Schematic diagrams of sigma-1 receptor constructs. Diagram is modified from Aydar et al. (27). A, wild-type, aa 1–223 (cell line 11) and Kozak-modified, aa 1–223 with Gln-2 changed to Glu-2 (cell line 41); B, N-terminal construct, aa 1–100 (cell line K3); C, C-terminal construct, aa 102–223 (cell line sg101).

Screening of cell lines for stable expression of target sigma-1 receptor mRNA sequences. Total RNA was extracted from cells by the guanidinium thiocyanate method and reverse-transcribed as described under “Experimental Procedures.” For PCR screening, 2 μl of reverse-transcribed cDNA and 1 μl of dNTPs were added to a conventional buffer mix containing 10 pmol of the appropriate 5′ and 3′ cDNA primers and 1 unit of Taq polymerase, and the reaction was carried out as described under “Experimental Procedures.” The specific 5′ and 3′ cDNA primers used to amplify the individual sigma-1 receptor-related sequences are shown in Table 2. The lanes of the gels are labeled as follows: Total = amplification of total sigma-1-related mRNA (endogenously expressed + expression from vector); Vector = amplification of sigma-1-related sequence carried on the designated vector; Endo = detection of endogenously expressed sigma-1-related sequence; Standard = amplification of a standard representing the sequence in question (control sequence). A, sigma-1 (wild type) PCR products in MCF-7 cells. The vector used here was an empty vector control. B, PCR products in line 41 cells (Kozak modification) and line 11 cells (wild type). C, PCR products in Line K3 cells (N-terminal fragment; aa 1–100). D, PCR products in line sg101 cells (C-terminal fragment; aa 102–223).

Western blot analysis of cell lines for stable expression of target sigma-1 receptor proteins. Extracts of each cell line were prepared and subjected to SDS-PAGE and Western blotting as described under “Experimental Procedures,” using commercially available anti-sigma-1 receptor antibody directed at either an N-terminal or C-terminal epitope of the receptor (Santa Cruz Biotechnology). Images shown are from chemiluminescence visualization. The 10–27-kDa region of the blot is shown. A, C-terminal directed antibody at 1:1000 dilution, detecting 25-kDa intact sigma-1 receptor (M7 = untransfected MCF-7 cells). B, C-terminal directed antibody at 1:100 dilution, detecting 13.7-kDa C-terminal fragment. C, N-terminal directed antibody at 1:200 dilution, detecting 11.4-kDa N-terminal fragment. D, membrane distribution and attachment of C-terminal fragment. Line sg101 cells were homogenized and fractionated into crude plasma membrane/mitochondrial, crude microsomal, and cytosolic fractions, as described under “Experimental Procedures.” The ER fraction was then treated with sequential exposure to chaotropic agents (200 mm KCl, 0.1% (w/v) sodium deoxycholate, and 1 m NaSCN) as described under “Experimental Procedures.” The treated membranes and the resultant wash fraction (Sol) were subjected to Western blot analysis using a C-terminal directed anti-sigma-1 receptor antibody (1:100 dilution) and compared with untreated membranes and cell cytosol (Cytos).

Saturation binding and Scatchard analysis of [³H](+)-pentazocine binding to membranes from MCF-7 cells and transfected MCF-7 cell lines. A total membrane fraction was prepared from each cell line as described under “Experimental Procedures.” Membranes were then incubated with [³H](+)-pentazocine under the conditions described under “Experimental Procedures” using either the “hot only” or “hot + cold” methods as indicated. Saturation isotherm (left) and Scatchard plot (right) are shown for each cell line. K_d and B_max values were determined by analysis using GraphPad Prism 4 (San Diego, CA). Values shown are averages of three experiments ± S.E., each carried out in triplicate. K_d and B_max values in each cell line are summarized in Table 3 for convenience. A, untransfected MCF-7 cells. To obtain the full range of concentrations up to 1,000 nm, a combination of hot only and hot + cold incubations were used. Four concentrations of [³H](+)-pentazocine up to 10.3 nm were used, followed by combination of 10.3 nm [³H](+)-pentazocine with four concentrations of unlabeled (+)-pentazocine up to 1,000 nm to give a total of eight points. The data were fit to both one-site and two-site binding models. The absolute sum of squares values revealed that the models were not statistically different, thus the simplest model is used. K_d = 393 ± 181 nm; B_max = 1,431 ± 281 fmol/mg protein. B, line 11 cells. Membranes were incubated with 12 concentrations of [³H](+)-pentazocine ranging from 0.1 to 53.1 nm. K_d = 7.43 ± 3.98 nm; B_max = 16,710 ± 4,710 fmol/mg protein. C, line 41 cells. Membranes were incubated with 12 concentrations of [³H](+)-pentazocine ranging from 0.1 to 60.2 nm. K_d = 3.88 ± 2.72 nm; B_max = 31,110 ± 4,480 fmol/mg protein. D, line sg101 cells. Membranes were incubated with labeled and unlabeled (+)-pentazocine up to a concentration of 1,000 nm, as described in A above. K_d = 493 ± 190 nm; B_max = 1,438 ± 253 fmol/mg protein. E, line K3 cells. Membranes were incubated with labeled and unlabeled (+)-pentazocine up to a concentration of 1,000 nm, as described in A above. The data were best fit to a two-site model, with K_d1 = 207 ± 45.5 nm; B_max1 = 654 ± 217 fmol/mg protein and K_d2 = > 1,000 nm; B_max2 > 3,000 fmol/mg protein.

