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1 Department of Internal Medicine 2 Department of Molecular Physiology and Biophysics 3 Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-6300, USA
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Abstract |
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(Received 12 August 2003;
accepted after revision 27 October 2003;
first published online 31 October 2003)
Corresponding author M. E. Anderson: 383 Preston Research Building, Vanderbilt University Medical Center, Nashville, TN 37232-6300, USA. Email: mark.anderson{at}vanderbilt.edu
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Introduction |
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Ca2+i can increase as a direct consequence of ICa-L, or through secondary release of Ca2+ from the SR in heart. Some studies suggest that SR Ca2+ release is more important than ICa-L for activating CaMK, because CaMK-dependent phosphorylation of the SR regulatory protein phospholamban is reduced 50–90% when SR Ca2+ release is prevented by ryanodine (Kuschel et al. 1999; Bartel et al. 2000). On the other hand, Ca2+i from ICa-L is sufficient for engaging Ca2+–CaM-dependent ICa-L inactivation mechanisms in non-cardiac cells (Zuhlke et al. 1999; Peterson et al. 1999) where SR Ca2+ release is not anticipated to contribute to Ca2+i. Furthermore, it remains unknown whether Ca2+i for Ca2+–CaM-dependent ICa-L inactivation and for activating CaMK is primarily from ICa-L or the SR in cardiomyocytes during dynamic changes in cell membrane potential, as occur in the working heart.
We controlled CaMK activity with a Ca2+–CaM-independent form of CaMK and a CaMK inhibitory buffer (IB), previously shown to prevent ICa-L increases by endogenous CaMK (Wu et al. 2001a), and inhibited Ca2+–CaM with an inhibitory peptide (Wu et al. 2001b) or Ba2+ substitution to separately control CaMK and Ca2+–CaM activity in ventricular myocytes, in order to differentially test the effects of Ca2+i from ICa-L and SR for regulating ICa-L availability. ICa-L was measured in cardiomyocytes using a non-steady-state inactivation protocol that mimicked time and voltage conditions present during the cardiac action potential. Our findings support the novel concept that CaMK regulation of ICa-L in cardiomyocytes depends upon cell membrane potential. Both Ca2+i from ICa-L and the SR can recruit Ca2+–CaM for ICa-L inactivation, but SR Ca2+ release is required for CaMK effects, while SR Ca2+ release also predominates for Ca2+–CaM-dependent inactivation at strong depolarizations. A new model of voltage- and Ca2+i-dependent ICa-L regulation is proposed.
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Methods |
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Whole-cell mode configuration was used for voltage clamping isolated rabbit ventricular myocytes according to previously published methods (Wu et al. 1999a). Ventricular myocytes were isolated from New Zealand White rabbits killed by pentobarbital (50 mg kg-1, I.V.) overdose prior to excising the heart. The Vanderbilt University Animal Care Committee approved all experiments. Cells were held at –80 mV for >5 min for adequate dialysis with pipette solution before initiating experiments. ICa-L was conditioned by stepping the cell membrane from –80 mV to +40 mV in 10 mV increments from 30 to 500 ms at 0.1 Hz, and peak ICa-L was measured at a test potential of +10 mV (Fig. 1) and expressed as relative current. In some experiments ICa-L inactivation was quantified as the fraction of residual inward current present at the end of 30 ms (R30) and 80 ms (R80) conditioning pulses. All experiments were performed at 24°C. Adding Cs+ and TEA and reducing Na+ and K+ in the pipette and bath solutions eliminated Na+ and K+ currents. A Ca2+-activated Cl- conductance (ICl,Ca), known to be activated by SR Ca2+ release in rabbit ventricular myocytes (Wu & Anderson, 2000) at cell membrane potentials more positive than +20 mV (Wu et al. 1999b), was most clearly seen as a transient