum) [500-600 μM (Barrero et al., 1997)] in HeLa cells. [Ca²⁺]_lum must also be substantially depleted in hepatocytes (>45% or 95%) (Beecroft and Taylor, 1997; Combettes et al., 1996) and in A7r5 cells by >70% (Parys et al., 1993) before Ins(1,4,5)P₃R sensitivity changes are detected. In each case, control of Ins(1,4,5)P₃R activity by Ca²⁺ binding to the lumenal aspect of the receptor is unlikely to explain `quantal' Ca²⁺ release when [Ca²⁺]_lum exceeds 55, 5 or 30% of the normal steady-state value, respectively, in these tissues.

On the other hand, lumenal Ca²⁺ binding as a control of Ins(1,4,5)P₃R has not been demonstrated in other studies in permeabilized cells [e.g. portal vein (Hirose and Iino, 1994) or hepatocytes (Combettes et al., 1992)]. Decreases in [Ca²⁺]_lum failed to reduce the sensitivity of Ins(1,4,5)P₃-mediated Ca²⁺ release or alter Ca²⁺ leakage when pumps were blocked in permeabilized cells from avian supraorbital nasal gland (Shuttleworth, 1992). Indeed, single-channel Ins(1,4,5)P₃R activity, measured in planar lipid bilayers, increased when [Ca²⁺] at the lumenal aspect of the channel declined (Bezprozvanny and Ehrlich, 1994). In the latter study, [Ca²⁺]_lum exceeding 1 mM inhibited Ins(1,4,5)P₃R activity (Bezprozvanny and Ehrlich, 1994) (see also Thrower et al., 2000). Together, these results suggest that regulation of Ins(1,4,5)P₃Rs by Ca²⁺ at the lumenal aspect of the channel might, at best, operate over a limited range of [Ca²⁺]_lum.

Ca²⁺ depletion per se also reduces the amount of this ion available for release by reducing the Ins(1,4,5)P₃R Ca²⁺ current. As a consequence, the extent of any positive-feedback process at Ins(1,4,5)P₃Rs in promoting further release of the ion will be reduced (Iino and Endo, 1992). The effect of lumenal [Ca²⁺] on Ins(1,4,5)P₃R activity, therefore, might arise from an interaction of released SR Ca²⁺ with cytosolic Ca²⁺-binding sites on Ins(1,4,5)P₃Rs – that is, lumenal control might be exerted at the cytoplasmic aspect of the channel (Horne and Meyer, 1995; Iino and Endo, 1992). For example, Ins(1,4,5)P₃-mediated Ca²⁺ release was dependent on SR lumenal [Ca²⁺] at low (e.g. 50 nM) but not high (405 nM) [Ca²⁺]_c, suggesting that increased [Ca²⁺]_c could mimic lumenal control in RBL cells (Horne and Meyer, 1995). Ca²⁺ buffers (BAPTA and fluo-3) also prevented the regulation of release by SR lumenal [Ca²⁺] (Horne and Meyer, 1995; Iino and Endo, 1992). This contribution of lumenal Ca²⁺ might, through CICR, be operative over the normal steady-state lumenal [Ca²⁺], although the relevance of the process to `quantal' Ca²⁺ release remains to be explored.

In view of the importance of `quantal' Ca²⁺ release as a control of Ins(1,4,5)P₃-mediated Ca²⁺ release, the kinetics of the release process itself have been examined. Single voltage-clamped colonic myocytes were used. This tissue is particularly useful as Ca²⁺ release through Ins(1,4,5)P₃Rs does not activate the ryanodine receptor (RyR) (Flynn et al., 2001; MacMillan et al., 2005; McCarron et al., 2004), which would otherwise complicate analysis of the results. Photolysis of caged Ins(1,4,5)P₃ or the muscarinic agonist carbachol (CCh), applied by pressure ejection from a `puffer' pipette, was used to evoke Ca²⁺ release through Ins(1,4,5)P₃Rs. Localized (10 μm) subcellular photolysis of caged Ins(1,4,5)P₃ enabled both the site and kinetics of Ins(1,4,5)P₃-mediated Ca²⁺ release to be determined accurately. Our results suggest that `quantal' release cannot be explained by the presence of multiple compartments within the SR store but, rather, by the latter's control of the amount of Ca²⁺ released by each [Ins(1,4,5)P₃] and SR [Ca²⁺]. This control, and the ability of the SR to detect changes in [Ca²⁺], might not reside within the SR but, rather, at the cytoplasmic aspect of Ins(1,4,5)P₃Rs by means of a Ca²⁺-dependent positive-feedback CICR loop. The extent of the positive feedback is itself determined by the SR [Ca²⁺]. As the SR [Ca²⁺] falls during release, positive feedback declines, to be restarted only when multiple neighbouring Ins(1,4,5)P₃Rs are activated almost simultaneously by increased [Ins(1,4,5)P₃]. The reduced unitary Ins(1,4,5)P₃R current, which accompanies the decline in Ca²⁺ release, is offset by multiple, synchronous, neighbouring Ins(1,4,5)P₃R openings, thus explaining `quantal' Ca²⁺ release.

Results

Ins(1,4,5)P₃ production through muscarinic receptor activation (using 50 μM CCh) released Ca²⁺ from the SR to increase [Ca²⁺]_c (Fig. 1A). When the CCh concentration was then increased tenfold (500 μM), a more substantial Ca²⁺ release from the SR occurred (Fig. 1A). To ensure that CCh indeed evoked this [Ca²⁺]_c increase by releasing the ion from the SR, the experiments were carried out in a Ca²⁺-free bathing solution. Under these conditions, [Ca²⁺]_c declined from its resting F/F₀ value of 1 to 0.97±0.008 (n=6; P<0.05). CCh (50 μM) subsequently increased [Ca²⁺]_c to 1.14±0.061 F/F₀ (n=6; P<0.05) and, in the continued presence of this concentration of agonist, the [Ca²⁺]_c then returned to 0.98±0.018, not significantly different from baseline values obtained before CCh (50 μM; n=6; P>0.05). When [CCh] was increased tenfold (500 μM), [Ca²⁺]_c increased to a F/F₀ value of 1.63±0.153, an average increase of 3.5±1.1 fold above that obtained at the lower CCh concentration (n=6; P<0.05). Therefore, in the continued presence of a low concentration of agonist (50 μM), the SR was not totally depleted but retained significant quantities of Ca²⁺ available for release by the higher concentration (500 μM) of agonist.

Essentially similar results were obtained in the presence of external Ca²⁺ (Fig. 1B). Here, CCh (50 μM) increased [Ca²⁺]_c from a resting value of 0.99±0.003 to 1.7±0.16 (n=5; P<0.05). In the continued presence of CCh (50 μM), resting [Ca²⁺]_c was restored (1.0±0.06; n=5). A tenfold higher [CCh] (500 μM) then increased [Ca²⁺]_c to an F/F₀ value of 2.2±0.3 (n=5; P<0.01), an increase of 2.9±0.7 fold above that occurring at the lower [CCh]. Clearly, mechanisms other than a total depletion of SR [Ca²⁺] regulate the amount of Ca²⁺ available for release at lower (50 μM) [CCh]. This observation – that is, failure to deplete totally the SR Ca²⁺ available for release – characterizes the `quantal' release process (Meyer and Stryer, 1990; Muallem et al., 1989) and enables a graded [Ins(1,4,5)P₃]-dependent Ca²⁺ release from the SR to occur (Fig. 1) and the active life of the SR as a repository of Ca²⁺ to be prolonged.

Fig. 1.

