Association of immunophilins with mammalian TRPC channels

Drosophila photoreceptor channels TRP and TRPL are held in a large signalplex by the scaffolding protein, INAD. Immunophilin FKBP59, another member of the signalplex, binds to both INAD and TRPL. Mutation P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709), eliminates TRPL interaction with FKBP59. The first leucylprolyl (LP) dipeptide in this region is conserved in mammalian TRPC channel proteins. However, the second LP is changed to isoleucylprolyl (IP) in TRPC1, -C4, and -C5, and valylprolyl (VP) in TRPC3, -C6, and -C7. The purpose of the present study was to determine if mammalian FKBP12 or FKBP52 interact with TRPC channel proteins. Using TRPC-specific antibodies, immunoprecipitations from Sf9 cells individually co-expressing each of the TRPC proteins along with the immunophilins showed that TRPC3, -C6, and -C7 interact with FKBP12, whereas TRPC1, -C4, and -C5 interact with FKBP52. The binding of FKBP12 and FKBP52 was specific and could be displaced by the immunosuppressant drug FK506, at concentrations of 0.5 and 10 microm, respectively. To evaluate TRPC-immunophilin interactions in vivo, immunoprecipitations were performed using membrane lysates of rat cerebral cortex. FKBP12 co-immunoprecipitated with TRPC3, -C6, and -C7 from rat brain, whereas FKBP52 was found to associate with TRPC1, -C4, and -C5. The association of immunophilins with the TRPC channels in rat brain lysates could be displaced by FK506. Receptor-mediated activation of TRPC6, stably expressed in HEK cells, was significantly inhibited by FK506, which also disrupted interaction between TRPC6 and the endogenous immunophilin found in HEK cells. Pro to Gln mutations in the first LP dipeptide in the putative FKBP binding domain eliminated FKBP12 and FKBP52 interaction with TRPC3 and -C6, and TRPC1 and -C4, respectively. However, mutual swap of VP and IP in TRPC3 and TRPC5 did not alter the association or the selectivity of the channels for their respective immunophilin binding partner. These results suggest that immunophilins are TRPC channel accessory proteins that play an important role in the mechanism of channel activation following receptor stimulation.     

Molecular cloning and embryonic expression of dFKBP59, a novel Drosophila FK506-binding protein

A Drosophila cDNA encoding a structural homolog of mammalian FKBP59 (also identified as FKBP52), a member of the FK506-binding protein (FKBP) class of immunophilins, was isolated. The gene dFKBP59 corresponding to this cDNA has been characterized and mapped to the 30D3-4 region. The predicted amino acid sequence of this cDNA shows that the dFKBP59 protein contains one highly conserved FKBP12-like domain followed by two others with less conservation. Northern hybridization reveals that the dFKBP59 mRNA is expressed throughout the Drosophila life-cycle. In contrast to its mammalian homologs, in situ hybridization detected dFKBP59 expression in specific tissues: the lymph glands, Garland cells and oenocyte cells, which are all specialized tissues in which intensive exocytic/endocytic cycling takes place. Garland cells and oenocytes (also called Drosophila nephrocytes) function in taking up waste material from the hemolymph. Finally, I have mapped an enhancer trap element within the 5' region of dFKBP59 which may help in future studies to address the question of its function during Drosophila development.     

Open channel block by Ca2+ underlies the voltage dependence of drosophila TRPL channel

The light-activated channels of Drosophila photoreceptors transient receptor potential (TRP) and TRP-like (TRPL) show voltage-dependent conductance during illumination. Recent studies implied that mammalian members of the TRP family, which belong to the TRPV and TRPM subfamilies, are intrinsically voltage-gated channels. However, it is unclear whether the Drosophila TRPs, which belong to the TRPC subfamily, share the same voltage-dependent gating mechanism. Exploring the voltage dependence of Drosophila TRPL expressed in S2 cells, we found that the voltage dependence of this channel is not an intrinsic property since it became linear upon removal of divalent cations. We further found that Ca(2+) blocked TRPL in a voltage-dependent manner by an open channel block mechanism, which determines the frequency of channel openings and constitutes the sole parameter that underlies its voltage dependence. Whole cell recordings from a Drosophila mutant expressing only TRPL indicated that Ca(2+) block also accounts for the voltage dependence of the native TRPL channels. The open channel block by Ca(2+) that we characterized is a useful mechanism to improve the signal to noise ratio of the response to intense light when virtually all the large conductance TRPL channels are blocked and only the low conductance TRP channels with lower Ca(2+) affinity are active.     

