FKBP12.6 and cADPR regulation of Ca2+ release in smooth muscle cells

Yong-Xiao Wang,1 Yun-Min Zheng,1 Qi-Bing Mei,4 Qinq-Song Wang,1 Mei Lin Collier,2 Sidney Fleischer,3 Hong-Bo Xin,4 and Michael I. Kotlikoff4

1Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208; 2Department of Animal Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104; 3Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235; and 4Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853

Submitted 19 March 2003 ; accepted in final form 15 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular Ca2+ release through ryanodine receptors (RyRs) plays important roles in smooth muscle excitation-contraction coupling, but the underlying regulatory mechanisms are poorly understood. Here we show that FK506 binding protein of 12.6 kDa (FKBP12.6) associates with and regulates type 2 RyRs (RyR2) in tracheal smooth muscle. FKBP12.6 binds to RyR2 but not other RyR or inositol 1,4,5-trisphosphate receptors, and FKBP12, known to bind to and modulate skeletal RyRs, does not associate with RyR2. When dialyzed into tracheal myocytes, cyclic ADP-ribose (cADPR) alters spontaneous Ca2+ release at lower concentrations and produces macroscopic Ca2+ release at higher concentrations; neurotransmitter-evoked Ca2+ release is also augmented by cADPR. These actions are mediated through FKBP12.6 because they are inhibited by molar excess of recombinant FKBP12.6 and are not observed in myocytes from FKBP12.6-knockout mice. We also report that force development in FKBP12.6-null mice, observed as a decrease in the concentration/tension relationship of isolated trachealis segments, is impaired. Taken together, these findings point to an important role of the FKBP12.6/RyR2 complex in stochastic (spontaneous) and receptor-mediated Ca2+ release in smooth muscle.

FK506 binding protein 12.6; ryanodine receptor type 2; calcium sparks; calcium-activated chloride currents


CA2+ RELEASE FROM THE SARCOPLASMIC RETICULUM (SR) or endoplasmic reticulum (ER) in muscle and nonmuscle cells is a highly evolved process involving complex signaling between the plasmalemmal and SR or ER membranes (3, 50). This process is particularly well developed in striated muscle, where the release of Ca2+ from the SR is essential for the development of force and underlies cardiac and skeletal muscle contraction. In cardiac myocytes, Ca2+ influx through voltage-dependent Ca2+ channels activates ryanodine receptors (RyR) (Ca2+ release channels) and then triggers further Ca2+ release from the SR, a process termed Ca2+-induced Ca2+ release (CICR), which entails a tight linkage between Ca2+ ions permeating L-type Ca2+ channels in the sarcolemma and then activating Ca2+-sensitive type 2 RyR (RyR2) channels on the SR membrane (10, 36).

The extent to which an analogous process exists in smooth muscle and the physiological relevance of this form of Ca2+ release have been controversial. In many vascular and nonvascular smooth muscle cells, full activation of voltage-dependent Ca2+ currents (ICa) does not evoke intracellular Ca2+ release (16, 22, 29-31). However, CICR has been reported in myocytes from the taenia caeci (25), intestine (69), urinary bladder (12, 18, 26), portal vein (21), and coronary artery (17, 19), although differing in essential ways from the cardiac form of calcium release (12). Thus CICR takes a nonobligate form in smooth muscle, reflecting a "loose coupling" between the L-type Ca2+ channel and RyR, enabling the L-type channel to conduct Ca2+ without gating RyR, if the net flux of ions is not sufficient (12). However, even in smooth muscle tissues in which CICR does not appear to constitute a component of excitation-contraction coupling, calcium release mediated by RyR appears to play an important physiological role in vasodilation (6) through the "spontaneous" gating of RyR, resulting in Ca2+ sparks and the coupling to calcium-activated potassium channels (42). Moreover, the extent to which the RyR calcium release system amplifies inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release remains unknown.

Here we address factors that regulate RyR-mediated Ca2+ release in smooth muscle, including the association of FK506 binding proteins of 12 and 12.6 kDa (FKBP12 and FKBP12.6) with RyR and the role of cADPR, which may bind to and regulate the interaction of FKBPs and RyR (51). We show that FKBP12.6 selectively associates with RyR2 and regulates Ca2+ release and contraction in smooth muscle tissue and that cADPR modulates RyR Ca2+ release through its interaction with FKBP12.6. Finally, we present evidence suggesting that RyR may amplify neurotransmitter-induced Ca2+ release through IP3R, suggesting a broad role of the cADPRFKBP12.6-RyR2 in excitation-contraction coupling.


    MATERIAL AND METHODS
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Measurements of membrane currents and whole cell intracellular calcium. Unless otherwise noted, electrophysiological experiments were conducted in single equine tracheal smooth muscle cells prepared as previously described (58, 59). Membrane currents were recorded on an EPC9 system (HEKA Elektronik, Lambrecht/Pfalz, Germany) using nystatin-perforated or standard patch-clamp methods (15). For the simultaneous measurement of spatially averaged whole cell intracellular calcium ([Ca2+]i), cells were loaded with fura 2, and a single-excitation wavelength fluorescence method was used to obtain high-bandwidth resolution (59). Fura 2-loaded cells were excited at 380 nM and emitted light (510 ± 10 nM) detected by a photomultiplier tube. The bath solution was (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 10 HEPES, 1.8 CaCl2, and 10 glucose (pH 7.4). In perforated patch-clamp experiments, the patch pipette solution was (in mM) 130 CsCl, 5 MgCl2, 3 EGTA, 1 CaCl2, and 10 HEPES (pH 7.3). The pipette solution contained (in mM) 130 CsCl, 1.2 MgCl2, 1 ATP-Mg, and 10 HEPES (pH 7.3) for the standard patch-clamp experiments. All experiments were performed at 35°C. Experiments in wild-type and FKBP12.6-knockout mouse myocytes (Fig. 6) were conducted on cells isolated from the pulmonary artery because the yield of viable murine tracheal myocytes was not sufficient for experiments.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6. Cyclic ADP-ribose (cADPR) induces Ca2+ release through effects on RyRs. A: dialysis of 0.1 µM cADPR had little effect on STIC (spontaneous local Ca2+ release) frequency or amplitude (top trace), whereas 1 µM increased the frequency and amplitude of STICs (middle trace) and 10 µM induced a macroscopic inward Ca2+-activated Cl- current [ICl(Ca)] (bottom trace). Tracheal myocytes were voltage clamped at -60 mV and dialyzed with cADPR in Cs+ intracellular solution (at the start of trace shown). B: FK506 induces Ca2+ release and blocks subsequent cADPR effect in tracheal myocytes. In cell-attached mode (top trace, patch held at -20 mV, K+ solution), exposure of a cell to FK506 (50 µM) evoked a burst of activity of single Ca2+-activated K+ channels, indicating Ca2+ release. In the continued presence of FK506, patch rupture (voltage clamped at -60 mV) and dialysis of cADPR did not induce Ca2+ release, which would be seen by an outward Ca2+-activated current in this experiment. In the converse experiment (middle trace), application of FK506 failed to evoke a Ca2+ response after dialysis of cADPR and Ca2+ release (Cs+ solution). Preincubation of cADPR with a molar excess of FKBP12.6 protein (bottom trace) prevented cADPR-induced Ca2+ release. In this experiment, the cell was voltage clamped at -60 mV. The patch pipette was tip-loaded with FKBP12.6 (50 µM) alone and then back-filled with FKBP12.6 plus cADPR (10 µM) to dialyze FKBP12.6 into the cell before cADPR exposure. Application of caffeine (10 mM) induced a typical ICl(Ca) in the cell. C: cADPR dialysis activates Ca2+ release in wild-type, but not FKBP12.6-null, mouse myocytes. Dialysis of cADPR (10 µM) activated typical oscillations of ICl(Ca) (top trace), indicating repeated Ca2+ release. Conversely, in FKBP12.6-/- myocytes, cADPR dialysis failed to induce ICl(Ca), whereas application of caffeine indicates presence of releasable Ca2+. Both cells were voltage clamped at -60 mV and dialyzed with Cs+ intracellular solution.