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Quantal Ca²⁺ release in a single smooth muscle cell. (A) In a Ca²⁺-free solution, carbachol (CCh; 50 μM; Ac) increased [Ca²⁺]_c (Ab); in its continued presence, [Ca²⁺]_c returned to near-basal levels. A tenfold higher concentration of CCh (500 μM; Ac) evoked a further release of Ca²⁺. [Ca²⁺]_c changes are represented by the colour changes in the frames i-v (Aa; blue low and yellow/red high [Ca²⁺]_c) and by the fluorescence transients (F/F₀; Ab). The images in the frames in Aa were taken before 50 μM CCh (i), during 50 μM CCh (ii,iii) and during 500 μM CCh (iv,v). The time-points at which images were obtained are indicated by their respective numerals (i-v) above the Ca²⁺ transients (Ab); these numerals correspond to those in Aa. The scale bar in the bright-field image of the cell (Aa) applies to all frames. (B) Essentially identical results were obtained in the presence of external Ca²⁺ except that [Ca²⁺]_c oscillations occurred with 500 μM CCh. These presumably arose, at least in part, from Ca²⁺ entry. Figures in Ac and Bb refer to concentrations of CCh applied to the cell by pressure ejection. In these experiments, fluorescence was measured in a region that encompassed the entire cell.

Several explanations (Beecroft and Taylor, 1997; Bootman et al., 1992; Muallem et al., 1989) for `quantal' release rely on there being a particular structural arrangement of the SR in which multiple stores exist with different Ca²⁺ release characteristics in response to low and high [Ins(1,4,5)P₃]. To examine whether the Ins(1,4,5)P₃-sensitive stores existed as multiple separate elements or as a continuum on the lumenal aspect of the SR, one small (10 μm diameter) area of the SR was depleted of Ca²⁺ by the repeated photolysis of caged Ins(1,4,5)P₃ in a Ca²⁺-free solution and the consequence of this depletion on the amount of Ca²⁺ available for release from the remainder of the SR was measured (Fig. 2). Were it to comprise separate elements, depleting one small area of the SR would have little effect on the total amount of Ca²⁺ available for release elsewhere in the SR. In the event, depletion at one site also depleted the entire SR of Ca²⁺, suggesting that the Ins(1,4,5)P₃-sensitive Ca²⁺ store indeed existed as a single lumenally continuous entity throughout the SR through which Ca²⁺ could diffuse freely. Thus photolysing caged Ins(1,4,5)P₃ at a 10 μm diameter site at one end of the cell (Fig. 2, photolysis site 1) increased [Ca²⁺]_c, as revealed by the increase in the F/F₀ ratio (Fig. 2; from a resting F/F₀ value of 1 to 3.8±0.8; n=4; P<0.05). The peak amplitude of the [Ca²⁺]_c increase declined from the release site (measured at 10 μm intervals; Fig. 2C) to 7% of peak values at the second photolysis site. The latter value suggests that relatively little Ins(1,4,5)P₃ reached the second photolysis site.

Fig. 2.

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Localized depletion of the Ins(1,4,5)P₃-sensitive Ca²⁺ store depletes the entire store of Ca²⁺. At –70 mV, locally photolysed Ins(1,4,5)P₃ (↑, B) at a 10-μm-diameter region (photolysis site 1; bright spot in A, left-hand panel; see also patch electrode, left side) evoked Ca²⁺ transients (B). Results from photolysis site 1 are indicated by the red bars below the [Ca²⁺]_c traces in B. When repositioned to site 2 (A, right-hand panel), subsequent photolysis ∼90 seconds later produced reproducible [Ca²⁺]_c increases (B). Results from photolysis site 2 are indicated by the blue line below the [Ca²⁺]_c trace (B). In a Ca²⁺-free solution [containing EGTA (1 mM) and MgCl₂ (3 mM); unfilled line above the [Ca²⁺]_c trace], the [Ca²⁺]_c increase evoked by Ins(1,4,5)P₃ at photolysis site 2 (A) declined in amplitude as the store was depleted of Ca²⁺ (B). When the store content had been substantially reduced at photolysis site 2 (A) (as revealed by the smaller Ca²⁺ transients, B), Ins(1,4,5)P₃ was liberated by photolysis at site 1 (A). Again, as at photolysis site 2, the response was now almost abolished compared with that of the control. On restoring external Ca²⁺ (B, right-hand side), the Ca²⁺ increase evoked by Ins(1,4,5)P₃ at photolysis site 1 was restored towards control values. These results suggest that the SR is lumenally continuous and within it Ca²⁺ can diffuse freely throughout. [Ca²⁺]_c measurements (B) have been derived from fluorescence intensity changes occurring in a circle of diameter 5 μm in the center of the photolysis region. Thus, those results from photolysis site 1 are from a circle of diameter 5 μm positioned at site 1; results from photolysis site 2 are from a circle of diameter 5 μm at site 2. (C) Local photolysed Ins(1,4,5)P₃ (↑) at photolysis site 1 (A, left-hand panel) increased [Ca²⁺]_c (C, right-hand panel), which was maximal at, and decreased with each 10 μm increment away from, the release site (C, right panel); region 1 is the photolysis site. [Ca²⁺]_c measurements were made at lines of width 1 pixel. The position of each measurement (regions 1-9) line is shown (C, left panel) at a width of two pixels to facilitate visualization. The second photolysis site lies between regions 8-9 – that is, ∼75 μm away from photolysis site 1.

Subsequent (∼90 seconds later) photolysis of caged Ins(1,4,5)P₃ at a 10 μm diameter site at the other end of the same cell (Fig. 2, photolysis site 2) also evoked reproducible increases in [Ca²⁺]_c (from a F/F₀ value of 1 to 2.2±0.1; n=4; P<0.05). Repeated application of Ins(1,4,5)P₃ at photolysis site 2 in a Ca²⁺-free solution depleted the SR of Ca²⁺, as revealed by the decrease in the [Ca²⁺]_c transient (F/F₀ changed insignificantly to 1.1±0.1 from a resting value of 1; n=4; P>0.05). Significantly, when the Ins(1,4,5)P₃-evoked Ca²⁺ transient had been reduced at photolysis site 2, the Ca²⁺ increase evoked by Ins(1,4,5)P₃ at photolysis site 1 was also reduced (F/F₀ changed insignificantly to 1.1±0.1 from a resting value of 1; n=4). Thus, releasing Ca²⁺ at one site of the SR reduced the amount of ion available for release from other distant sites. As a control, at the end of the experiment, Ca²⁺ was restored to the bathing solution and, under these conditions, Ins(1,4,5)P₃-evoked Ca²⁺ transients recovered (F/F₀ 2.6±0.9; P<0.05). Together, these results confirm that, as far as Ca²⁺ release is concerned, the SR functions as a single element throughout which Ca²⁺ can diffuse freely.

To confirm that the SR store was lumenally continuous, the possibility that the entire SR could be refilled from one small site was investigated (Fig. 3). The SR was depleted of Ca²⁺, as evidenced by the abolition of transients evoked by repeated application of Ins(1,4,5)P₃ to one small area of the cell in a Ca²⁺-free bathing solution. Ca²⁺ was then readmitted to refill the SR but made available only to one small region of the cell distant from the site of depletion (Fig. 3) by sealing on an `on cell' patch. The Ca²⁺ available in the `on cell' electrode was the only source of the ion for the entire cell. Under these conditions, the entire SR was found to have been replenished with Ca²⁺. This included that site previously depleted of Ca²⁺, despite the cell not having received any direct influx of the ion there. Thus, in controls, the Ca²⁺ rise evoked by photolysis of caged Ins(1,4,5)P₃ was 1.7±0.44 (ΔF/F₀; n=3; P<0.05). This rise decreased to 0.03±0.03 (ΔF/F₀; n=3; P>0.05) in a Ca²⁺-free bathing solution. When the Ca²⁺-containing (30 mM) electrode was sealed onto the cell distant (62±8 μm; n=3) from the site of photolysis, Ins(1,4,5)P₃-mediated Ca²⁺ release was restored after ∼3 minutes (from 0.03±0.03 ΔF/F₀ to 0.6±0.15 ΔF/F₀; n=3; P<0.05). Together, these experiments demonstrate that the entire Ins(1,4,5)P₃-sensitive store can, first, be depleted from a single site and, second, be replenished from a single site, and confirm that the Ins(1,4,5)P₃-sensitive store is a single, lumenally continuous entity throughout which Ca²⁺ can freely diffuse. Thus, involvement of multiple separate stores within the SR appears unlikely as a means of explaining `quantal' release in colonic myocytes.