FK506-binding protein (FKBP12) regulates ryanodine receptor-evoked Ca2+ release in colonic but not aortic smooth muscle

In smooth muscle, the ryanodine receptor (RyR) mediates Ca(2+) release from the sarcoplasmic reticulum (SR) Ca(2+) store. Release may be regulated by the RyR accessory FK506-binding protein (FKBP12) either directly, as a result of FKBP12 binding to RyR, or indirectly via modulation of the activity of the phosphatase calcineurin or kinase mTOR. Here we report that RyR-mediated Ca(2+) release is modulated by FKBP12 in colonic but not aortic myocytes. Neither calcineurin nor mTOR are required for FKBP12 modulation of Ca(2+) release in colonic myocytes to occur. In colonic myocytes, co-immunoprecipitation techniques established that FKBP12 and calcineurin each associated with the RyR2 receptor isoform (the main isoform in this tissue). Single colonic myocytes were voltage clamped in the whole cell configuration and cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) increases evoked by the RyR activator caffeine. Under these conditions FK506, which displaces FKBP12 (to inhibit calcineurin) and rapamycin, which displaces FKBP12 (to inhibit mTOR), each increased the [Ca(2+)](c) rise evoked by caffeine. Notwithstanding, neither mTOR nor calcineurin are required to potentiate caffeine-evoked Ca(2+) increases evoked by each drug. Thus, the mTOR and phosphatidylinositol 3-kinase inhibitor, LY294002, which directly inhibits mTOR without removing FKBP12 from RyR, did not alter caffeine-evoked [Ca(2+)](c) transients. Nor did inhibition of calcineurin by cypermethrin, okadaic acid or calcineurin inhibitory peptide block the FK506-induced increase in RyR-mediated Ca(2+) release. In aorta, although RyR3 (the main isoform), FKBP12 and calcineurin were each present, RyR-mediated Ca(2+) release was unaffected by either FK506, rapamycin or the calcineurin inhibitors cypermethrin and okadaic acid in single voltage clamped aortic myocytes. Presumably failure of FKBP12 to associate with RyR3 resulted in the immunosuppressant drugs (FK506 and rapamycin) being unable to alter the activity of RyR. The effects of these drugs are therefore, apparently dependent on an association of FKBP12 with RyR. Together, removal of FKBP12 from RyR augmented Ca(2+) release via the channel in colonic myocytes. Neither calcineurin nor mTOR are required for the FK506- or rapamycin-induced potentiation of RyR Ca(2+) release to occur. The results indicate that FKBP12 directly inhibits RyR channel activity in colonic myocytes but not in aorta.     

Removal of FKBP12.6 does not alter the conductance and activation of the cardiac ryanodine receptor or the susceptibility to stress-induced ventricular arrhythmias

The 12.6-kDa FK506-binding protein (FKBP12.6) is considered to be a key regulator of the cardiac ryanodine receptor (RyR2), but its precise role in RyR2 function is complex and controversial. In the present study we investigated the impact of FKBP12.6 removal on the properties of the RyR2 channel and the propensity for spontaneous Ca(2+) release and the occurrence of ventricular arrhythmias. Single channel recordings in lipid bilayers showed that FK506 treatment of recombinant RyR2 co-expressed with or without FKBP12.6 or native canine RyR2 did not induce long-lived subconductance states. [(3)H]Ryanodine binding studies revealed that coexpression with or without FKBP12.6 or treatment with or without FK506 did not alter the sensitivity of RyR2 to activation by Ca(2+) or caffeine. Furthermore, single cell Ca(2+) imaging analyses demonstrated that HEK293 cells co-expressing RyR2 and FKBP12.6 or expressing RyR2 alone displayed the same propensity for spontaneous Ca(2+) release or store overload-induced Ca(2+) release (SOICR). FK506 increased the amplitude and decreased the frequency of SOICR in HEK293 cells expressing RyR2 with or without FKBP12.6, indicating that the action of FK506 on SOICR is independent of FKBP12.6. As with recombinant RyR2, the conductance and ligand-gating properties of single RyR2 channels from FKBP12.6-null mice were indistinguishable from those of single wild type channels. Moreover, FKBP12.6-null mice did not exhibit enhanced susceptibility to stress-induced ventricular arrhythmias, in contrast to previous reports. Collectively, our results demonstrate that the loss of FKBP12.6 has no significant effect on the conduction and activation of RyR2 or the propensity for spontaneous Ca(2+) release and stress-induced ventricular arrhythmias.     