 

Recordings of Ca2+ sparks. Measurements of Ca2+ sparks in fluo 4-AM-loaded myocytes were performed with a high-speed laser scanning confocal head (Noran Oz), as previously described (12). X-Y images were collected every 8.3 ms (256 x 240 pixels) and analyzed using Noran software.

RT-PCR and affinity chromatography. Total RNA from various muscles was isolated using Absolutely RNA RT-PCR Miniprep Kit (Stratagene, La Jolla, CA). First-strand cDNA was synthesized from RNA using Stratascript reverse transcriptase (Stratagene). The resultant cDNA template was amplified with the following forward and reverse oligonucleotide primers: 5'-CCC GAA GCG CGG CCA GAC CTG-3' and 5'-TAG GTC AAC ACA CAT ACA GAA G-3' for FKBP12, and 5'-ATG GGC GTG GAG ATC GAG AC-3' and 5'-GTA GCT CCA TAG GCC ACA TCA-3' for FKBP12.6 (44). The amplified products were electrophoresed on agarose gel and visualized by ethidium bromide staining.

SR preparations were made from equine tracheal smooth muscle and incubated with GST-FKBP12 or GST-FKBP12.6 fusion proteins in homogenization buffer containing 3 M sucrose and 5 mM imidazole, as previously described (67). The mixture was centrifuged to remove free GST-FKBP12 and GST-FKBP12.6 fusion proteins and incubated with glutathione Sepharose 4B resin. The resin was washed and eluted, and the eluted proteins were separated by SDS-PAGE and blotted for immunodetection with specific RyR and IP3R antibodies.

Smooth muscle tension measurements. Mice were euthanized with pentobarbital sodium, and the trachea were rapidly excised. The trachealis muscle was carefully dissected free of surrounding tissues and transversely sectioned. Individual transverse segments of 3- to 4-mm length were placed in 2-ml tissue bath (Radnoti), with one end of the strips fixed to a small clip and the other end to a high-sensitivity force transducer (Harvard Apparatus). The bath solution contained (in mM) 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose (pH 7.4), aerated with 95% O2-5% CO2 at 35°C. The strips were set at a resting tone of 0.5 g and equilibrated for 45 min. Force signals in response to methacholine (mACH) were monitored and recorded PowerLab/4SP (AD Instruments). Tissues were blotted and weighed following the experiments, and data are expressed as force per milligram of tissue.

Statistics. Measurements before and after FK506 or dialysis were compared using the paired Student's t-test. Force generation in control and knockout mice was compared by the unpaired Student t-test. All data are presented as means ± SE.


    RESULTS
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FK506 induces Ca2+ release from the SR. To explore the role of FK506 binding proteins (FKBPs) on the regulation of Ca2+ release in smooth muscle cells, we first examined the effect of FK506, which binds to and removes FKBPs from RyR (7, 27, 54), on spatially averaged [Ca2+]i and inward Ca2+-activated Cl- currents [ICl(Ca)]. Freshly isolated equine tracheal smooth muscle cells were loaded with fura 2-AM and voltage clamped at -60 mV using the permeabilized, whole cell patch-clamp method, with patch pipettes filled with Cs+ to block K+ currents. As shown in Fig. 1, application of FK506 (10 µM) significantly increased the frequency and amplitude of spontaneous transient inward currents (STICs), which are due to the gating of Ca2+-activated Cl- channels (34), but did not alter global [Ca2+]i (Fig. 1A). In a total of nine cells, the mean frequency increased from 0.9 ± 0.2 to 2.1 ± 0.3 Hz (P < 0.05), and the mean amplitude increased by 116 ± 8% (71 ± 11 vs. 164 ± 17 pA) (P < 0.05) after application of FK506. Cells in which spontaneous currents were not observed (most myocytes under these recording conditions) were exposed to a higher concentration of FK506 (50 µM) to examine the effect on spatially averaged [Ca2+]i. As shown in Fig. 1B, exposure to 50 µM FK506 resulted in sufficient Ca2+ release to increase [Ca2+]i and activated a macroscopic ICl(Ca) (Fig. 1B). The mean peak [Ca2+]i increase and mean amplitude of ICl(Ca) were 691 ± 27 nM and 469 ± 36 pA, respectively (n = 6). The augmentation of Ca2+ spark frequency and amplitude was not due to an increase in SR Ca2+ associated with FK506 exposure, because prior exposure to 50 µM FK506 consistently decreased the Ca2+ transient associated with exposure to caffeine (Fig. 2C). In four experiments, the peak [Ca2+]i evoked by exposure to 10 mM caffeine was 697 ± 46 nM before and 381 ± 23 nM after FK506 treatment for 5 min (P < 0.05).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1. FK506 induces an increase in intracellular Ca2+ ([Ca2+]i) and inward Ca2+-activated Cl- currents in isolated tracheal smooth muscle cells. A: FK506 (10 µM) significantly increased the frequency and amplitude of spontaneous inward transient currents (STICs, bottom trace) in a spontaneously active tracheal myocyte, without any change altering the spatially averaged whole cell [Ca2+]i (top trace). B: exposure of a tracheal myocyte to a higher concentration of FK506 (50 µM) induced an increase in whole cell [Ca2+]i and an inward macroscopic Ca2+-activated Cl- current. Holding voltage = -60 mV in both experiments shown. C: FK506 releases Ca2+ from the sarcoplasmic reticulum (SR), as seen by the marked decrease in Ca2+ released from the SR with serial caffeine pulses.