outward current in response to voltage command steps to +30 and +40 mV. ICl,Ca was eliminated by niflumic acid (10–20 µM, data not shown) and thapsigargin (Fig. 6A), and was not present at the test command potential of +10 mV. Thus, ICl,Ca was not likely to have significantly contributed to ICa-L measurements because the relationship of relative current (see below) to conditioning potential was not affected by niflumic acid (data not shown), and the relationship between relative current and R30 and R80 was not altered during positive voltage commands in IB (see below, Fig. 5A and B) or IB and thapsigargin (Fig. 7A and B). However, we cannot rule out the possibility that ICl,Ca did contribute to CaMK and Ca2+–CaM effects, especially at voltage command steps to +30 and +40 mV (Wu et al. 1999b). Elimination of the residual current by nifedipine (10 µM) or Cd2+ (100 µM) confirmed that the identity of active inward current was ICa-L (data not shown). The control pipette (intracellular) solution was (mM): CsCl 120.0, EGTA 10.0, Hepes 10.0, tetraethylammonium chloride (TEA) 10.0, phosphocreatine 5.0, CaCl2 3.0, MgATP 1.0, NaGTP 1.0, and pH was adjusted to 7.2 with 1.0 N CsOH. The ability of endogenous CaMK to facilitate ICa-L was eliminated by addition of an inhibitory buffer (IB) solution (see below) (Wu et al. 1999a, 2001a). The bath (extracellular) solution was NMDG 137.0, CsCl 25.0, Hepes 10.0, glucose 10.0, CaCl2 (or BaCl2) 1.8, MgCl2 0.5, and pH was adjusted to 7.4 with 12 N HCl. Ryanodine (10 µM) or thapsigargin (1 µM) were added to the bath solution for some experiments. Myocyte contraction was eliminated under these conditions (data not shown).
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A recombinant monomeric truncation mutant of the mouse CaMK 
isoform (amino acid residues 1–380) was expressed using baculovirus and then purified using CaM agarose affinity chromatography (Brickey et al. 1990). This CaMK was stored in IB (50 mM Hepes, pH 7.5, 1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol, 10% (v/v) ethylene glycol) and activated by autophosphorylation in a 100 µl reaction containing 50 mM Hepes, pH 7.5, 2 mM magnesium acetate, 1.5 mM CaCl2, 18 µM CaM, 2 mM DTT and 100 µM ATP
S. The reaction was initiated by addition of the (1–380) CaMK (9 µmol l-1 final subunit concentration), incubated at 30°C for 10 min, and stopped by the addition of EDTA (10 mM). IB was used without added CaMK, or after inactivation of enzymatically active CaMK by heating, to observe CaMK-independent effects of manipulating Ca2+–CaM and SR Ca2+ release. IB prevents ICa-L facilitation by endogenous CaMK (Wu et al. 2001a) and was used to separate CaMK from Ca2+–CaM activity and SR Ca2+ release. Ca2+–CaM-dependent autophosphorylation of the CaMK produces a constitutively active species that can phosphorylate substrates in the absence of Ca2+–CaM. Ca2+-independent activity was typically 35–50% of total activity in the presence of Ca2+–CaM using the peptide substrates syntide-2 or autocamtide. Constitutively active CaMK and IB were diluted 10-fold in the pipette solution (0.9 µM final) for use in voltage clamp studies and its activity confirmed in vitro, as described (Wu et al. 1999a). This dilution was chosen to approximate the physiological CaM kinase activity in heart (
1–2 µM) derived from percentage yield calculations during purification (Iwasa et al. 1986; Gupta & Kranias, 1989).
The CaMK inhibitory peptide AC3-I (KKALHRQEAVDCL, IC503 µM) (Braun & Schulman, 1995) (Macromolecular Resources, Fort Collins, CO, USA) is a modified CaMK substrate, which inhibits endogenous and thiophosphorylated constitutively active CaMK. AC3-I was included in the pipette solution at a final concentration of 20 µM. The CaM binding peptide 290–309 (Calbiochem) is modelled on the CaM binding domain of CaMK, and inhibits Ca2+–CaM signalling generally, and was added to the pipette solution at a final concentration of 50 µM. CaMK, 290–309 and AC3-I were dialysed into cells for 5–10 min prior to initiating experiments.