Fig. 3.

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Ca²⁺ can move through the SR to replenish a site previously depleted of the ion. (A-C) At –70 mV, locally photolysed Ins(1,4,5)P₃ (↑, C) at a 10-μm-diameter region [bright spot in A, left-hand panel; see also whole-cell electrode (left side) and the Ca²⁺-containing shadow of the electrode (right side, `Ca²⁺ electrode')] increased [Ca²⁺]_c (B,C). The [Ca²⁺]_c images (B) are derived from the time-points indicated by the corresponding roman numerals in C. [Ca²⁺]_c changes in B are represented by colour; blue low and red high [Ca²⁺]_c (i-xii). A second photolysis of Ins(1,4,5)P₃ ∼90 seconds later at the same site (C) generated an approximately comparable [Ca²⁺]_c increase. In a Ca²⁺-free solution (containing 1 mM EGTA and 3 mM MgCl₂), the [Ca²⁺]_c increase evoked by Ins(1,4,5)P₃ declined and was abolished as the store became depleted of Ca²⁺. [Ca²⁺]_c changes as before in B are represented by colour: (v-vii; note the cell position was moved by the solution exchange, A middle panel). When the Ca²⁺-containing electrode (`Ca²⁺ electrode') was subsequently sealed onto the cell (A, right-hand panel; C, blue bar) the local [Ca²⁺]_c, at the site of the Ca²⁺ electrode attachment, increased, as indicated by the colour changes as before (B, right-hand panels, ix-xii), presumably as a consequence of store-operated Ca²⁺ entry. [Ca²⁺]_c was at basal levels by 30 μm from the patch pipette, the photolysis site was 77 μm from the pipette. [Ca²⁺]_c at the photolysis site remained low (B,C). The Ca²⁺ increase to Ins(1,4,5)P₃ at the photolysis region (A) was subsequently increased towards that of the control (C). The position of the region of measurement is shown as a white line in B (i,v,ix), left-hand corner. Measurements were made from a 1-pixel line; the line is drawn at a 2-pixel width to facilitate its visualization.

The mechanisms underlying `quantal' release themselves were then explored. Ins(1,4,5)P₃-mediated Ca²⁺ release might be regulated by the [Ca²⁺] within the lumen of the SR and it may be that this release declines and stops as the SR Ca²⁺ content falls (see Introduction and references cited). To examine whether the Ins(1,4,5)P₃-sensitive store stops responding to Ins(1,4,5)P₃ before the SR is depleted of Ca²⁺, the store was `depleted' in a Ca²⁺-free solution by repetitively applying Ins(1,4,5)P₃ at a concentration that produced a submaximal response. Depletion was evidenced by the reduced peak amplitude of the [Ca²⁺]_c transient (Fig. 4). After the SR had been apparently `depleted' in this way, a concentration of Ins(1,4,5)P₃ that produced a maximal response was applied – evoking a substantial release of Ca²⁺ (Fig. 4). In these experiments, the maximum increase in [Ca²⁺]_c evoked by Ins(1,4,5)P₃ was 1.32±0.24 (ΔF/F₀; n=7). The photolysis lamp energy was reduced to produce a submaximal increase (0.81±0.19 ΔF/F₀) 61±5.3% of the maximum. In a Ca²⁺-free solution, repetitive submaximal [Ins(1,4,5)P₃] depleted the SR, and the magnitude of the [Ca²⁺]_c increase to the reduced lamp energy declined (0.02±0.1 ΔF/F₀) to 3.0±1.2% of its control submaximal value (n=7). Once `depleted' in this way, the lamp energy was returned to that producing a maximum response, and a [Ca²⁺]_c increase occurred (0.48±0.13 ΔF/F₀) that was 35±6.3% of the control (n=7; Fig. 4). This result demonstrated that the Ins(1,4,5)P₃-sensitive store contained significant residual quantities of Ca²⁺ even when apparently `depleted'. A lack of available SR Ca²⁺ cannot therefore account for the inability of submaximal [Ins(1,4,5)P₃] to evoke Ca²⁺ release.

Moreover, the result suggests that Ca²⁺ release could be regulated by the [Ca²⁺] within the lumen of the SR. A decline in lumenal Ca²⁺ might act on Ins(1,4,5)P₃Rs to alter the sensitivity of the Ins(1,4,5)P₃R to Ins(1,4,5)P₃. Any decrease in lumenal [Ca²⁺] would be accompanied by a reduced driving force for Ca²⁺ release because of the decreased concentration gradient of the ion. The reduction in driving force would be apparent as a decrease in the velocity of release. If, furthermore, lumenal regulation limits and terminates release, then the velocity of Ca²⁺ release should slow during the release process with the fall in SR [Ca²⁺]. The kinetics of the Ins(1,4,5)P₃-mediated Ca²⁺ release process were therefore examined to determine whether they could explain the mechanisms of lumenal regulation of Ca²⁺ release. Contrary to expectations, in response to photolysed caged Ins(1,4,5)P₃, the rate of evoked Ca²⁺ release did not decrease initially but rather increased (accelerated) during the release process (Fig. 5). The acceleration suggested that positive feedback was occurring during the release process itself.

Fig. 4.

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Submaximal [Ins(1,4,5)P₃] does not deplete the SR store of Ca²⁺. At –70 mV, high [Ins(1,4,5)P₃] producing maximal responses (pink area) and lower [Ins(1,4,5)P₃] producing submaximal responses (blue area; produced by lowering the flash-lamp energy), each evoked approximately reproducible increases in [Ca²⁺]_c. In a Ca²⁺-free solution (containing 1 mM EGTA and 3 mM MgCl₂; for the duration of the filled bar), the submaximal [Ca²⁺]_c increases declined, then disappeared. The absence of a response to [Ins(1,4,5)P₃] was not due to depletion of the store. Increasing Ins(1,4,5)P₃ (pink; right side) evoked further Ca²⁺ release. Another mechanism, other than depletion of the store of Ca²⁺, for example `lumenal' regulation of Ins(1,4,5)P₃R, might have accounted for the loss of response to Ins(1,4,5)P₃ (see main text). The time between each Ins(1,4,5)P₃ challenge was ∼1 minute, except after the introduction of the Ca²⁺-free solution, which took ∼3 minutes to equilibrate.

Interestingly, the extent of acceleration of release decreased with declining store content, as evidenced by the slowing of the rate of rise of a series of Ins(1,4,5)P₃-evoked Ca²⁺ transients in a single cell, each evoked during a decrease in SR content (Fig. 5). Thus as the SR content fell, peak velocity (d[Ca²⁺]_c/dt) also decreased from 3.4±1.5 to 0.15±0.08 (μM per second; n=3; P<0.05). This decline was not a consequence of nonlinear Ca²⁺ buffering – that is, an increased Ca²⁺ buffer capacity at lower compared with higher [Ca²⁺]_c. Nor was it the result of a function of the [Ca²⁺]_c over which the velocities were determined; differences in the velocities of Ca²⁺ release were maintained when examined as functions of [Ca²⁺]_c rather than of time (Fig. 5E). Indeed, acceleration of release might be an important determinant of the peak [Ca²⁺]_c achieved by Ins(1,4,5)P₃-mediated Ca²⁺ release (Fig. 5F). Here, the velocity of release and the peak [Ca²⁺]_c achieved were plotted for four transients obtained at different SR [Ca²⁺] (from the protocol in Fig. 5A). The calibrated signals from the four transients each from three cells are shown (Fig. 5F); the relationship between the maximum velocity of release and the peak [Ca²⁺]_c achieved was approximately linear (Fig. 5F).