 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. FK506 increases Ca2+ sparks through activation of ryanodine receptors. A: confocal images of a fluo 4-AM-loaded equine tracheal myocytes were obtained at 8.3-ms intervals for 10 s before (left) and after application of FK506 (50 µM, right). Ca2+ sparks were observed in 5 different regions; the frequency and amplitude of sparks were significantly increased after exposure to FK506. B: ryanodine receptors (RyRs), but not inositol 1,4,5-trisphosphate receptors (IP3Rs), mediate FK506-induced Ca2+ release in smooth muscle cells. Ca2+ release (assayed by measurement of the Ca2+-activated Cl- current) was not affected by dialysis of heparin, which completely blocks IP3R-mediated Ca2+ release (top). Conversely, the RyR blocker ruthenium red (50 µM) prevented FK506, but not methacholine, Ca2+ release (bottom). Both myocytes were voltage clamped at -60 mV and dialyzed with Cs+ intracellular solution.

 

Because STICs represent the activation of Ca2+-activated Cl- channels by Ca2+ sparks (70), we next examined the effect of FK506 on the frequency and amplitude of spontaneous Ca2+ sparks. Consistent with the effect on STICs, brief application of FK506 (50 µM, 5 or 10 s) increased the frequency and amplitude of Ca2+ sparks in fluo 4-AM-loaded tracheal smooth muscle cells. An example of these experiments is shown in Fig. 2A. The average frequency of Ca2+ sparks was increased from 0.4 ± 0.1 to 0.9 ± 0.1 Hz, and the average peak amplitude (F/F0) of Ca2+ sparks increased from 0.5 ± 0.1 to 0.8 ± 0.1 (n = 4, P < 0.05). The decay time of Ca2+ sparks was also significantly increased; the time constant ({tau}) of Ca2+ spark decay was 61 ± 12 ms before application of FK506 and 123 ± 17 ms after application of FK506 (n = 4, P < 0.05). Dialysis of ruthenium red (50 µM, Fig. 2B) for 5 min completely blocked the FK506-induced ICl(Ca) (Ca2+ release), whereas dialysis of heparin (10 mg/ml), which inhibits IP3R, was without effect, indicating that FK506 activated Ca2+ release from RyR. Similar observations were made in a total of 11 cells for ruthenium red and 6 cells for heparin experiments. These data are consistent with a mechanism in which FK506 binds to FKBPs and removes these proteins from the RyR complex, resulting in altered RyR gating (37, 48, 56, 66).

FKBP12.6 selectively binds RyR2 in smooth muscle cells. We next sought to determine whether FKBPs are physically associated with RyR in tracheal smooth muscle. As shown in Fig. 3A, RT-PCR experiments indicated expression of both FKBP12 and FKBP12.6 mRNAs in smooth muscle. SR membranes purified from equine trachealis muscle were incubated with FKBP12 and FKBP12.6 GST fusion proteins (55, 67) and were purified with glutathione Sepharose resin, and the eluted complex was separated on agarose and probed with subtypespecific, anti-RyR and -IP3R antibodies. As shown in Fig. 3B, RyR2, but not RyR1 or RyR3, proteins were detected associated with GST-FKBP12.6. By contrast, FKBP12-GST fusion protein was not associated with any RyR subtypes. Consistent with this finding, RyR2, but not RyR1 and RyR3, was directly detected in Western blots from airway muscle SR preparations. In control experiments, RyR1 and RyR3 were expressed in diaphragm muscle SR, and RyR1 was associated with GSTFKBP12 in diaphragm SR membranes (Fig. 3C). No association of IP3R (IP3R1, IP3R2, or IP3R3) with GST-FKBP12.6 or GST-FKBP12 was observed (Fig. 3D).



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 3. FKBP12 and FKBP12.6 are expressed in smooth muscle, but FKBP12.6 selectively binds to type 2 RyRs (RyR2s). A: FKBP12 and FKBP12.6 mRNAs are expressed in smooth muscle. Total RNAs were isolated from equine trachealis, heart, and bladder muscles and amplified with oligonucleotide primers specific for FKBP12 and FKBP12.6. B: GST-FKBP12.6 fusion protein is found to associate with RyR2, but not RyR1 and RyR3 of tracheal muscle, whereas GST-FKBP12 fusion protein is not bound to any subtypes of RyRs. Fusion proteins were incubated with SR preparations from tracheal muscle and complexed proteins separated by SDS-PAGE. RyRs were detected by specific antibody against RyR1, RyR2, or RyR3. T, tracheal muscle SR alone; T+F12.6, tracheal muscle SR + GSTFKBP12.6; and T+F12, tracheal muscle SR+GSTFKBP12. C: GST-FKBP12 fusion protein is tightly bound to diaphragm muscle RyR1. D, diaphragm muscle SR alone; D+F12, diaphragm muscle SR + GSTFKBP12. D: no association of either GST-FKBP12 or FKBP12.6 fusion protein with IP3R1, IP3R2, or IP3R3 was detected. IP3Rs were detected by specific anti-IP3R1, -IP3R2, or -IP3R3 antibody.

 

FKBP12.6 modulates RyR function in smooth muscle. To determine whether FKBP12 or FKBP12.6 proteins regulate Ca2+ release in smooth muscle cells, voltage-clamped tracheal myocytes were dialyzed with recombinant FKBPs added to the patch pipette, and the effects on STICs (an index of spontaneous Ca2+ release) were examined. As shown in Fig. 4, STIC activity decreased in a time-dependent manner after dialysis of FKBP12.6. The average frequency and amplitude of STICs within the 30 s after membrane rupture were used as control and compared with a 30-s period recorded 5 min after dialysis. FKBP12.6 dialysis significantly reduced the frequency and amplitude of STICs, indicating a decrease in the activity of Ca2+ release channels. In a total of seven cells, the average frequency of STICs was decreased from 1.1 ± 0.3 (control) to 0.8 ± 0.2 Hz (5 min after dialysis) (P < 0.05), whereas the average amplitude of STICs was decreased from 83 ± 10 to 57 ± 6 pA (P < 0.05). By contrast, dialysis of FKBP12 was without effect on either the frequency or amplitude of STICs (n = 6). In control experiments, no change in the frequency or amplitude of STICs was observed after cell dialysis (without FKBP12 and FKBP12.6) for 5 min (data not shown). Taken together with the FK506 and FKBP-GST fusion protein experiments described above, these results indicate that interactions between FKBP12.6, but not FKBP12, and RyRs play an important role in the regulation of Ca2+ release channels in smooth muscle cells and likely account for the effect of FK506 in promoting Ca2+ release.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4. FKBP12.6, but not FKBP12, functionally regulates Ca2+ release in smooth muscle cells. Dialysis of FKBP12.6, but not FKBP12 (50 µM) significantly reduced the frequency and amplitude of STICs (spontaneous localized Ca2+ release) in tracheal myocytes. Both cells were voltage clamped at -60 mV and dialyzed with Cs+ intracellular solution.