Translocation of the Drosophila transient receptor potential-like (TRPL) channel requires both the N- and C-terminal regions together with sustained Ca2+ entry

In Drosophila photoreceptors the transient receptor potential-like (TRPL), but not the TRP channels undergo light-dependent translocation between the rhabdomere and cell body. Here we studied which of the TRPL channel segments are essential for translocation and why the TRP channels are required for inducing TRPL translocation. We generated transgenic flies expressing chimeric TRP and TRPL proteins that formed functional light-activated channels. Translocation was induced only in chimera containing both the N- and C-terminal segments of TRPL. Using an inactive trp mutation and overexpressing the Na(+)/Ca(2+) exchanger revealed that the essential function of the TRP channels in TRPL translocation is to enhance Ca(2+)-influx. These results indicate that motifs present at both the N and C termini as well as sustained Ca(2+) entry are required for proper channel translocation.     

Phosphorylation of the insect immunophilin FKBP46 by the Spodoptera frugiperda homolog of casein kinase II

Immunophilins are a family of conserved proteins found in both prokaryotes and eukaryotes, that exhibit peptidylprolyl isomerase (PPIase) activity. Members of this family bind to immunosuppressive drugs and on this basis are divided into two classes: FKBPs bind to FK506 and rapamycin, while cyclophilins bind to cyclosporin A. In this paper, we report on insect immunophilin FKBP46 and its associated kinase. The insect FKBP46 belongs to the high-molecular-weight immunophilins and shares many characteristic features with its mammalian counterparts, but its functional role remains unclear. Here, we show that FKBP46 is phosphorylated by a protein kinase present in the nucleus of both insect Spodoptera frugiperda (Sf9) and human Jurkat cells. This protein kinase is immunoreactive with polyclonal antiserum raised against Drosophila melanogaster casein kinase II (CKII). We have cloned, overexpressed and characterized a new member of the CKII family derived from Spodoptera frugiperda cells. Recombinant Sf9 CKII alpha subunit shares 75% identity to human, chicken and Drosophila melanogaster homologs, whereas the Sf9 CKII beta subunit is 77% identical to rat, chicken and human. Moreover, we demonstrate that the insect immunophilin FKBP46 can be phosphorylated by human and Sf9 casein kinase II. Finally, we show that FKBP46 interacts with DNA, and this interaction is not prevented by phosphorylation.     

Reversible phosphorylation of the signal transduction complex in Drosophila photoreceptors

In the Drosophila visual cascade, the transient receptor potential (TRP) calcium channel, phospholipase Cbeta (no-receptor-potential A), and an eye-specific isoform of protein kinase C (eye-PKC) comprise a multimolecular signaling complex via their interaction with the scaffold protein INAD. Previously, we showed that the interaction between INAD and eye-PKC is a prerequisite for deactivation of a light response, suggesting eye-PKC phosphorylates proteins in the complex. To identify substrates of eye-PKC, we immunoprecipitated the complex from head lysates using anti-INAD antibodies and performed in vitro kinase assays. Wild-type immunocomplexes incubated with [(32)P]ATP revealed phosphorylation of TRP and INAD. In contrast, immunocomplexes from inaC mutants missing eye-PKC, displayed no phosphorylation of TRP or INAD. We also investigated protein phosphatases that may be involved in the dephosphorylation of proteins in the complex. Dephosphorylation of TRP and INAD was partially suppressed by the protein phosphatase inhibitors okadaic acid, microcystin, and protein phosphatase inhibitor-2. These phosphatase activities were enriched in the cytosol of wild-type heads, but drastically reduced in extracts prepared from glass mutants, which lack photoreceptors. Our findings indicate that INAD functions as RACK (receptor for activated PKC), allowing eye-PKC to phosphorylate INAD and TRP. Furthermore, dephosphorylation of INAD and TRP is catalyzed by PP1/PP2A-like enzymes preferentially expressed in photoreceptor cells.     

