A 17-aa IP₃R peptide displaces cytochrome c from IP₃R. (A) A peptide encompassing the minimal cytochrome c binding domain (amino acids 2621-2636) with a C-terminal cysteine (IP3RCYT) was tested for the ability to disrupt cytochrome c/IP₃R binding. Cytochrome c (80 nM) was incubated with 100 nM GST-FL and varying concentrations of IP3RCYT. Complexes were immobilized on GST-Sepharose and analyzed for cytochrome c binding by Western blotting. A Ponceau stain of the nitrocellulose blot is shown to illustrate equal loading of GST-FL. (B) Densitometric analysis of cytochrome c pull-down and subsequent graphing of percent binding (vs. no peptide) demonstrates an IC₅₀ value of 106 nM for IP3RCYT. Data were fit to the monophasic Hill equation y = min + (max-min)/1 + (x/IC₅₀)^Hillslope. The experiment was repeated three times with essentially identical results.

Extrinsic apoptotic signaling via FasL is associated with cytochrome c binding to IP₃R and calcium mobilization. (A) Jurkat cells were treated with 1 ng/ml FasL vesicles for 0, 12, or 24 h, and the subcellular distribution of cytochrome c (CytC), cytochrome c oxidase (CytOx), IP₃R, heme oxygenase-2 (HO2), and lactate dehydrogenase (LDH) was monitored in the mitochondrial-enriched 10,000 × g pellet (P2), the 100,000 × g cytosol (S3), or the light membrane-enriched 100,000 × g pellet (P3). (B) Immunoprecipitation of IP₃R from P3 fraction solubilized in buffer A with anti-IP₃R or preimmune serum. Samples were immunoblotted for IP₃R and cytochrome c. (C) DEVD cleavage activity of the S3 fraction. (D and E) Calcium mobilization in response to FasL examined in individual fura 2-acetoxymethyl ester-loaded Jurkat cells. A representative single cell (D) and 50-cell average (E) from one experiment are shown. Approximately 30% of cells responded with an immediate and oscillatory rise in cytoplasmic calcium in response to FasL. All data are from the same experiment and are representative of at least three separate determinations.

Cytochrome c binding determinants of IP₃R. (A) The IP₃R domain structure includes an N-terminal IP₃ binding domain (IP3), the modulatory domain (MOD), and the C-terminal transmembrane domain (TM). Previously we demonstrated that cytochrome c binds IP₃R within amino acids 2589-2749 encompassing the full-length C terminus (FL) (20). Three 80-aa fragments of FL (CT1, CT2, and CT3) were generated to facilitate cytochrome c mapping. Rat type I IP₃R amino acid boundaries are indicated. (B) HEK 293 lysates expressing FL, CT1, CT2, and CT3 were subjected to in vitro pull-down with cytochrome c agarose. Only CT1 (amino acids 2589-2669) bound cytochrome c agarose. Blot is anti-HA. (C) Schematic diagram of ≈20-aa deletion constructs of FL within amino acids 2589-2669. (D) Binding of deletion constructs to cytochrome c agarose. IP₃R fragment 2621 binds, whereas 2637, 2653, and CT2 do not. Construct 2605 had minimal binding but expressed at very low levels and was susceptible to degradation (data not shown), suggesting improper folding. Experiments were performed exactly as in B. (E) Sequence and physicochemical properties of amino acids 2621-2636. Arrows indicate a cluster of glutamic acid residues. (F) Effect on cytochrome c binding of mutations replacing glutamic acids residues 2628 and 2629 to glutamine (E2628/2629Q) or glutamic acid residues 2633 and 2634 to glutamine (E2633/2634Q). Both mutations eliminate binding to cytochrome c agarose.

A cell permeant fluorescent derivative of IP3RCYT blocks caspase activation and cell death in HeLa cells. (A) Schematic diagram of IP3RCYT peptide conjugated to BODIPY 577/618 via maleimide linkage to the C-terminal cysteine. (B) Phase and confocal fluorescent images of HeLa cells incubated with or without 400 nM BODIPY-IP3RCYT. Image capture times were equal. Note the diffuse cytoplasmic localization. (C) HeLa cells pretreated for 30 min with varying concentrations of BODIPY-IP3RCYT were stimulated with 1 μM STS for 6 h. Caspase activity was measured fluorometrically by using Z-DEVD-AMC as a substrate. Data were fit by using the Hill equation. Maximal inhibition was ≈30% of basal caspase activity (Inset). (D) HeLa cells pretreated with 400 nM BODIPY-IP3RCYT were stimulated with 1 μM STS for 6 h. Viability was assessed by trypan blue staining of nuclei. Cells were counted blind to the treatment, and at least 5,000 cells were scored over three separate experiments. *, P < 0.01.

Fas-mediated cell death is attenuated by BODIPY-IP3RCYT. (A) Phase and confocal fluorescent images of Jurkat cells incubated with or without 400 nM BODIPY-IP3RCYT. Image capture times were equal. (B and C) Jurkat cells pretreated with 400 nM BODIPY-IP3RCYT or vehicle for 30 min and 1 ng/ml FasL vesicles were added for the indicated amount of time. Trypan blue staining (B) and DEVD-cleavage activity (C) were assayed as in Fig. 3. *, P < 0.05.