Statistics
The null hypothesis was evaluated with Student's t test or ANOVA, as appropriate. Bonferroni's correction was applied for multiple comparisons.
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Results |
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Brief, positive conditioning prepulses progressively increased relative ICa-L availability (Fig. 1), while IB significantly reduced relative ICa-L availability (Fig. 2A–C), compared to control pipette solution, suggesting that endogenous CaMK can increase ICa-L under action potential plateau conditions. The increase in ICa-L in response to brief (30–130 ms), positive conditioning prepulses was lost at longer (500 ms) prepulse durations when Ca2+ was the charge carrier (Fig. 2D), but persisted when Ba2+ was substituted for Ca2+ (Fig. 2E), in cells dialysed with IB. The persistence of Ca2+-dependent reduction in ICa-L availability in IB indicates that Ca2+–CaM-dependent inactivation is operative under these experimental conditions. These findings show that Ca2+ critically determines ICa-L availability during time and voltage conditions present during the ventricular action potential plateau, and serve as a starting point for dissecting the role of Ca2+–CaM, CaMK and SR Ca2+ release in determining ICa-L availability in cardiac myocytes.
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Previous work has shown that IB prevented CaMK mediated ICa increases in cardiac myocytes (Wu et al. 1999a, 2001a), strongly suggesting that CaMK inhibition was a critical feature of IB actions at ICa-L. On the other hand, the persistence of Ca2+-dependent ICa-L inactivation (Fig. 2A, D and E) suggested IB did not disrupt Ca2+–CaM signalling, generally. We created a topographical surface plot of relative ICa-L availability to better illustrate the effects of IB over a wide range of conditioning times and voltages (Fig. 2B). These plots reveal the functional targeting of significant IB actions to time and voltage durations relevant to the action potential plateau (P < 0.05 for all intergroup comparisons from conditioning potentials between 0 and +40 mV and conditioning pulse durations from 30 to 130 ms). In order to more thoroughly test the concept that IB was selective for CaMK and that reduction in ICa-L at action potential plateau potentials in IB was due to CaMK, we dialysed a Ca2+ independent form of CaMK into myocytes in the presence of IB (Fig. 3B). Ca2+–CaM-independent CaMK significantly restored reduced ICa-L availability in IB (Fig. 3B), indicating that inhibition of endogenous CaMK was the critical IB effect. The effects of Ca2+–CaM-independent CaMK on ICa-L were specifically due to the enzymatic activity of the exogenous kinase, as they were significantly reduced by coadministration of the CaMK inhibitory peptide AC3-I (data not shown), and eliminated by heat inactivation of the added CaMK (Fig. 3B). CaMK effects were confined to brief conditioning pulses (30–130 ms), and were lost after longer conditioning prepulses (190–500 ms, Fig. 4), confirming that CaMK actions on ICa-L were targeted to time and voltage domains relevant to the cardiac action potential plateau.
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Ca2+–CaM reduces ICa-L availability at action potential plateau potentials
The finding that cells dialysed with IB retained Ca2+-dependent inactivation (Figs 2A, D and E) suggested that Ca2+–CaM remained operative under these experimental conditions. Cellular dialysis with the Ca2+–CaM binding peptide 290–309 did significantly increase relative ICa-L availability. In contrast to CaMK that was only effective after positive conditioning potentials (Figs 3B and 5), 290–309 was effective after weakly and strongly depolarizing conditioning potentials (Figs 3A and 5). These data show that endogenous Ca2+–CaM was a significant signal transduction element for grading ICa-L in the presence of IB. However, 290–309 did not fully increase ICa-L availability to levels present with Ba2+ as charge carrier (Fig. 2E), perhaps indicating that a constitutively bound pool of CaM (Erickson et al. 2001; Pitt et al. 2001) was inaccessible to the peptide.
In contrast to the effects of 290–309 on ICa-L availability, peak ICa-L during the conditioning pulse in IB + 290–309 (4.1 ± 0.4 pA pF-1, n= 5) was significantly less than peak ICa-L recorded in IB alone (7.1 ± 0.9 pA pF-1, n= 7) or in IB + CaMK (8.1 ± 0.5 pA pF-1, n= 5), whereas IB + 290–309 did not decrease peak ICa-L compared to control solution (6.2 ± 0.5 pA pF-1, n= 5).