Ca(2+)-dependent interaction between FKBP12 and calcineurin regulates activity of the Ca(2+) release channel in skeletal muscle

Calcineurin is a Ca(2+) and calmodulin-dependent protein phosphatase with diverse cellular functions. Here we examined the physical and functional interactions between calcineurin and ryanodine receptor (RyR) in a C2C12 cell line derived from mouse skeletal muscle. Coimmunoprecipitation experiments revealed that the association between RyR and calcineurin exhibits a strong Ca(2+) dependence. This association involves a Ca(2+) dependent interaction between calcineurin and FK506-binding protein (FKBP12), an accessory subunit of RyR. Pretreatment with cyclosporin A, an inhibitor of calcineurin, enhanced the caffeine-induced Ca(2+) release (CICR) in C2C12 cells. This effect was similar to those of FK506 and rapamycin, two drugs known to cause dissociation of FKBP12 from RyR. Overexpression of a constitutively active form of calcineurin in C2C12 cells, DeltaCnA(391-521) (deletion of the last 131 amino acids from calcineurin), resulted in a decrease in CICR. This decrease in CICR activity was partially recovered by pretreatment with cyclosporin A. Furthermore, overexpression of an endogenous calcineurin inhibitor (cain) or an inactive form of calcineurin (DeltaCnA(H101Q)) in C2C12 cells resulted in up-regulation of CICR. Taken together, our data suggest that a trimeric-interaction among calcineurin, FKBP12, and RyR is important for the regulation of the RyR channel activity and may play an important role in the Ca(2+) signaling of muscle contraction and relaxation.     

TRP, TRPL and Cacophony Channels Mediate Ca(2+) Influx and Exocytosis in Photoreceptors Axons in Drosophila

In Drosophila photoreceptors Ca(2+)-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, we studied the distribution and physiological properties of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca(2+) channel cacophony is also present there. Measurements in the lamina with the Ca(2+) fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca(2+) increments mediated by cacophony. Additional Ca(2+) influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca(2+) also contributes to exocytosis, since this process was reduced after Ca(2+)-store depletion. Therefore, several Ca(2+) pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves cacophony; this is complemented by internal Ca(2+) release and the activation of TRP and TRPL coupled to Ca(2+) depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons.     

Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor.

FK506-binding protein (FKBP12) has been found to be associated with the skeletal muscle ryanodine receptor (RyR1) (calcium release channel), whereas FKBP12.6, a novel isoform of FKBP, is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To assess the amino acid residues of FKBP12.6 that are critical for selective binding to RyR2, the residues of FKBP12.6 that differ with FKBP12 were mutated to the respective residues of FKBP12. RyR2 of cardiac sarcoplasmic reticulum, prelabeled by exchange with [35S]FKBP12.6, was used as assay system for binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W) of FKBP12.6 was found to lack selective binding to the cardiac RyR2, comparable with that of FKBP12.0. In complementary studies, mutations of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three amino acid residues (Gln31, Asn32, and Phe59) of human FKBP12.6 account for the selective binding to cardiac RyR2.     

The INAD scaffold is a dynamic, redox-regulated modulator of signaling in the Drosophila eye

INAD is a scaffolding protein that regulates signaling in Drosophila photoreceptors. One of its PDZ domains, PDZ5, cycles between reduced and oxidized forms in response to light, but it is unclear how light affects its redox potential. Through biochemical and structural studies, we show that the redox potential of PDZ5 is allosterically regulated by its interaction with another INAD domain, PDZ4. Whereas isolated PDZ5 is stable in the oxidized state, formation of a PDZ45 "supramodule" locks PDZ5 in?the reduced state by raising the redox potential of?its Cys606/Cys645 disulfide bond by ～330?mV. Acidification, potentially mediated via light and PLCβ-mediated hydrolysis of PIP(2), disrupts the interaction between PDZ4 and PDZ5, leading to PDZ5 oxidation and dissociation from the TRP Ca(2+) channel, a key component of fly visual signaling. These results show that scaffolding proteins can actively modulate the intrinsic redox potentials of their disulfide bonds to exert regulatory roles in signaling.     