Interestingly, as the SR content declined, the time required for the peak rise in [Ca²⁺]_c to occur was significantly delayed (Fig. 5G). The latter result suggests that Ins(1,4,5)P₃-induced inactivation of Ins(1,4,5)P₃R did not terminate Ca²⁺ release. Had Ins(1,4,5)P₃-induced inactivation of Ins(1,4,5)P₃R terminated Ca²⁺ release, then the peak [Ca²⁺]_c rise would be expected to occur at approximately the same time regardless of transient amplitude.

The question arises as to the basis of the positive-feedback processes itself. A decreased lumenal SR Ca²⁺ content, an increased [Ca²⁺]_c or cooperative channel opening might, singly or collectively, explain this process. If an increased [Ca²⁺]_c is responsible, the positive feedback will be attenuated when these increases are limited, for example by enhanced cytoplasmic Ca²⁺ buffering. On the other hand, if a decline in lumenal [Ca²⁺] underlies the acceleration of release, positive feedback should be unaltered when cytoplasmic Ca²⁺ buffering is increased. To investigate these possibilities, the cytoplasmic Ca²⁺ buffer capacity was increased by the Ca²⁺ chelator BAPTA in its membrane-permeable (AM) form (Fig. 6). In these experiments, as controls, a plasma membrane depolarization, followed by a [Ins(1,4,5)P₃] that produced a maximal increase and then one that gave a submaximal [Ca²⁺]_c increase, were each applied to the cell. Next, BAPTA-AM (25-50 μM) was introduced into the bathing fluid (∼7 minutes were allowed for its hydrolysis), and the same depolarization and Ins(1,4,5)P₃ challenges repeated (Fig. 6). In BAPTA-AM, the rate of [Ca²⁺]_c increase to each challenge was reduced compared with that of controls, as expected from the increased buffering power of the cytoplasm produced by the chelator (Fig. 6). An examination of the relationship between the expected [Ca²⁺]_c increase calculated from the time integrated I_Ca (see Materials and Methods) and the measured rise in [Ca²⁺]_c derived from the fluorescence measurements confirmed the increased buffering conferred by BAPTA-AM. As the depolarization-evoked [Ca²⁺]_c rise in colonic myocytes is derived exclusively from influx through voltage-dependent Ca²⁺ channels (I_Ca) with no Ca²⁺ release through CICR being involved (Bradley et al., 2004; Bradley et al., 2002), it was possible to analyze and compare the relationship between the [Ca²⁺]_c (from the fluorescence measurements) for a given Ca²⁺ influx (calculated from I_Ca) in the presence and absence of BAPTA-AM (Fig. 6D). As to whether or not the reduced Ins(1,4,5)P₃ response derived exclusively from the buffering capacity of BAPTA-AM or from other sources, such as a decline in Ins(1,4,5)P₃R activity as a result of diminished Ca²⁺-dependent feedback, Ins(1,4,5)P₃-evoked Ca²⁺ transients were then examined when the buffering provided by BAPTA was taken into account. To do this and determine the approximate contribution of buffering to the reduction of the Ins(1,4,5)P₃ response, depolarization-evoked [Ca²⁺]_c increases were examined. A scaling factor was derived from the ratio of the amplitudes of the attenuated depolarization-evoked Ca²⁺ transients in the presence and absence of BAPTA. This scaling factor was used to compensate for the contribution of the chelator to the Ca²⁺ buffering activity of the cell in assessing the amount of [Ca²⁺]_c rise evoked by depolarization. The scaling factor was, in effect, an estimation of the contribution of the chelator to the increased buffer capacity produced by BAPTA-AM, and its use allowed a comparison of the amplitude of the depolarization-evoked [Ca²⁺]_c rises independently of the buffer capacity of the cell to be made. The scaling factor was now applied to the reduced Ins(1,4,5)P₃ responses obtained in the presence of BAPTA-AM. As Fig. 6E,F shows, the maximal and submaximal Ins(1,4,5)P₃-evoked [Ca²⁺]_c transients were significantly reduced (to 14±10% and 12±8%, respectively; n=4; P<0.05) even when scaled (mean scaling factor 4.9±1.2) to take account of the contribution of the increased buffering capacity. In other words, buffering alone could not explain the reduction in the Ins(1,4,5)P₃ response. BAPTA-AM also reduced significantly the increase in the velocity of the rise in [Ca²⁺]_c compared with that of controls (3.2±0.2 μM per second in control, 0.039±0.006 μM per second in BAPTA; n=4; P<0.05) – that is, the rate of Ca²⁺ release was constant rather than increased (Fig. 6F) during the release process in the presence of the chelator. As an increased cytoplasmic buffer capacity prevented the acceleration of Ca²⁺ release, the positive feedback occurring during release is, it is proposed, cytoplasmic in origin and essential for achieving the peak rise in [Ca²⁺]_c produced by Ins(1,4,5)P₃. Thus, Ca²⁺ released from open Ins(1,4,5)P₃ channels might promote further Ca²⁺ release at nearby Ins(1,4,5)P₃Rs in a CICR-like process. Depletion of the store by BAPTA in these experiments is unlikely to explain the reduction in Ca²⁺ release as the measurements are steady state and as the basal [Ca²⁺]_c was approximately similar in both control (130±16 nM) and BAPTA-AM treated cells (113±17 nM; n=4).

Fig. 5.

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Ins(1,4,5)P₃-sensitive Ca²⁺ release at different SR Ca²⁺ contents. At –70 mV, photolysed Ins(1,4,5)P₃ increased [Ca²⁺]_c (↑, A). A second photolysis of Ins(1,4,5)P₃ ∼90 seconds later at the same site generated an approximately comparable [Ca²⁺]_c increase (A). In a Ca²⁺-free bath solution (containing 1 mM EGTA and 3 mM MgCl₂), this [Ca²⁺]_c increase declined in amplitude and rate of rise as the store was depleted of Ca²⁺ (A). The velocity of release increased during the release process, as revealed by the increasing steepness of the slope during release (B,C), and acceleration increased (D). C and D are the first and second derivatives, respectively, of the upstroke of the transients numbered 1-4 in A. As the increase in velocity is also evident when the first derivative of the upstroke is plotted against [Ca²⁺]_c (E) rather than time (C), nonlinear Ca²⁺ buffering does not provide an explanation for these results. Had it done so, the velocities derived from each transient would have been similar when examined as a function of [Ca²⁺]_c. The numbered traces in B-E correspond to the Ca²⁺ transients numbered in A. The amplitudes of the transient (B) have been scaled and normalized to facilitate comparison. One explanation for these results is that Ins(1,4,5)P₃-mediated Ca²⁺ release is itself facilitated by Ca²⁺ released via the channel in a positive-feedback process. As lumenal [Ca²⁺] declines (in the Ca²⁺-free solution) and with it Ca²⁺ release, so does the extent of the Ca²⁺-dependent positive feedback. (F) The peak velocity of release (a measure of the extent of positive feedback) (C) determines the peak [Ca²⁺]_c achieved after Ins(1,4,5)P₃-mediated Ca²⁺ release. Here, the peak velocity is plotted against the peak [Ca²⁺]_c obtained from the same cell. In this figure, the results from three separate cells, each indicated by the different-coloured symbols, are shown and the [Ca²⁺]_c was calibrated as described in Materials and Methods. (G) Ins(1,4,5)P₃-evoked Ca²⁺ release from the transients numbered 1-4 in A that have been scaled to facilitate comparison of their time-course. As the peak [Ca²⁺]_c achieved decreases (see A), the time required to reach their peak increased (G). This result would suggest that Ins(1,4,5)P₃-mediated inactivation of Ins(1,4,5)P₃R is unlikely to explain the termination of release as the [Ins(1,4,5)P₃] is similar in each case.