 

Enhancement of neurotransmitter-induced smooth muscle contraction in FKBP12.6-null mice. To further explore the physiological function of FKBP12.6 in smooth muscle cells, we examined whether neurotransmitter-induced smooth muscle contraction was altered in FKBP12.6-knockout mice (66). As shown in Fig. 5A, cholinergic contractions in isolated tracheal strips were significantly enhanced in FKBP12.6-null mice. After exposure to mACH (3 µM), peak muscle tension was 64 ± 8 mg in tracheal strips from FKBP12.6-/- mice and 39 ± 3 mg in control mice of the same genetic background and approximate age (n = 12, P < 0.05). Similarly, the peak tension induced by 30 µM mACH in FKBP12.6-/- tracheal strips was greater than in control tracheal strips (60 ± 6 vs. 97 ± 12 mg). Several mechanisms could account for augmented cholinergic responses in FKBP12.6-null mice, including a depolarization of the muscle associated with increased basal [Ca2+]i, augmented SR Ca2+ stores, and amplification of the IP3 Ca2+ release by dysregulated RyRs (65).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. FKBP12.6 and RyRs regulate excitation/contraction coupling in smooth muscle. A: FKBP12.6 gene inactivation results in enhanced smooth muscle contraction. Trachealis muscle segments from FKBP12.6-null and ageand strain-matched control mice were exposed to 3 or 30 µM methacholine (mACH) to generate muscle tension. Contraction was significantly enhanced in FKBP12.6-null mice, suggesting dysregulation of the Ca2+ release process. Numbers in parentheses indicate the number of experiments. *P < 0.05 compared with control. B: block of RyR with ruthenium red alters muscarinic Ca2+ release in single smooth muscle cells. Recordings at left show mACH-induced Ca2+-activated Cl- currents (Ca2+ release) from single equine tracheal myocytes dialyzed without (control) and with ruthenium red (50 µM) for 5 min. Block of RyR blunts Ca2+ release at submaximal levels of receptor stimulation, indicating an amplifying effect of RyR on IP3-mediated Ca2+ release. Both cells were voltage clamped at -60 mV and dialyzed with Cs+ intracellular solution to block K- currents. Graph at right summarizes the effect of ruthenium red on mACH-induced Ca2+-activated Cl- currents (Ca2+ release). *P < 0.05 vs. control.

 

Consistent with the findings that FKBP12.6 regulates Ca2+ release and cell contraction through RyRs, dialysis of ruthenium red in isolated myocytes markedly inhibited mACH-induced Ca2+ release. Examples of mACH-induced ICl(Ca) in a cell dialyzed with ruthenium red (50 µM) for 5 min and a control cell are shown in Fig. 5B. The average amplitudes of mACH-induced ICl(Ca) from six cells dialyzed with ruthenium red and five control cells were 185 ± 27 and 415 ± 74 pA, respectively (P < 0.05). These results suggest that FKBP12.6 plays a physiological role in neurotransmitter-induced Ca2+ release and associated cell contraction in smooth muscle. Similar findings have been reported in colonic myocytes (2).

cADPR modulates Ca2+ release in smooth muscle through interactions with FKBP12.6 proteins. cADPR is an endogenous NAD+ metabolite that has been shown to promote Ca2+ release from RyR in a variety of mammalian cells, including cardiac, pancreatic, and neurosecretory PC12 cells (11, 41, 44). We examined the effect of dialysis of cADPR into equine trachealis smooth muscle cells on Ca2+ release by monitoring STICs. As shown in Fig. 6A, dialysis of cADPR at 1 µM produced a concentration-dependent increase in the amplitude and frequency of STICs, which reflect spontaneous calcium release events. The mean frequency and amplitude of STICs were 1.3 ± 0.2 Hz and 21 ± 3 pA in the 30 s after rupture of the membrane patch and 2.2 ± 0.3 Hz and 43 ± 5 pA within a 30-s period after dialysis for 5 min (n = 6, P < 0.05). At a higher concentration (10 µM), cADPR induced a macroscopic ICl(Ca), indicating global Ca2+ release (mean current was 447 ± 59 pA, n = 5).

To determine the role of FKBPs in cADPR-mediated Ca2+ release, we first examined whether preexposure to FK506 blocked cADPR-induced Ca2+ release in equine airway myocytes by using on-cell recordings of spontaneous transient outward currents to monitor Ca2+ release, followed by cell dialysis. Figure 6B, top, shows a typical example of these experiments. Extracellular application of FK506 to a myocyte evoked a burst increase in activity of single Ca2+-activated K+ channels in a membrane patch from an on-cell recording, indicating stimulation of RyR-mediated Ca2+ release (42). In the continued presence of FK506, patch rupture and dialysis of cADPR (10 µM) failed to induce any ICl(Ca) (Ca2+ release). Similar results were obtained in five other myocytes. Consistent with this result, initial dialysis of cADPR (10 µM) induced ICl(Ca) and blocked subsequent FK506 responses (Fig. 6B, middle; n = 6). Thus cADPR and FK506 appear to compete at the level of SR Ca2+ release.

We next examined whether predialysis of a molar excess of FKBP12.6 could block the Ca2+ release actions of cADPR. To dialyze FKBP12.6 into the cell before the effect of cADPR, the patch pipette was tip-loaded with FKBP12.6 (50 µM) alone and back-filled with FKBP12.6 plus cADPR (10 µM). As shown in Fig. 6B, bottom, after dialysis of FKBP12.6, cADPR failed to induce a ICl(Ca), indicating no Ca2+ release, whereas application of caffeine (10 mM) induced a normal ICl(Ca) (Ca2+ release) in the same cell. Similar observations were made in four other cells.