Activation of the Drosophila TRP and TRPL channels requires both Ca2+ and protein dephosphorylation

The Transient Receptor Potential (TRP) proteins constitute a large and diverse family of channel proteins, which is conserved through evolution. TRP channel proteins have critical functions in many tissues and cell types, but their gating mechanism is an enigma. In the present study patch-clamp whole-cell recordings was applied to measure the TRP- and TRP-like (TRPL)-dependent currents in isolated Drosophila ommatidia. Also, voltage responses to light and to metabolic stress were recorded from the eye in vivo. We report new insight into the gating of the Drosophila light-sensitive TRP and TRPL channels, by which both Ca2+ and protein dephosphorylation are required for channel activation. ATP depletion or inhibition of protein kinase C activated the TRP channels, while photo-release of caged ATP or application of phorbol ester antagonized channels openings in the dark. Furthermore, Mg(2+)-dependent stable phosphorylation event by ATPgammaS or protein phosphatase inhibition by calyculin A abolished activation of the TRP and TRPL channels. While a high reduction of cellular Ca2+ abolished channel activation, subsequent application of Ca2+ combined with ATP depletion induced a robust dark current that was reminiscent of light responses. The results suggest that the combined action of Ca2+ and protein dephosphorylation activate the TRP and TRPL channels, while protein phosphorylation by PKC antagonized channels openings.     

N-terminal region of FKBP12 is essential for binding to the skeletal ryanodine receptor

It is known that the two types of FK506-binding proteins FKBP12 and FKBP12.6 are tightly associated with the skeletal (RyR1) and cardiac ryanodine receptors (RyR2), respectively, and their interactions are important for channel functions of the RyR. In the case of cardiac muscle, three amino acid residues (Gln-31, Asn-32, and Phe-59) of FKBP12.6 could be essential for the selective binding to RyR2 (Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Fleischer, S. (1999) J. Biol. Chem. 274, 15315-15319). In this study to identify amino acid residues of FKBP12 that are important for the selective binding to RyR1, we mutated 9 amino acid residues of FKBP12 that differ from the counterparts of FKBP12.6 (Q3E, R18A, E31Q, D32N, M49R, R57A, W59F, H94A, and K105A), and we examined binding properties of these mutants to RyR1 by in vitro binding assay by using glutathione S-transferase-fused proteins of the mutants and Triton X-100-solubilized, FKBP12-depleted rabbit skeletal sarcoplasmic reticulum vesicles. Among the nine mutants tested, only Q3E and R18A lost their selective binding ability to RyR1. Furthermore, co-immunoprecipitation of RyR1 with 33 various mutants for the 9 positions produced by introducing different size, charge, and hydrophobicity revealed that an integration of the hydrogen bonds by the irreplaceable Gln-3 and the hydrophobic interactions by the residues Arg-18 and Met-49 could be a possible mechanism for the binding of FKBP12 to RyR1. Therefore, these results suggest that the N-terminal regions of FKBP12 (Gln-3 and Arg-18) and Met-49 are essential and unique for binding of FKBP12 to RyR1 in skeletal muscle.     

Selective association of TRPC channel subunits in rat brain synaptosomes

TRPC genes encode a ubiquitous family of ion channel proteins responsible for Ca(2+) influx following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. These channels may be localized to large multimeric signaling complexes via association with PDZ-containing scaffolding proteins. Based on sequence homology, the TRPC channel family can be divided into two major subgroups: TRPC1, -C4, and -C5 and TRPC3, -C6, and -C7. Although TRPC channels are thought to be tetramers, the actual subunit composition remains unknown. To determine subunit arrangement, individual TRPC channel pairs were heterologously expressed in Sf9 insect cells and immunoprecipitated using affinity-purified rabbit polyclonal antibodies specific for each channel subtype. Reciprocal co-immunoprecipitations showed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate but that cross-association between the two major subgroups does not occur. Additionally, the interaction between each TRPC channel and the PDZ-containing protein, INAD (protein responsible for the inactivation-no-after-potential Drosophila mutant), was examined. TRPC1, -C4, and -C5 co-immunoprecipitated with INAD, whereas TRPC3, -C6, and -C7 did not. To define channel subunit interactions in vivo, immunoprecipitations were performed from isolated rat brain synaptosomal preparations. The results revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur. These results demonstrate that TRPC channels are present in nerve terminals and provide the first direct evidence for selective assembly of channel subunits in vivo.     