SR Ca2+ release selectively reduces ICa-L after brief, positive conditioning pulses
The results of experiments so far show that the CaMK-dependent component of Ca2+i signalling to L-type Ca2+ channels is voltage dependent (Figs 3B and 4); however, they do not distinguish between ICa-L and SR Ca2+ release as dominant sources of signalling Ca2+i for grading ICa-L. Both ryanodine and thapsigargin significantly increased ICa-L after positive conditioning potentials in the presence of IB, indicating that Ca2+-induced Ca2+ release is present under these experimental conditions (Fig. 6A–C). Reduction in SR Ca2+ release by either ryanodine or thapsigargin targeted ICa-L increases over similar cell membrane potential (Fig. 6B and C) and temporal domains (Fig. 6D) as CaMK (Figs 3B and 4), suggesting the possibility that activation of endogenous CaMK is predominately due to SR Ca2+ release, and that Ca2+–CaM-dependent inactivation relies on SR Ca2+i under action potential plateau conditions.
CaMK actions at ICa-L are determined by SR Ca2+ release
Previous studies suggest SR Ca2+ is important for activating endogenous CaMK in cardiac myocytes (Kuschel et al. 1999; Wu et al. 1999a, 2001b; Bartel et al. 2000), so we reasoned that Ca2+-independent CaMK would circumvent the requirement for activation of endogenous CaMK by SR Ca2+ release. Surprisingly, thapsigargin-treated cells did not show increases in ICa-L availability at action potential plateau potentials in response to Ca2+-independent CaMK, but did show a significant depolarizing shift in ICa-L availability in response to weaker depolarizations (Fig. 6E). This result was in striking contrast to the significant increases in ICa-L after positive conditioning pulses when myocytes with intact SR Ca2+ release were supplemented with Ca2+-independent CaMK (Figs 3B and 4). In contrast to CaMK-dependent increases in R30, R80 and relative ICa-L at +20 mV (Fig. 5A and B), CaMK replacement was ineffective for increasing these parameters after thapsigargin at +20 mV (Fig. 7A and B). CaMK replacement also failed to evoke a consistent response in R80 and relative current after a –20 mV conditioning step (Fig. 7D). These data underscore the close relationship between SR Ca2+ release and L-type Ca2+ channel function, and suggest the possibility that the Ca2+-independent form of CaMK acts to increase ICa-L availability through a SR-dependent mechanism.
SR Ca2+ release significantly determines Ca2+–CaM responses after strong depolarizations
In contrast to CaMK (Fig. 3B), Ca2+–CaM effects on ICa-L appear to be voltage independent because they operate over a broad range of physiological conditioning potentials (Fig. 3A). SR Ca2+ release does contribute to Ca2+-dependent ICa-L inactivation (Balke & Wier, 1991; Wu et al. 2001b), and reduction of SR Ca2+ release by ryanodine (Fig. 6B) or thapsigargin (Fig. 6C) significantly increased relative ICa-L in cells dialysed with IB only after positive conditioning steps, suggesting that the source of activator Ca2+i for Ca2+–CaM-dependent inactivation may vary in a voltage-dependent manner in cardiac myocytes. In order to better understand the contribution of ICa-L and SR to Ca2+–CaM for regulating ICa-L responses, we dialysed 290–309 into myocytes after thapsigargin. Ca2+–CaM inhibition with 290–309 significantly increased relative ICa-L (Fig. 6F) and R30 (Fig. 7C) and R80 (Fig. 7D) at –20 mV, but not at +20 mV (Fig. 7A and B), supporting previous findings that ICa-L is sufficient for Ca2+–CaM-dependent inactivation, but suggesting that normal SR Ca2+ release significantly determines Ca2+–CaM signalling to L-type Ca2+ channels at strongly depolarized conditioning potentials, present during the action potential plateau.