To further confirm the hypothesis that cADPR releases Ca2+ through interactions with FKBPs, we examined cADPR effects in pulmonary artery smooth muscle cells isolated from control and FKBP12.6 knockout mice. In these myocytes, spontaneous chloride currents were never observed. However, as shown in Fig. 6C, dialysis of 10 µM cADPR evoked macroscopic Ca2+ release events in wild-type myocytes, as shown by the activation of large ICl(Ca). Dialysis of 10 µM cADPR failed to activate any inward currents in FKBP12.6-/- myocytes (n = 6), whereas currents were observed in all 5 myocytes recorded from control mice. Thus FKBP12.6 is necessary for cADPR Ca2+ release in smooth muscle cells.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we report evidence that FKBP12.6 proteins associate with RyR and functionally regulate Ca2+ release in smooth muscle. FBKP12.6 regulates spontaneous Ca2+ release through its interaction with RyR2 and appears to modulate Ca2+ release in smooth muscle through the amplification of phospholipase-linked (IP3R-mediated) SR Ca2+ release under conditions of submaximal ligand binding (Fig. 5). cADPR similarly regulates Ca2+ release in tracheal myocytes, an action that requires FKBP12.6 proteins. These findings expand the context of RyR-mediated Ca2+ release in smooth muscle excitation-contraction coupling.

Recent studies indicate that FKBPs, e.g., FKBP12 and FKBP12.6, are tightly associated with and regulate the physiological functions of RyRs in skeletal and cardiac myocytes (37, 39, 40, 45, 47, 48, 55, 56, 64, 65, 68). Our finding that exposure of smooth muscle cells to FK506 increases the frequency, amplitude, and duration of spontaneous Ca2+ release events and associated STICs (Figs. 1 and 2) is generally consistent with observations of the amplifying effect of FK506 on cardiac Ca2+ transients (40, 64), although other actions of the drug may account for this effect (14). Other recent studies have shown that FK506 induces an increase in [Ca2+]i in intestinal myocytes (4) and increases the activity of STOCs due to the simultaneous opening of many Ca2+-activated K+ channels in bladder myocytes (63). We show that this action is inhibited by ruthenium red but not by heparin (Fig. 2B), indicating that FKBPs functionally stabilize RyR Ca2+ release channels in airway smooth muscle, similar to their actions in cardiac myocytes.

FKBP12 is tightly associated with and required for physiological functions of RyR1 in skeletal myocytes (7, 27, 38), whereas FKBP12.6 selectively associates with RyR2 in cardiac myocytes (28, 33, 53, 65). FKBPs have also been reported to modulate IP3R function (9). Because both RyR1 and RyR2 are reportedly expressed in smooth muscle (13, 43), and IP3 Ca2+ signaling is well developed in smooth muscle, we examined the expression of FKBP12 and FKBP12.6 and their association with RyR in this tissue. RT-PCR indicated the presence of FKBP12 and FKBP12.6 mRNA in smooth muscle, but incubation of FKBP-GST fusion proteins with smooth muscle SR indicated that FKBP12.6 selectively associates with RyR2 (Fig. 3). This result is consistent with the general view that FKBP12.6 preferentially associates with RyR2 (33, 39, 55, 62, 65), although binding has been reported to RyR1 (49) and RyR3 (8). Western blot analysis indicated little expression of RyR1 or RyR3 in SR membranes from trachealis, whereas RyR1 and RyR3 were detected in diaphragm, as expected. Thus the failure of FKBP12.6 fusion proteins to associate with RyR1 and RyR3 could be explained by very low expression of these two receptors in this muscle or by selective binding to RyR2. We did not detect binding of FKBP12 to RyR1 or RyR3 in trachealis, despite evidence of association in skeletal SR (Fig. 3), further suggesting that the level of expression of these proteins was too low to detect association. No evidence of association with IP3R1, IP3R2, or IP3R3, previously shown to be expressed in smooth muscle (52), was observed. Thus, collectively, our data indicate that FKBP12.6 associates with and functionally modulates RyR2, thereby playing an important role in regulation of Ca2+ release in smooth muscle. In agreement with this finding, dialysis of purified FKBP12.6, but not FKBP12 protein, into single smooth muscle cells significantly reduced the frequency and amplitude of STICs (Fig. 4).

Whereas RyR-mediated Ca2+ release occurs in some smooth muscle in the form of spontaneous Ca2+ sparks (42) and CICR (12), the role of these Ca2+-gated release channels in neurohormonal Ca2+ release is unclear, despite the obvious potential of this system to amplify IP3-mediated Ca2+ release from the SR. Here we present evidence that RyRs amplify SR release evoked by phospholipase-linked receptors and that this process is regulated by FKBP12.6 proteins. Smooth muscle strips from FKBP12.6-knockout mice displayed augmented contraction to submaximal mACH concentrations compared with age and strain-matched control mice (Fig. 5). These data are consistent with augmented RyR2-mediated Ca2+ release from FKBP12.6-null cardiac myocytes (66). Previous data have suggested that RyRs do not play a significant role in Ca2+ release evoked by neurotransmitter agonists (59). We reasoned, however, that such amplification is likely to occur under conditions of submaximal IP3 generation, where Ca2+ release after gating of a fraction of available IP3R might be augmented by gating of RyR through high local Ca2+ concentrations. Consistent with this hypothesis, we found a substantial augmentation of Ca2+ release in single myocytes at submaximal methacholine concentrations (Fig. 5). In agreement with our data, recent studies from cultured portal vein smooth muscle cells have shown that inhibition of RyRs by anti-RyR antibodies blocks Ca2+ release after {alpha}-adrenergic stimulation with norepinephrine (5). These findings, together with the fact that both RyRs and IP3Rs are functionally or structurally colocalized in the same SR in smooth muscle cells (1, 23, 24, 35, 57, 60, 61), suggest a key role of RyRs in the amplification of SR Ca2+ release by phospholipase C-linked neurotransmitter receptors. Targeted gene inactivation of FKBP12.6, which serves to stabilize the closed state of RyR2, likely augments this amplification by extending the duration of RyR openings (66), resulting in enhanced contractile responses.

Finally, our studies establish a molecular context for the reported Ca2+-mobilizing actions of cADPR in smooth muscle (46). cADPR promotes Ca2+ release in cardiac, pancreatic, and PC12 cells through the opening of RyR2 (11, 41, 44). Here we show that cADPR increases spontaneous Ca2+ release events and produces macroscopic Ca2+ release in a concentration-dependent manner and that this effect is blocked by previous exposure to FK506 or a molar excess of FKBP12.6 protein. Results from Noguchi et al. (44) indicate that prior exposure of pancreatic {beta}-cells to FK506 also prevents cADPR-induced Ca2+ release. Our data indicate that the effect of cADPR on RyR gating requires FKBP12.6 protein bound to RyR2 and suggest, but do not prove, a direct interaction between cADPR and FKBP12.6. Consistent with FKBP12.6 as the target for cADPR actions, we show that cADPR is not capable of inducing Ca2+ release in myocytes from FKBP12.6-null mice (Fig. 6C). The similarity between actions of FK506 (Fig. 1) and cADPR (Fig. 6) on Ca2+ release suggest that the action of cADPR is to bind FKBP12.6 and remove it from RyR2, similar to the known action of FK506 (28, 33).