To determine whether or not positive feedback and acceleration play a role in `quantal' release, the effect of an increased cytoplasmic Ca²⁺ buffering (using BAPTA-AM) on the release process was examined. As a first step, CCh at 50 μM, then at 500 μM, were each applied on two consecutive occasions to the same cell. When this sequence was repeated, the second application of each concentration of CCh evoked percentage Δ[Ca²⁺]_c increases that were not significantly different (P>0.05) from those produced by the first sequence [112±14% (50 μM) and 112±6% (500 μM; n=3)].

As Fig. 7 shows, depolarization (–70 mV to +10 mV), followed by CCh (50 then 500 μM) each increased [Ca²⁺]_c (Δ[Ca²⁺]_c 312±69 nM, 112±56 nM and 297±97 nM, respectively; n=5). A second similar depolarization after the CCh sequence, in the same cell, evoked a rise in [Ca²⁺]_c comparable to the first. After BAPTA-AM (25-50 μM), the [Ca²⁺]_c rises evoked by depolarization and by CCh (50 then 500 μM) were each reduced to 75±20 nM, 17±6 nM and 20±8 nM (n=5), respectively, of controls. This result was anticipated from the rise in cytoplasmic Ca²⁺ buffering provided by BAPTA. To determine whether the reduced CCh response derived exclusively from the increased buffering capacity provided by BAPTA-AM, the reduced CCh responses and the depolarization-evoked [Ca²⁺]_c increases were compared (as above). A scaling factor for the attenuated depolarization-evoked Ca²⁺ transients in BAPTA-AM (compared with control) was derived as described previously and applied to compensate for the contribution of the chelator to the Ca²⁺ buffering activity of the cell in assessing the amount of [Ca²⁺]_c rise evoked by depolarization. The scaling factor again represented the contribution of the chelator to the increased buffer capacity produced by BAPTA-AM. The scaling factor was also applied to the reduced CCh responses in the presence of BAPTA-AM. The responses to the CCh sequence were reduced significantly, even when the scaling factor was taken into account (Fig. 7F; 112±56 nM and 297±97 nM in control and 83±41 nM and 96±32 nM in BAPTA after scaling). Furthermore, the ratio of the first to the second [Ca²⁺]_c rises evoked by CCh (500 μM versus 50 μM) declined from 6.3±2.4 in control to 1.3±0.3 in BATPA-AM. These results suggest that the positive feedback occurring during release is important in enabling `quantal' release to proceed. The latter results are unlikely to be explained by the nonlinear nature of the buffering by BAPTA as the buffer power provided by the chelator decreases with increased [Ca²⁺]_c. For example, over the range of 100-150 nM, the buffer power (total Ca²⁺/free [Ca²⁺]) of BAPTA is 147, whereas, over the range 250-300 nM, the buffer power decreases to 80. Thus, while the [Ca²⁺]_c rises evoked by CCh were attenuated to a greater extent at higher [Ca²⁺]_c by BAPTA, the buffer power of the chelator was reduced at these [Ca²⁺]_c.

Fig. 6.

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BAPTA (AM form) prevents the acceleration of Ins(1,4,5)P₃-mediated Ca²⁺ release and limits the rise in [Ca²⁺]_c. Depolarization (–70 to +10 mV, 3 seconds) (B) activated I_Ca (C) and increased [Ca²⁺]_c (A). At –70 mV (B), local photolysis of Ins(1,4,5)P₃ [↑; concentrations producing maximum (red) or submaximum (blue) responses] increased [Ca²⁺]_c (A). Prior (7 minutes) introduction of BAPTA AM (50 μM) to the bathing solution reduced the Ins(1,4,5)P₃-evoked [Ca²⁺]_c rise (A, unfilled bar) owing to increased cytoplasmic Ca²⁺ buffering, as revealed by the reduced [Ca²⁺]_c rise for a similar Ca²⁺ influx (A,C). Note the smaller rise in measured [Ca²⁺]_c (from the fluorescence measurements) for a given calculated [Ca²⁺]_c increase (from the Ca²⁺ current) in BAPTA (D) compared with that of controls. Scaling the [Ca²⁺]_c transients obtained in BAPTA so that the depolarization-evoked transients in the presence and absence of the chelator are of comparable size allowed a compensation for the increased buffer capacity of the cell to be made (E; left-hand panel). Application of the same scaling factor to the Ins(1,4,5)P₃-evoked [Ca²⁺]_c increases revealed that the Ins(1,4,5)P₃-evoked [Ca²⁺]_c transients were substantially reduced in the presence of the chelator (E; middle and right panels). Significantly, when the cytoplasmic Ca²⁺ buffer capacity had been increased (with BAPTA), the increase in velocity of the [Ca²⁺]_c rise (d[Ca²⁺]_c/dt) seen in control was substantially reduced (F). Thus the rate of Ca²⁺ release was largely constant rather than increased (F) during the release process in the presence of the chelator – that is, the velocity increase arose as a result of a Ca²⁺-dependent positive feedback acting at the cytoplasmic aspect of the Ins(1,4,5)P₃R. BAPTA in its Ca²⁺-free form (BAPTA_Cafree) might itself directly inhibit Ins(1,4,5)P₃R (Richardson and Taylor, 1993). However, high concentrations of BAPTA are required – for example, increasing BAPTA_Cafree from 90 μM to 9 mM reduced Ins(1,4,5)P₃-mediated Ca²⁺ release by 8% (Bootman et al., 1995).

Discussion

Our results suggest that `quantal' Ca²⁺ release is a consequence of lumenal regulation of Ins(1,4,5)P₃-mediated Ca²⁺ release at Ins(1,4,5)P₃Rs. This regulation takes place at the cytoplasmic aspect of the receptor through a positive-feedback process, itself dependent on the SR Ca²⁺ content. Thus, Ca²⁺ released through Ins(1,4,5)P₃Rs then evokes a further release of the ion through a CICR-like process. As the SR Ca²⁺ content falls, so does the extent of positive feedback CICR and release stops. At low [Ins(1,4,5)P₃], when the SR [Ca²⁺] is high, the unitary Ins(1,4,5)P₃R currents are large. Under these conditions, few Ins(1,4,5)P₃Rs within an Ins(1,4,5)P₃R cluster are activated by the low [Ins(1,4,5)P₃], but the large Ins(1,4,5)P₃R Ca²⁺ current generates a positive feedback to evoke a more substantial Ca²⁺ release that involves several neighbouring Ins(1,4,5)P₃Rs. As Ca²⁺ release proceeds, the local SR-lumenal [Ca²⁺] near the channel cluster declines and with it the positive feedback. Release wanes then ceases, even in the continued presence of Ins(1,4,5)P₃. In these circumstances, Ca²⁺ release arising from activation of a single Ins(1,4,5)P₃R is insufficient to promote release from neighbouring Ins(1,4,5)P₃Rs within the cluster because of the inter-receptor distance (Fig. 8). Although Ins(1,4,5)P₃Rs continue to be activated, the small unitary Ca²⁺ currents so generated fail to promote positive feedback. With increasing [Ins(1,4,5)P₃], the probability of an increased number of receptors that are closely apposed to each other being activated almost simultaneously is enhanced. The resulting summated release has an extended radius of influence and activates additional Ins(1,4,5)P₃Rs to renew release. With continued release, the local [Ca²⁺] within the SR declines, and with it the unitary Ins(1,4,5)P₃R Ca²⁺ currents and the cycle continues as described above. The slow diffusion of lumenal Ca²⁺ (because of the higher stationary buffer capacity) compared with that in the cytoplasm means that each Ins(1,4,5)P₃R cluster might function independently. Nevertheless, the maintained stochastic activation of Ins(1,4,5)P₃Rs throughout the SR, by Ins(1,4,5)P₃, will result in the average SR [Ca²⁺] eventually falling to a level that terminates release of the ion within a cluster, so rendering the cell unable to generate a physiological response.

