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First published online 11 December 2007
doi: 10.1242/jcs.017541

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

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, John Arbuthnott Building, 27 Taylor Street, Glasgow G4 0NR, UK

View larger version (25K): [in this window] [in a new window]   Fig. 1. 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.

View larger version (39K): [in this window] [in a new window]   Fig. 2. 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.

View larger version (44K): [in this window] [in a new window]   Fig. 3. 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.

View larger version (20K): [in this window] [in a new window]   Fig. 4. 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.

View larger version (42K): [in this window] [in a new window]   Fig. 5. 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.

View larger version (32K): [in this window] [in a new window]   Fig. 6. 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 ICa (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 (BAPTACafree) might itself directly inhibit Ins(1,4,5)P3R (Richardson and Taylor, 1993). However, high concentrations of BAPTA are required – for example, increasing BAPTACafree from 90 µM to 9 mM reduced Ins(1,4,5)P3-mediated Ca2+ release by 8% (Bootman et al., 1995).

View larger version (25K): [in this window] [in a new window]   Fig. 7. BAPTA in the membrane-permeable (AM) form prevents quantal Ca2+ release. Depolarization (–70 to +10 mV, 3 seconds) (C) activated ICa (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).

View larger version (69K): [in this window] [in a new window]   Fig. 8. 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.