These findings and previous studies (20, 32, 46) suggest that cADPR-FKBP12.6-RyR2 may represent an important signaling pathway in smooth muscle. The finding that Ca2+ release evoked by stimulation of muscarinic receptors is significantly augmented by predialysis of cADPR at a concentration that itself does not produce Ca2+ release and that spontaneous Ca2+ sparks are markedly enhanced at relatively modest cADPR concentrations suggest that two processes central to the regulation of smooth muscle tone (e.g., IP3-mediated Ca2+ release and Ca2+ sparks/Ca2+-activated sarcolemmal ion channels) are subject to regulation by this process. Moreover, stimulation of neurotransmitter receptors in smooth muscle cells may itself activate ADP-ribosyl cyclase, generating cADPR and augmenting the role of RyR in Ca2+ release through the dissociation of FKBP12.6 from RyR2, resulting in a powerful, positive feedback system for Ca2+ release.

In summary, our study demonstrates that FKPB12.6, but not FKBP12, proteins functionally regulate Ca2+ release in smooth muscle cells. This protein associates with and modulates RyR2, but not other RyR isoforms or IP3Rs. cADPR modulates Ca2+ sparks and is capable of triggering global Ca2+ release in tracheal myocytes, and this action appears to occur through effects on the FKBP12.6/RyR2 complex. Altered contractile responses in FKBP12.6-null mice further suggests that this system may play an important modulatory role in neurotransmitter-induced Ca2+ release in smooth muscle.


    ACKNOWLEDGMENTS
 
We thank Dr. Richard J. H. Wojcikiewicz for providing the antibody against type-2 IP3 receptors. FK506 was kindly provided by Fujisawa Pharmaceutical Co., Ltd.

GRANTS

This work was supported by grants from National Institutes of Health (to Y.-X. Wang and M. I. Kotlikoff), American Heart Association (to Y.-X. Wang), and the Pennsylvania Thoracic Association (to Y.-X. Wang).


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. I. Kotlikoff, Dept. of Biomedical Sciences, Cornell Univ., VRT Box 11, Ithaca, NY 14853 (E-mail: mik7{at}cornell.edu), or Y.-X. Wang, Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: wangy{at}mail.amc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Amedee T, Large WA, and Wang Q. Characteristics of chloride currents activated by noradrenaline in rabbit ear artery cells. J Physiol 428: 501-516, 1990.[Abstract]

2. Bayguinov O, Hagen B, Bonev AD, Nelson MT, and Sanders KM. Intracellular calcium events activated by ATP in murine colonic myocytes. Am J Physiol Cell Physiol 279: C126-C135, 2000.[Abstract/Free Full Text]

3. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature 361: 315-325, 1993.[CrossRef][ISI][Medline]

4. Bielefeldt K, Sharma RV, Whiteis C, Yedidag E, and Abboud FM. Tacrolimus (FK506) modulates calcium release and contractility of intestinal smooth muscle. Cell Calcium 22: 507-514, 1997.[ISI][Medline]

5. Boittin FX, Macrez N, Halet G, and Mironneau J. Norepinephrine-induced Ca2+ waves depend on InsP3 and ryanodine receptor activation in vascular myocytes. Am J Physiol Cell Physiol 277: C139-C151, 1999.[Abstract/Free Full Text]

6. Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT, and Aldrich RW. Vasoregulation by the beta1 subunit of the calcium-activated potassium channel. Nature 407: 870-876, 2000.[CrossRef][ISI][Medline]

7. Brillantes AB, Ondrias K, Scott A, Kobrinsky E, Ondriasova E, Moschella MC, Jayaraman T, Landers M, Ehrlich BE, and Marks AR. Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell 77: 513-523, 1994.[ISI][Medline]

8. Bultynck G, De Smet P, Rossi D, Callewaert G, Missiaen L, Sorrentino V, De Smedt H, and Parys JB. Characterization and mapping of the 12 kDa FK506-binding protein (FKBP12)-binding site on different isoforms of the ryanodine receptor and of the inositol 1,4,5-trisphosphate receptor. Biochem J 354: 413-422, 2001.[CrossRef][ISI][Medline]

9. Cameron AM, Steiner JP, Roskams AJ, Ali SM, Ronnett GV, and Snyder SH. Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ flux. Cell 83: 463-472, 1995.[ISI][Medline]

10. Cannell MB, Cheng H, and Lederer WJ. The control of calcium release in heart muscle. Science 268: 1045-1049, 1995.[ISI][Medline]

11. Clementi E, Riccio M, Sciorati C, Nistico G, and Meldolesi J. The type 2 ryanodine receptor of neurosecretory PC12 cells is activated by cyclic ADP-ribose. Role of the nitric oxide/cGMP pathway. J Biol Chem 271: 17739-17745, 1996.[Abstract/Free Full Text]

12. Collier ML, Ji G, Wang Y, and Kotlikoff MI. Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release. J Gen Physiol 115: 653-662, 2000.[Abstract/Free Full Text]

13. Coussin F, Macrez N, Morel JL, and Mironneau J. Requirement of ryanodine receptor subtypes 1 and 2 for Ca2+-induced Ca2+ release in vascular myocytes. J Biol Chem 275: 9596-9603, 2000.[Abstract/Free Full Text]

14. DuBell WH, Wright PA, Lederer WJ, and Rogers TB. Effect of the immunosupressant FK506 on excitation-contraction coupling and outward K+ currents in rat ventricular myocytes. J Physiol 501: 509-516, 1997.[Abstract]

15. Fleischmann BK, Murray RK, and Kotlikoff MI. Voltage window for sustained elevation of cytosolic calcium in smooth muscle cells. Proc Natl Acad Sci USA 91: 11914-11918, 1994.[Abstract/Free Full Text]

16. Fleischmann BK, Wang YX, Pring M, and Kotlikoff MI. Voltage-dependent calcium currents and cytosolic calcium in equine airway myocytes. J Physiol 492: 347-358, 1996.[Abstract]