Dissecting independent channel and scaffolding roles of the Drosophila transient receptor potential channel

Drosophila transient receptor potential (TRP) serves dual roles as a cation channel and as a molecular anchor for the PDZ protein, INAD (inactivation no afterpotential D). Null mutations in trp cause impairment of visual transduction, mislocalization of INAD, and retinal degeneration. However, the impact of specifically altering TRP channel function is not known because existing loss-of-function alleles greatly reduce protein expression. In the current study we describe the isolation of a set of new trp alleles, including trp(14) with an amino acid substitution juxtaposed to the TRP domain. The trp(14) flies stably express TRP and display normal molecular anchoring, but defective channel function. Elimination of the anchoring function alone in trp(Delta)(1272), had minor effects on retinal morphology whereas disruption of channel function caused profound light-induced cell death. This retinal degeneration was greatly suppressed by elimination of the Na(+)/Ca(2+) exchanger, CalX, indicating that the cell death was due primarily to deficient Ca(2+) entry rather than disruption of the TRP-anchoring function.     

Role of FKBP12.6 in cADPR-induced activation of reconstituted ryanodine receptors from arterial smooth muscle

cADP ribose (cADPR) serves as second messenger to activate the ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) and mobilize intracellular Ca(2+) in vascular smooth muscle cells. However, the mechanisms mediating the effect of cADPR remain unknown. The present study was designed to determine whether FK-506 binding protein 12.6 (FKBP12.6), an accessory protein of the RyRs, plays a role in cADPR-induced activation of the RyRs. A 12.6-kDa protein was detected in bovine coronary arterial smooth muscle (BCASM) and cultured CASM cells by being immunoblotted with an antibody against FKBP12, which also reacted with FKBP12.6. With the use of planar lipid bilayer clamping techniques, FK-506 (0.01-10 microM) significantly increased the open probability (NP(O)) of reconstituted RyR/Ca(2+) release channels from the SR of CASM. This FK-506-induced activation of RyR/Ca(2+) release channels was abolished by pretreatment with anti-FKBP12 antibody. The RyRs activator cADPR (0.1-10 microM) markedly increased the activity of RyR/Ca(2+) release channels. In the presence of FK-506, cADPR did not further increase the NP(O) of RyR/Ca(2+) release channels. Addition of anti-FKBP12 antibody also completely blocked cADPR-induced activation of these channels, and removal of FKBP12.6 by preincubation with FK-506 and subsequent gradient centrifugation abolished cADPR-induced increase in the NP(O) of RyR/Ca(2+) release channels. We conclude that FKBP12.6 plays a critical role in mediating cADPR-induced activation of RyR/Ca(2+) release channels from the SR of BCASM.     

Coordination of an Array of Signaling Proteins through Homo- and Heteromeric Interactions Between PDZ Domains and Target Proteins

The rapid activation and feedback regulation of many G protein signaling cascades raises the possibility that the critical signaling proteins may be tightly coupled. Previous studies show that the PDZ domain containing protein INAD, which functions in Drosophila vision, coordinates a signaling complex by binding directly to the light-sensitive ion channel, TRP, and to phospholipase C (PLC). The INAD signaling complex also includes rhodopsin, protein kinase C (PKC), and calmodulin, though it is not known whether these proteins bind to INAD. In the current work, we show that rhodopsin, calmodulin, and PKC associate with the signaling complex by direct binding to INAD. We also found that a second ion channel, TRPL, bound to INAD. Thus, most of the proteins involved directly in phototransduction appear to bind to INAD. Furthermore, we found that INAD formed homopolymers and the homomultimerization occurred through two PDZ domains. Thus, we propose that the INAD supramolecular complex is a higher order signaling web consisting of an extended network of INAD molecules through which a G protein–coupled cascade is tethered.