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Discussion |
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The present studies use a combination of approaches to inhibit endogenous CaMK-dependent ICa-L increases (with IB) or control CaMK activity (with exogenous Ca2+–CaM independent CaMK) while SR Ca2+ release is preserved or eliminated (with ryanodine or thapsigargin) in cardiomyocytes. The central finding of these experiments is that CaMK increases in ICa-L are functionally targeted over time and voltage domains that are directly relevant to the cardiac action potential plateau. This finding suggests CaMK actions are analogous to protein kinase A, which can also regulate L-type Ca2+ channels by a voltage-dependent mechanism (Sculptoreanu et al. 1993). Previous investigations established that CaMK can directly increase L-type Ca2+ channel openings in excised membrane patches from ventricular myocytes (Dzhura et al. 2000), but these experiments did not test for temporal- or voltage-dependent features of CaMK signalling and could not measure effects of SR Ca2+ release. The dramatic loss of CaMK signalling effects after elimination of SR Ca2+ release (Figs 6E and F and 7) shows those CaMK actions are reliant upon SR Ca2+ release. One possible unifying explanation for these observations is that an important CaMK action on ICa-L in intact myocytes may be indirect, via modulation of SR Ca2+ release (Li et al. 1997; Wu et al. 2001a). The concept that CaMK exerts important actions on ICa-L by influencing SR Ca2+ release may reconcile earlier reports showing that ICa-L increases were linked to dynamic reduction in SR Ca2+ release (Delgado et al. 1999), but were eliminated by inactivation of SR Ca release (Wu et al. 1999a, 2001b). Interestingly CaMK slightly, but significantly, increased relative ICa-L availability at weakly depolarized potentials in the absence of SR Ca2+ release (Figs 6E and 7D), raising the possibility that CaMK may be capable of regulating ICa-L by a SR-independent pathway under these voltage clamp conditions. The finding that ICa-L availability responses to CaMK (Fig. 6E) and 290–309 (Fig. 6F) are very similar in thapsigargin, after weak and strong depolarizations, suggests that in the absence of SR Ca2+ release, CaMK and Ca2+–CaM may compete for shared molecular machinery, such as the L-type Ca2+ channel C terminus. Thus, the present studies add important new information to our understanding of how CaMK may contribute to ICa-L regulation in the working heart.
Ca2+–CaM, ICa-L and SR Ca2+ release
CaM is a ubiquitous Ca2+i-sensing protein that is required for Ca2+i-dependent ICa-L inactivation in cardiac L-type Ca2+ channels (Peterson et al. 1999; Zuhlke et al. 1999). CaMK and CaM colocalize with L-type Ca2+ channels and ryanodine receptors (Wu et al. 1999a; Pate et al. 2000; Balshaw et al. 2001; Pitt et al. 2001; Dzhura et al. 2002; Erickson et al. 2003), and are capable of regulating both of these proteins. Ca2+–CaM also activates other proteins, including CaMK, so that myriad effects potentially complicate interpretation of Ca2+–CaM inhibition experiments. The present experiments used IB dialysis and a Ca2+–CaM inhibitory peptide (290–309) to separately control Ca2+–CaM and CaMK signalling. These findings support the concept that Ca2+–CaM reduces available ICa-L in a voltage-independent manner (Fig. 3A). However, the critical source of Ca2+i for Ca2+–CaM is determined by cell membrane voltage because ryanodine (Fig. 6B) and thapsigargin (Fig. 6C) only increased ICa-L at plateau potentials and because 290–309 was ineffective at increasing ICa-L at plateau potentials in the absence of SR Ca2+ release (Figs 3A and 6F). In contrast, CaM sequestration with the 290–309 peptide enhanced ICa availability after weakly depolarizing prepulses, independent of SR Ca2+ release (Figs 6F and 7D and E), suggesting that ICa-L alone is a sufficient source of Ca2+i for Ca2+–CaM-dependent inactivation of ICa-L at weakly depolarized cell membrane potentials. The finding that Ca2+–CaM competition by 290–309 significantly increased relative currents and slowed ICa-L inactivation in IB (Fig. 5) is consistent with a recent report showing marked slowing of ICa-L inactivation and action potential prolongation in cardiomyocytes transfected with Ca2+-binding deficient, dominant-negative CaM mutants (Alseikhan et al. 2002). A potential limitation to studies with 290–309 is highlighted by the finding that peak ICa-L during the conditioning pulse was reduced in IB + 290–309 compared to IB alone, raising the possibility that an outward current, such as ICl,Ca, may be activated by 290–309 and complicate ICa-L measurements during this experimental condition. Taken together, these results reveal the interdependence of CaM, SR Ca2+ release and cell membrane potential and add to other recent work highlighting the importance of CaM as a Ca2+i-driven signalling element for regulating ICa in heart.