17. Ganitkevich VY and Isenberg G. Ca2+ entry through Na+-Ca2+ exchange can trigger Ca2+ release from Ca2+ stores in Na+-loaded guinea-pig coronary myocytes. J Physiol 468: 225-243, 1993.[Abstract]

18. Ganitkevich VY and Isenberg G. Contribution of Ca2+-induced Ca2+ release to the [Ca2+]i transients in myocytes from guinea-pig urinary bladder. J Physiol 458: 119-137, 1992.[Abstract]

19. Ganitkevich VY and Isenberg G. Efficacy of peak Ca2+ currents (ICa) as trigger of sarcoplasmic reticulum Ca2+ release in myocytes from the guinea-pig coronary artery. J Physiol 484: 287-306, 1995.[Abstract]

20. Geiger J, Zou AP, Campbell WB, and Li PL. Inhibition of cADP-ribose formation produces vasodilation in bovine coronary arteries. Hypertension 35: 397-402, 2000.[Abstract/Free Full Text]

21. Gregoire G, Loirand G, and Pacaud P. Ca2+ and Sr2+ entry induced Ca2+ release from the intracellular Ca2+ store in smooth muscle cells of rat portal vein. J Physiol 472: 483-500, 1993.[Abstract]

22. Guerrero A, Singer JJ, and Fay FS. Simultaneous measurement of Ca2+ release and influx into smooth muscle cells in response to caffeine. A novel approach for calculating the fraction of current carried by calcium. J Gen Physiol 104: 395-422, 1994.[Abstract]

23. Haeusler G, Richards JG, and Thorens S. Noradrenaline contractions in rabbit mesenteric arteries skinned with saponin. J Physiol 321: 537-556, 1981.[Abstract]

24. Hazama H, Nakajima T, Hamada E, Omata M, and Kurachi Y. Neruokinin A and Ca2+ current induce Ca2+-activated Cl- currents in guinea-pig tracheal myocytes. J Physiol 492: 377-393, 1996.[Abstract]

25. Iino M. Calcium-induced calcium release mechanism in guinea pig taenia caeci. J Gen Physiol 94: 363-383, 1989.[Abstract]

26. Imaizumi Y, Muraki K, and Watanabe M. Ionic currents in single smooth muscle cells from the ureter of the guinea-pig. J Physiol 411: 131-159, 1989.[Abstract]

27. Jayaraman T, Brillantes AM, Timerman AP, Fleischer S, Erdjument-Bromage H, Tempst P, and Marks AR. FK506 binding protein associated with the calcium release channel (ryanodine receptor). J Biol Chem 267: 9474-9477, 1992.[Abstract/Free Full Text]

28. Kaftan E, Marks AR, and Ehrlich BE. Effects of rapamycin on ryanodine receptor/Ca2+-release channels from cardiac muscle. Circ Res 78: 990-997, 1996.[Abstract/Free Full Text]

29. Kamishima T, Davies NW, and Standen NB. Mechanisms that regulate [Ca2+]i following depolarization in rat systemic arterial smooth muscle cells. J Physiol 522 285-295, 2000.[Abstract/Free Full Text]

30. Kamishima T and McCarron JG. Depolarization-evoked increases in cytosolic calcium concentration in isolated smooth muscle cells of rat portal vein. J Physiol 492: 61-74, 1996.[Abstract]

31. Kim SJ, Ahn SC, Kim JK, Kim YC, So I, and Kim KW. Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes. Am J Physiol Cell Physiol 273: C1947-C1956, 1997.[Abstract/Free Full Text]

32. Kuemmerle JF and Makhlouf GM. Agonist-stimulated cyclic ADP ribose. Endogenous modulator of Ca2+-induced Ca2+ release in intestinal longitudinal muscle. J Biol Chem 270: 25488-25494, 1995.[Abstract/Free Full Text]

33. Lam E, Martin MM, Timerman AP, Sabers C, Fleischer S, Lukas T, Abraham RT, O'Keefe SJ, O'Neill EA, and Wiederrecht GJ. A novel FK506 binding protein can mediate the immunosuppressive effects of FK506 and is associated with the cardiac ryanodine receptor. J Biol Chem 270: 26511-26522, 1995.[Abstract/Free Full Text]

34. Large WA and Wang Q. Characteristics and physiological role of the Ca2+-activated Cl- conductance in smooth muscle. Am J Physiol Cell Physiol 271: C435-C454, 1996.[Abstract/Free Full Text]

35. Leijten PA and van Breemen C. The effects of caffeine on the noradrenaline-sensitive calcium store in rabbit aorta. J Physiol 357: 327-339, 1984.[Abstract]

36. Lopez-Lopez JR, Shacklock PS, Balke CW, and Wier WG. Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science 268: 1042-1045, 1995.[ISI][Medline]

37. Marks AR. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol Heart Circ Physiol 272: H597-H605, 1997.[Abstract/Free Full Text]

38. Marx SO, Ondrias K, and Marks AR. Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors). Science 281: 818-821, 1998.[Abstract/Free Full Text]

39. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, and Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101: 365-376, 2000.[ISI][Medline]

40. McCall E, Li L, Satoh H, Shannon TR, Blatter LA, and Bers DM. Effects of FK-506 on contraction and Ca2+ transients in rat cardiac myocytes. Circ Res 79: 1110-1121, 1996.[Abstract/Free Full Text]

41. Meszaros LG, Bak J, and Chu A. Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca2+ channel. Nature 364: 76-79, 1993.[CrossRef][ISI][Medline]

42. Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, and Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633-637, 1995.[Abstract]

43. Neylon CB, Richards SM, Larsen MA, Agrotis A, and Bobik A. Multiple types of ryanodine receptor/Ca++ release channels are expressed in vascular smooth muscle. Biochem Biophys Res Commun 215: 814-821, 1995.[CrossRef][ISI][Medline]

44. Noguchi N, Takasawa S, Nata K, Tohgo A, Kato I, Ikehata F, Yonekura H, and Okamoto H. Cyclic ADP-ribose binds to FK506-binding protein 12.6 to release Ca2+ from islet microsomes. J Biol Chem 272: 3133-3136, 1997.[Abstract/Free Full Text]

45. Ono K, Yano M, Ohkusa T, Kohno M, Hisaoka T, Tanigawa T, Kobayashi S, Kohno M, and Matsuzaki M. Altered interaction of FKBP12.6 with ryanodine receptor as a cause of abnormal Ca2+ release in heart failure. Cardiovasc Res 48: 323-331, 2000.[CrossRef][ISI][Medline]

46. Prakash YS, Kannan MS, Walseth TF, and Sieck GC. Role of cyclic ADP-ribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle. Am J Physiol Cell Physiol 274: C1653-C1660, 1998.[Abstract/Free Full Text]