Fig. 7.

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BAPTA in the membrane-permeable (AM) form prevents quantal Ca²⁺ release. Depolarization (–70 to +10 mV, 3 seconds) (C) activated I_Ca (D) and increased [Ca²⁺]_c (A). At –70 mV (C), CCh (50 μM; B) produced a small, and CCh (500 μM) a substantial, [Ca²⁺]_c increase (A). Approximately 7 minutes after the introduction of BAPTA AM (25 μM; A, unfilled bar) to the bathing solution, the depolarization-evoked [Ca²⁺]_c rise was significantly reduced owing to increased cytoplasmic buffering, as revealed by the reduced [Ca²⁺]_c rise for a similar Ca²⁺ influx (A,D), as was the CCh-evoked [Ca²⁺]_c rise (A,B). Scaling up the [Ca²⁺]_c transients obtained in BAPTA so that the depolarization-evoked transients in the presence and absence of the chelator are of comparable size allowed a compensation for the increased buffer capacity of the cell to be made (E). Application of the same scaling factor to the CCh-evoked [Ca²⁺]_c increases allowed comparison of the CCh-evoked [Ca²⁺]_c transients in the presence of the chelator (G-I). When the cytoplasmic Ca²⁺ buffer capacity had been increased (with BAPTA), the lower concentration of CCh was affected to a smaller extent than the higher CCh concentration (inset shown on an expanded scale; note the colour coding). The change in noise in G (red trace) during CCh (500 μM) occurred because of a decrease in data sampling rate (from 10 Hz to 1 Hz).

Our proposal for `quantal' Ca²⁺ release assumes that Ins(1,4,5)P₃Rs fall under the control of lumenal Ca²⁺. Evidence that this might be so is the observation that Ca²⁺ release evoked by low [Ins(1,4,5)P₃] in a Ca²⁺-free medium ceases as the SR Ca²⁺ content declines even when this store retains a significant residual quantity of Ca²⁺. However, the sensor for regulation of Ins(1,4,5)P₃R by lumenal Ca²⁺, it is proposed, resides at the cytoplasmic rather than the lumenal aspect of the channel and involves a Ca²⁺-dependent CICR-like positive-feedback process. That conclusion is derived from an examination of the rising phase of Ca²⁺ release transients and the effect of Ca²⁺ buffers on them. Ca²⁺ release followed a sigmoidal time course – initially slow, then accelerated, followed by slowing. During Ca²⁺ release, as the SR [Ca²⁺] declined, the driving force on Ca²⁺ (and so the single Ins(1,4,5)P₃ channel Ca²⁺ current) declined. Viewed as the amount of Ca²⁺ released (or as an increase in [Ca²⁺]_c), the declining unitary current should have expressed itself as a slowing of the rate of [Ca²⁺]_c rise with time. Since the rate of release increased (rather than decreased) as release proceeded, it seems likely that the number of open channels increased during the release process, i.e. a positive-feedback process might be operative during Ca²⁺ release in which release of the ion apparently activates further Ca²⁺ release. In support, limiting the [Ca²⁺]_c rise, by use of the chelator BAPTA, attenuated the rate and amount of Ins(1,4,5)P₃-mediated Ca²⁺ release. Thus, Ca²⁺ released from Ins(1,4,5)P₃Rs might activate neighbouring receptors to evoke additional release. In colonic myocytes, the RyR makes no contribution to the [Ca²⁺]_c increase evoked by Ins(1,4,5)P₃ (Flynn et al., 2001; MacMillan et al., 2005; McCarron et al., 2002; McCarron et al., 2004): CICR at RyRs does not contribute to the positive-feedback process.

Fig. 8.

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Smooth muscle quantal Ca²⁺ release. Ins(1,4,5)P₃-evoked Ca²⁺ release is exquisitely sensitive to the SR Ca²⁺ content and the development of positive feedback. With low agonist [Ins(1,4,5)P₃] and a replete SR (top left-hand panel), Ins(1,4,5)P₃-mediated Ca²⁺ release from an activated (purple) Ins(1,4,5)P₃R (IP₃R) induces a release of Ca²⁺ from the SR, resulting in a reduction in SR lumenal [Ca²⁺] (blue low, red high). This release produces a large rise in [Ca²⁺]_c (red colour) that overlaps neighbouring quiescent (yellow) Ins(1,4,5)P₃Rs. The rise in [Ca²⁺]_c then stimulates adjacent Ins(1,4,5)P₃Rs and a CICR-like process there (top right-hand panel). During release, the SR Ca²⁺ content declines and with it the unitary Ins(1,4,5)P₃R Ca²⁺ current. As a result, the local [Ca²⁺] rise at the cytoplasmic aspect of Ins(1,4,5)P₃Rs is reduced (top right-hand panel). The extent of activation of neighbouring Ins(1,4,5)P₃Rs through CICR declines and eventually ceases – even in the continued presence of the agonist. With increasing concentrations of agonist (lower panel), the probability of coincidental activation of two or more neighbouring Ins(1,4,5)P₃Rs increases. While activation of single receptors cannot generate substantial CICR, the local [Ca²⁺] near neighbouring Ins(1,4,5)P₃Rs as a result of two receptors being activated is again sufficient to generate CICR and the positive feedback results in significant Ca²⁺ release. Thus, the extent of Ca²⁺ release is determined by the positive-feedback facility. The declining unitary currents, as a result of Ca²⁺ release, are offset by an increased number of Ins(1,4,5)P₃Rs being activated simultaneously to generate CICR and renew the release process.

For lumenal regulation to be expressed at the cytoplasmic aspect of the channel, the local change in SR-lumenal [Ca²⁺] during release must alter the unitary Ins(1,4,5)P₃R current. The unitary current amplitude over the physiological range of lumenal [Ca²⁺] is unclear. A reliable estimate at various lumenal [Ca²⁺] could not be obtained when [Ca²⁺], used as the charge carrier, fell below 20 mM because of the brief open times of the channels and small current amplitudes (Bezprozvanny and Ehrlich, 1994). Notwithstanding, extrapolating from results obtained at various lumenal [Ba²⁺], the unitary Ins(1,4,5)P₃R current varies approximately linearly with physiological changes in lumenal [Ca²⁺] (Bezprozvanny and Ehrlich, 1994). Indeed, as determined from computer simulations (Thul and Falcke, 2004), sufficient local lumenal [Ca²⁺] depletion might occur on the opening of a single Ins(1,4,5)P₃R to alter the unitary Ins(1,4,5)P₃R current. During current flow via a single Ins(1,4,5)P₃R, a local decline in lumenal [Ca²⁺] extended to alter the Ca²⁺ available for release at neighbouring Ins(1,4,5)P₃Rs in the cluster (Thul and Falcke, 2004). The total Ca²⁺ current of an Ins(1,4,5)P₃R cluster was approximately proportional to the square root of the number of open channels in a cluster rather than the sum of the single channel currents (Thul and Falcke, 2004). Thus, as each Ins(1,4,5)P₃R opens, local lumenal [Ca²⁺] declines and with it unitary Ins(1,4,5)P₃R currents. The extent of positive feedback arising from Ca²⁺ release through Ins(1,4,5)P₃Rs will decrease as release progresses, and CICR generated by Ca²⁺ release might so provide a lumenal Ca²⁺ sensor.