The relationship between ICa-L inactivation and availability
The present experiments show that it is possible to distinguish between SR Ca2+, CaM and CaMK signalling effects on relative ICa-L availability in cardiac myocytes. Both CaM (Fig. 3A) and CaMK (Fig. 3B) can separately regulate ICa-L availability under non-steady-state conditions. Relative ICa-L availability is closely related to ICa-L inactivation after positive conditioning pulses (Figs 5A and B, and 7A and B). This relationship is consistent with the concept that ICa-L inactivation (R30 and R80) during the conditioning pulse directly determines ICa-L availability during the subsequent test pulse. Experiments to inhibit or replace CaMK (Fig. 5), eliminate SR Ca2+ release, or reduce Ca2+–CaM (Fig. 7) did not alter this relationship at +20 mV. In contrast, R30 was significantly less than relative ICa-L after CaMK inhibition with IB (Fig. 5C), suggesting that CaMK could reduce this measure of ICa-L inactivation at –20 mV without altering the pool of L-type Ca2+ channels available for opening in response to the +10 mV test pulse. SR Ca2+ also reduced R30 and R80 during CaMK inhibition at –20 mV (compare IB in Fig. 5C and D with IB + thapsigargin in Fig. 7C and D). However, Ca2+i from ICa-L was sufficient for significant Ca2+–CaM actions on R30 and R80 at –20 mV, because these measures were both increased by 290–309 in the combined presence of IB and thapsigargin (Fig. 7D).
A model for voltage- and Ca2+i-dependent regulation of ICa-L in heart
The recent finding that the L-type Ca2+ channel C terminus undergoes significant voltage-dependent movement (Kobrinsky et al. 2003) provides a potentially important context for understanding our findings. The C terminus is now accepted to be richly endowed with Ca2+i sensing machinery, and three distinct Ca2+–CaM binding domains have been identified (Zuhlke et al. 1999; Pate et al. 2000; Pitt et al. 2001). On the other hand, the C terminus is also capable of binding activated CaMK (Hudmon et al. 2002). Given that both Ca2+–CaM and CaMK can converge upon the C terminus and given that the C terminus is significantly mobile over the cell membrane potential ranges used in our study, it is possible that the C terminus could be variably positioned to differentially respond to Ca2+i from ICa-L (when positioned near the pore region) or the SR (when positioned outside of the pore region). Based upon these considerations and upon our finding that SR Ca2+ release was required for complete Ca2+–CaM and CaMK actions at +20 mV (Figs 5 and 7), we hypothesize that the C terminus moves away from the pore region during strong depolarizations. Because Ca2+i from ICa-L was sufficient for Ca2+–CaM at –20 mV, we further hypothesize that the C terminus is close enough to the pore region during weak depolarizations to sense Ca2+i directly from ICa-L (Fig. 8).
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P < 0.01 and 
P < 0.001 for panels B–D. E, addition of Ca2+-independent CaMK failed to increase relative ICa-L in cells dialysed with IB and treated with thapsigargin after positive conditioning prepulses (IB + CaMK + Thaps). F, the Ca2+–CaM inhibitory peptide 290–309 failed to increase relative ICa-L in cells dialysed with IB and treated with thapsigargin (IB + Thaps + 290–309) after positive conditioning prepulses. 