47. Prestle J, Janssen PM, Janssen AP, Zeitz O, Lehnart SE, Bruce L, Smith GL, and Hasenfuss G. Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca2+ leak from the sarcoplasmic reticulum and increases contractility. Circ Res 88: 188-194, 2001.[Abstract/Free Full Text]

48. Sabatini DM, Lai MM, and Snyder SH. Neural roles of immunophilins and their ligands. Mol Neurobiol 15: 223-239, 1997.[ISI][Medline]

49. Sanborn BM, Dodge K, Monga M, Qian A, Wang W, and Yue C. Molecular mechanisms regulating the effects of oxytocin on myometrial intracellular calcium. Adv Exp Med Biol 449: 277-286, 1998.[ISI][Medline]

50. Somlyo AP and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994.[CrossRef][ISI][Medline]

51. Takasawa S, Nata K, Yonekura H, and Okamoto H. Cyclic ADP-ribose in insulin secretion from pancreatic beta cells. Science 259: 370-373, 1993.[ISI][Medline]

52. Tasker PN, Michelangeli F, and Nixon GF. Expression and distribution of the type 1 and type 3 inositol 1,4,5-trisphosphate receptor in developing vascular smooth muscle. Circ Res 84: 536-542, 1999.[Abstract/Free Full Text]

53. Timerman AP, Jayaraman T, Wiederrecht G, Onoue H, Marks AR, and Fleischer S. The ryanodine receptor from canine heart sarcoplasmic reticulum is associated with a novel FK-506 binding protein. Biochem Biophys Res Commun 198: 701-706, 1994.[CrossRef][ISI][Medline]

54. Timerman AP, Ogunbumni E, Freund E, Wiederrecht G, Marks AR, and Fleischer S. The calcium release channel of sarcoplasmic reticulum is modulated by FK-506-binding protein. Dissociation and reconstitution of FKBP-12 to the calcium release channel of skeletal muscle sarcoplasmic reticulum. J Biol Chem 268: 22992-22999, 1993.[Abstract/Free Full Text]

55. Timerman AP, Onoue H, Xin HB, Barg S, Copello J, Wiederrecht G, and Fleischer S. Selective binding of FKBP12.6 by the cardiac ryanodine receptor. J Biol Chem 271: 20385-20391, 1996.[Abstract/Free Full Text]

56. Valdivia HH. Modulation of intracellular Ca2+ levels in the heart by sorcin and FKBP12, two accessory proteins of ryanodine receptors. Trends Pharmacol Sci 19: 479-482, 1998.[CrossRef][ISI][Medline]

57. Wang YX, Dhulipala PD, Li L, Benovic JL, and Kotlikoff MI. Coupling of M2 muscarinic receptors to membrane ion channels via phosphoinositide 3-kinase gamma and atypical protein kinase C. J Biol Chem 274: 13859-13864, 1999.[Abstract/Free Full Text]

58. Wang YX, Dhulipala PK, and Kotlikoff MI. Hypoxia inhibits the Na+/Ca2+ exchanger in pulmonary artery smooth muscle cells. FASEB J 14: 1731-1740, 2000.[Abstract/Free Full Text]

59. Wang YX and Kotlikoff MI. Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase. Proc Natl Acad Sci USA 94: 14918-14923, 1997.[Abstract/Free Full Text]

60. Wang YX and Kotlikoff MI. Muscarinic signaling pathway for calcium release and calcium-activated chloride current in smooth muscle. Am J Physiol Cell Physiol 273: C509-C519, 1997.[Abstract/Free Full Text]

61. Wang YX and Kotlikoff MI. Signalling pathway for histamine activation of non-selective cation channels in equine tracheal myocytes. J Physiol 523: 131-138, 2000.[Abstract/Free Full Text]

62. Ward SM, Gadbut AP, Tang D, Papageorge AG, Wu L, Li G, Barnett JV, and Galper JB. TGFbeta regulates the expression of G{alpha}i2 via an effect on the localization of ras. J Mol Cell Cardiol 34: 1217-1226, 2002.[CrossRef][ISI][Medline]

63. Weidelt T and Isenberg G. Augmentation of SR Ca2+ release by rapamycin and FK506 causes K+-channel activation and membrane hyperpolarization in bladder smooth muscle. Br J Pharmacol 129: 1293-1300, 2000.[Abstract/Free Full Text]

64. Xiao RP, Valdivia HH, Bogdanov K, Valdivia C, Lakatta EG, and Cheng H. The immunophilin FK506-binding protein modulates Ca2+ release channel closure in rat heart. J Physiol 500: 343-354, 1997.[Abstract]

65. Xin HB, Rogers K, Qi Y, Kanematsu T, and Fleischer S. Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor. J Biol Chem 274: 15315-15319, 1999.[Abstract/Free Full Text]

66. Xin HB, Senbonmatsu T, Cheng DS, Wang YX, Copello JA, Ji GJ, Collier ML, Deng KY, Jeyakumar LH, Magnuson MA, Inagami T, Kotlikoff MI, and Fleischer S. Oestrogen protects FKBP12.6.null mice from cardiac hypertrophy. Nature 416: 334-338, 2002.[CrossRef][ISI][Medline]

67. Xin HB, Timerman AP, Onoue H, Wiederrecht GJ, and Fleischer S. Affinity purification of the ryanodine receptor/calcium release channel from fast twitch skeletal muscle based on its tight association with FKBP12. Biochem Biophys Res Commun 214: 263-270, 1995.[CrossRef][ISI][Medline]

68. Yano M, Ono K, Ohkusa T, Suetsugu M, Kohno M, Hisaoka T, Kobayashi S, Hisamatsu Y, Yamamoto T, Kohno M, Noguchi N, Takasawa S, Okamoto H, and Matsuzaki M. Altered stoichiometry of FKBP12.6 vs. ryanodine receptor as a cause of abnormal Ca2+ leak through ryanodine receptor in heart failure. Circulation 102: 2131-2136, 2000.[Abstract/Free Full Text]

69. Zholos AV, Baidan LV, and Shuba MF. Some properties of Ca2+-induced Ca2+ release mechanism in single visceral smooth muscle cell of the guinea-pig. J Physiol 457: 1-25, 1992.[Abstract]

70. ZhuGe R, Sims SM, Tuft RA, Fogarty KE, and Walsh JV Jr. Ca2+ sparks activate K+ and Cl- channels, resulting in spontaneous transient currents in guinea-pig tracheal myocytes. J Physiol 513: 711-718, 1998.[Abstract/Free Full Text]