The binding of multiple Ins(1,4,5)P₃ molecules to Ins(1,4,5)P₃R sites (Meyer et al., 1990) could also underlie the sigmoidal rise in [Ca²⁺]_c during Ins(1,4,5)P₃ release (Meyer et al., 1990). However, the inhibition of the sigmoidal relationship by increased Ca²⁺ buffering in the cytoplasm, in the present study, suggests that cooperativity in Ins(1,4,5)P₃ binding is an unlikely explanation for this relationship. Thus, our results agree with previous studies that show little or no cooperativity in Ins(1,4,5)P₃ binding or Ca²⁺ release when [Ca²⁺]_c was prevented from changing (Finch et al., 1991; Iino and Endo, 1992; Watras et al., 1991). The sigmoidal release characteristics might arise as a consequence of the stimulatory effects of Ca²⁺ released through Ins(1,4,5)P₃R (Marshall and Taylor, 1993), rather than co-operative Ins(1,4,5)P₃, binding (Meyer et al., 1990).

Unique structural features in the SR have also been proposed to explain `quantal' Ca²⁺ release (Hirose and Iino, 1994; Muallem et al., 1989; Parker and Ivorra, 1990; Shin et al., 2001). For example, cells might contain a series of Ca²⁺ stores, each with different sensitivities to Ins(1,4,5)P₃ such that, at any given concentration of the inositide, only some stores will be activated to release Ca²⁺ (Hirose and Iino, 1994; Muallem et al., 1989; Parker and Ivorra, 1990). Yet, structural aspects per se are unlikely to explain `quantal' release in the present study. The entire store was lumenally continuous and Ca²⁺ could diffuse freely throughout (see also Mogami et al., 1997; Park et al., 2000; Verkhratsky, 2005). Thus, the entire SR could be depleted and refilled from one small region of the store, suggesting that a lumenally continuous SR structure existed. Clearly, distinctions of structure in the SR, if they exist, do not appear to affect release of SR Ca²⁺.

The conclusion that the SR is a lumenally continuous entity was derived in part by photolysis of caged Ins(1,4,5)P₃ at one site in the cell and determining the extent of SR depletion at a distant site. While the diffusion of some Ins(1,4,5)P₃ to the end of the cell distant from the site of photolysis cannot be excluded completely, it is unlikely to explain the present findings. A large concentration of Ins(1,4,5)P₃ is released at the photolysis site. This concentration would decrease substantially with diffusion from the release site and by metabolism of the inositide. Indeed, after photolysis at one site, only a small rise in [Ca²⁺]_c occurred at the second site. In these circumstances, it seems unlikely that any small Ins(1,4,5)P₃ concentration that reached the second photolysis site could evoke a depletion of the SR that was comparable to that which occurred at photolysis site 1, where Ins(1,4,5)P₃ was released. If multiple stores existed, photolysis site 2 would have retained a substantial Ca²⁺ reserve (because of the lower concentration of Ins(1,4,5)P₃ reaching the site from photolysis site 1) and a significant release of Ca²⁺ would have been expected when Ins(1,4,5)P₃ is subsequently released there.

Ca²⁺- or Ins(1,4,5)P₃-dependent inactivation of Ins(1,4,5)P₃Rs is also unlikely to contribute to the termination of Ins(1,4,5)P₃-mediated Ca²⁺ release. If Ca²⁺-dependent inactivation terminated release (McCarron et al., 2004; Oancea and Meyer, 1996) and contributed to the `quantal' release process, the Ca²⁺ chelator, BAPTA, would have been expected to have potentiated the Ins(1,4,5)P₃-evoked [Ca²⁺]_c increase. In the event, BAPTA decreased this value. If Ins(1,4,5)P₃ inactivated Ins(1,4,5)P₃Rs to prevent release, then, at constant [Ins(1,4,5)P₃], release should have stopped at approximately the same time regardless of the amplitude of the [Ca²⁺]_c rise. However, as the amplitude of the [Ca²⁺]_c rise declined (in either BAPTA or in Ca²⁺-free solution), the time-course of release became more prolonged. Mechanisms other than Ins(1,4,5)P₃-induced inactivation would appear to be responsible for terminating Ins(1,4,5)P₃-mediated Ca²⁺ release (see also Hirose and Iino, 1994; Meyer and Stryer, 1990; Tanimura and Turner, 1996).

Studies of `quantal' Ca²⁺ release in permeabilized cells (e.g. Missiaen et al., 1994) generally include EGTA or BAPTA in the bathing medium. Their inclusion was necessary to control [Ca²⁺] but will limit the extent of positive feedback at Ins(1,4,5)P₃Rs. In these experiments, `quantal' release persisted in the presence of the chelators. BAPTA and EGTA might be unable to control completely [Ca²⁺] near an Ins(1,4,5)P₃R cluster and might only attenuate the process (present results). Very high concentrations (100 mM BAPTA) are required to abolish positive feedback (Horne and Meyer, 1995). Alternatively, it could be that, when one aspect (e.g. positive feedback) of `quantal' release is attenuated, additional compensatory components may dominate, or different mechanisms (e.g. regulation of Ins(1,4,5)P₃Rs by Ca²⁺ acting at the lumenal aspect of the channel) might explain `quantal' release in various cell types. That is, more than one mechanism may be involved in `quantal' Ca²⁺ release. Ca²⁺ release via Ins(1,4,5)P₃Rs is regulated by intralumenal modulators. Chromagranin, a Ca²⁺ storage protein, binds to the lumenal aspect of Ins(1,4,5)P₃R to increase channel activity (Thrower et al., 2003). ERp44, a binding protein that belongs to the thioredoxin family, interacts with the lumenal aspect of Ins(1,4,5)P₃Rs to inhibit channel activity (Higo et al., 2005). ERp44 binding to Ins(1,4,5)P₃Rs is itself regulated by lumenal [Ca²⁺] as well as the redox state and pH (Higo et al., 2005). Changes in the lumenal environment, such as [Ca²⁺], might be transmitted to Ins(1,4,5)P₃Rs via ERp44. Lumenal modulators may enable tuning of the control of Ins(1,4,5)P₃Rs to occur and provide alternative mechanisms by which `quantal' Ca²⁺ release could occur in other cell types.

The present results from smooth muscle cells suggest that the SR is lumenally continuous throughout and that lumenal [Ca²⁺] regulates the amount of Ins(1,4,5)P₃R activity and Ca²⁺ release. This regulation is exerted at the cytoplasmic rather than at the lumenal aspect of the channel. A positive-feedback facilitation of Ca²⁺ release, which is itself dependent on SR lumenal [Ca²⁺], determines the peak [Ca²⁺]_c achieved following Ins(1,4,5)P₃-mediated Ca²⁺ release. As the SR content declines, so does the extent of positive feedback and peak [Ca²⁺]_c. Release is terminated when the positive-feedback loop is broken by the decline in the [Ca²⁺] located near to Ins(1,4,5)P₃Rs. Ins(1,4,5)P₃R clusters will continue to be activated randomly throughout the SR to release Ca²⁺ until the average SR [Ca²⁺] is unable to support release. Ca²⁺ release is renewed by an increased [Ins(1,4,5)P₃]. In this case, the coincidental activation of several neighbouring Ins(1,4,5)P₃Rs within a cluster offsets the declining unitary current to renew positive feedback and Ca²⁺ release and account for `quantal' Ca²⁺ release.

Materials and Methods

Cell isolation

Male guinea-pigs (500-700 g) were humanely killed by cervical dislocation followed by immediate exsanguination in accordance with the guidelines of the Animal (Scientific Procedures) Act UK 1986. A segment of intact distal colon (∼5 cm) was transferred to oxygenated (95% O₂, 5% CO₂) physiological saline solution comprising: 118.4 mM NaCl, 25 mM NaHCO₃, 4.7 mM KCl, 1.13 mM NaH₂PO₄, 1.3 mM MgCl₂, 2.7 mM CaCl₂ and 11 mM glucose (pH 7.4). Following removal of the mucosa from the tissue, single smooth muscle cells, largely from circular muscle, were enzymatically dissociated (McCarron and Muir, 1999). All experiments were carried out at room temperature (20±2°C).

Electrophysiology

Membrane currents were measured using conventional tight-seal whole-cell recording methods. The extracellular solution contained: 80 mM Na glutamate, 40 mM NaCl, 20 mM tetraethylammonium chloride (TEA), 1.1 mM MgCl₂, 3 mM CaCl₂, 10 mM HEPES and 30 mM glucose (pH 7.4 with NaOH). The pipette solution contained: 85 mM (Cs)₂SO₄, 20 mM CsCl, 1 mM MgCl₂, 30 mM HEPES, 3 mM MgATP, 2.5 mM pyruvic acid, 2.5 mM malic acid, 1 mM NaH₂PO₄, 5 mM creatine phosphate, 0.5 mM guanosine phosphate and 0.025 mM caged inositol trisphosphate [Ins(1,4,5)P₃] trisodium salt. Whole-cell currents were measured using an Axopatch 200B (Axon Instruments, Union City, CA), low-pass filtered at 500 Hz (8-pole bessel filter; Frequency Devices, Haverhill, MA), digitally sampled at 1.5 kHz using a Digidata interface and pClamp (version 8; Axon Instruments) and stored for analysis. In some experiments, a second patch clamp electrode with bath solution (containing 30 mM Ca²⁺) was used. The electrode was sealed `on cell' in a `giga-seal' configuration. Here, currents were measured using a second Axopatch 200B (Axon Instruments, Union City, CA) and low-pass filtered at 500 Hz. The membrane potential of the second `on cell' electrode was 0 mV so that the entire cell, including the on `cell patch', was maintained at the value set by the whole-cell electrode.

Imaging

Cells were loaded with fluo3 AM (10 μM) and wortmannin (10 μM; to prevent contraction) for at least 20 minutes before the start of the experiment. Two-dimensional [Ca²⁺]_c images were obtained using a wide-field digital imaging system. Single cells were illuminated at 488 nm (bandpass 14 nm) from a monochromator (Polychrome IV, T.I.L.L. Photonics, Martinsried, Germany) and imaged through an oil-immersion objective (×40 UV 1.3 NA; Nikon UK, Surrey, UK). Excitation light was passed via a fibre-optic guide through a 485 bandpass (15 nm) filter and a field-stop diaphragm and reflected off a 505 nm long-pass dichroic mirror. Emitted light was guided through a 535 nm barrier filter (bandpass 45 nm) to an intensified, cooled, frame transfer CCD camera (Pentamax Gen IV, Roper Scientific, Trenton, NJ) operating in `virtual chip' mode with program WinView32 (Roper Scientific, Trenton NJ). Full-frame images (150×150 pixels), with a pixel size of 563 nm at the cell, were acquired at 20 or 100 frames per second. In some experiments, to attain longer periods of data acquisition, the software programme Metafluor (Molecular Devices, Wokingham, UK) was used. Here, the sampling was ∼10 frames per second during, and slowed to 1 frame per second between, Ca²⁺ transients. Ca²⁺ imaging data were recorded on a personal computer. Electrophysiological measurements and imaging data were synchronized by recording, on pClamp, a transistor transistor logic (TTL) output from the CCD camera, which reported its readout status together with the electrophysiological information.

Localized flash photolysis

The output of a xenon flashlamp (Rapp Optoelecktronic, Hamburg, Germany), used to uncage Ins(1,4,5)P₃, was passed through a UG-5 filter to select ultraviolet light, focused and merged into the excitation light path through a fibre-optic bundle and long-pass dichroic mirror at the lens part of the epi-illumination attachment of the microscope. The diameter of the fibre optic together with the lens magnification determined the area (spot size ∼10 μm) of Ins(1,4,5)P₃ photolysis (McCarron et al., 2004).

Data analysis

Images were analyzed using the program Metamorph 5.1 (Molecular Devices, Wokingham, UK). Fluorescence signals were measured in regions of interest comprising circles of diameter 5 μm or lines of width 1 pixel across the cell, unless otherwise indicated, and were expressed as ratios (F/F₀ or ΔF/F₀) of fluorescence counts (F) relative to baseline (control) values (taken as 1) before stimulation (F₀). Original fluorescence recordings were not filtered, smoothed or averaged. Where fluo-3 fluorescence signals were converted into [Ca²⁺]_c (nM), the K_d for fluo-3 was 390 nM, R_min was assumed to be 0 and R_max was determined from in vitro calibrations (Cannell et al., 1994).

The amount of Ca²⁺ estimated from the Ca²⁺ transient and measured by fluo-3 is termed `measured' Ca²⁺, whereas that arising from I_Ca is termed the `calculated' increase in [Ca²⁺]_c. The latter was determined using the equation: – ∫I_Cadt/2FV where – ∫I_Cadt is total charge entry, F the Faraday constant and V the cell volume. I_Ca was integrated by measuring the area under the curve, with reference to the current level after inactivation of I_Ca with a 3 second depolarization or elimination of I_Ca with 100 μM cadmium chloride. The rate of the [Ca²⁺]_c increase (d[Ca²⁺]_c/dt) was obtained by averaging the slopes of two adjacent data points.

Summarized results are expressed as means±s.e.m. of n cells. A paired or unpaired Student's t test was applied to the raw data, as appropriate; P<0.05 was considered significant.

Drugs and chemicals

Drugs were applied either by hydrostatic pressure ejection or addition to the extracellular solution, as stated in the text. With pressure ejection, the concentration of the drug at the cell will be significantly lower than that in the pipette owing to dilution in the bathing solution. Submaximal Ins(1,4,5)P₃-evoked [Ca²⁺]_c increases were obtained by decreasing the energy of the photolysis lamp. Concentrations in the text refer to the salts, where appropriate. Fluo-3 AM ester was purchased from TefLabs (Teflabs, Austin, TX) and caged Ins(1,4,5)P₃-trisodium salt from SiChem GmbH (Bremen, Germany). BAPTA-AM (Cambridge Bioscience, Cambridge, UK) was introduced via the bathing solution and ∼7 minutes were allowed for hydrolysis. All other reagents were obtained from Sigma (Poole, UK).

Acknowledgements

This work was funded by the Wellcome Trust (078054/Z/05/Z) and British Heart Foundation (PG/06/016), the support of which is gratefully acknowledged.

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2008

`Quantal' Ca²⁺ release at the cytoplasmic aspect of the Ins(1,4,5)P₃R channel in smooth muscle

Introduction

Results

Discussion

Materials and Methods

Cell isolation

Electrophysiology

Imaging

Localized flash photolysis

Data analysis

Drugs and chemicals

Acknowledgements

References

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`Quantal' Ca2+ release at the cytoplasmic aspect of the Ins(1,4,5)P3R channel in smooth muscle

Introduction

Results

Discussion

Materials and Methods

Cell isolation

Electrophysiology

Imaging

Localized flash photolysis

Data analysis

Drugs and chemicals

Acknowledgements

References

Email alerts

Cited by

Call for papers: Cell Biology of Mitochondria

Focal adhesion kinase signalling – tumour vulnerabilities and clinical opportunities

JCS-FocalPlane Training Grants

Biologists @ 100 - join us in Liverpool in March 2025

Interview with Journal of Cell Science Editor Rob Parton

Social media

Other journals from The Company of Biologists

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`Quantal' Ca²⁺ release at the cytoplasmic aspect of the Ins(1,4,5)P₃R channel in smooth muscle

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The Company of Biologists