Effect of sigma ligands on BDK-induced calcium release in untransfected MCF-7 cells. MCF-7 cells were loaded with fura-2 and stimulated with 60 nm BDK with or without pretreatment with various sigma ligands, and changes in [Ca²⁺]_i were measured as described under “Experimental Procedures.” The sigma ligands used were (+)-pentazocine (100 nm), BD1063 (30 μm), and the combination of (+)-pentazocine (100 nm) and BD1063 (30 μm). The sigma ligands were preincubated with cells for 16 min prior to rapid injection of BDK. A, representative time versus [Ca²⁺]_i traces. The individual traces were normalized by subtracting the base line. Each point is the averaged value from 6 wells. For clarity, the error bars have been removed. Standard error across the 6 wells was within 1.09%. The experiment for each curve was repeated 4–7 times with similar results. B, comparison of peak values from time versus [Ca²⁺]_i traces. Each value represents the mean of peak [Ca²⁺]_i from traces from six separate experiments, normalized relative to base line. Values shown are ± S.E. Relative to the control pretreated with no ligands, none of the ligands produced effects significantly different from control as analyzed by Student's t test. p = 0.1926, 0.8081, and 0.4472 for 100 nm PTZ; 30 μm BD1063; and 100 nm PTZ + 30 μm BD1063, respectively, relative to the control. p < 0.05 indicates significance.

Effect of BDK on [Ca²⁺]_i in cell lines expressing the sigma-1 receptor and its truncated forms. The various cell lines were loaded with Fura-2 as described under “Experimental Procedures.” After recording base line, BDK was rapidly injected to a final concentration of 60 nm, and [Ca²⁺]_i levels recorded and analyzed as described under “Experimental Procedures.” A, representative time versus [Ca²⁺]_i traces for the different cell lines are shown, with all values normalized to base line. Each point is the averaged value from 6 wells. For clarity, the error bars have been removed. Standard error across the 6 wells was within 5.0%. Experiment was repeated six times. B, comparison of peak values from time versus [Ca²⁺]_i traces. Each value represents the mean of peak [Ca²⁺]_i values from six separate experiments, ± S.E. Student's t test was used to analyze differences in the various transfected cell lines relative to untransfected MCF-7 cells (labeled M7). With the exception of cells transfected with empty vector, all of the constructs showed highly significant differences, relative to untransfected control: p < 0.0001 for 41, sg101, 11, and K3 cell lines. p = 0.0766 for cells transfected with empty vector. p < 0.05 indicates significance. Asterisks indicate a significant difference: * = p < 0.05; ** = p < 0.01; *** = p < 0.0001.

Dose-response curves for BDK-induced calcium release in untransfected and transfected MCF-7 cell lines. MCF-7 cell lines were loaded with fura-2 and stimulated with 0.75, 1.5, 3.0, 7.5, 37.5, 75, 150, 300, and 600 nm BDK (without pretreatment with any sigma ligands), and changes in [Ca²⁺]_i were measured as described under “Experimental Procedures.” Values shown are the averages of data from 6 to 12 wells. Error bars are eliminated for clarity. Curves were fitted using GraphPad Prism 4 (San Diego, CA), and ED₅₀ values were determined. ED₅₀ values were as follows: untransfected MCF-7 (M-7), 85.5 nm; line 41, 3.28 nm; line 11, 12.1 nm; line sg101, 7.69 nm; and line K3, 3.07 nm. A similar maximal response was attained with all cell lines, with the exception of line K3, which exhibited a lower maximal response.

Effect of sigma ligands on BDK-induced calcium release in cell lines expressing the sigma-1 receptor and its truncated forms. Cells were loaded with Fura-2 and treated with or without sigma receptor ligands before stimulation with 60 nm BDK as described under “Experimental Procedures” and the legend of Fig. 5. Data were treated as described in the legend to Fig. 5. For each lettered panel, the graph on the left shows representative time versus [Ca²⁺]_i traces for each condition. The graph on the right is a comparison of peak values from the time versus [Ca²⁺]_i traces shown as a bar graph, with each value representing the mean of peak [Ca²⁺]_i values from six separate experiments, ± S.E. Student's t test was used to analyze differences in the various conditions relative to the control (no sigma ligand). Asterisks over bars indicate a significant difference: * = p < 0.05; ** = p < 0.01; *** = p < 0.0001. PTZ = (+)-pentazocine. A, line 11 cells as follows: p = 0.0058, <0.0001, and 0.0136 for 100 nm PTZ, 30 μm BD1063, and 100 nm PTZ + 30 μm BD1063, respectively, relative to the control. B, line 41 cells as follows: p = 0.0056, 0.0024, and 0.7573 for 100 nm PTZ, 30 μm BD1063, and 100 nm PTZ + 30 μm BD1063, respectively, relative to the control. C, line K3 cells as follows: p = 0.006, 0.0002, and 0.0008 for 100 nm PTZ, 30 μm BD1063, and 100 nm PTZ + 30 μm BD1063, respectively, relative to the control. D, line sg101 cells as follows: none of the sigma ligands had a significant effect on BDK-induced calcium changes. p = 0.3494, 0.2343, and 0.6947 for 100 nm PTZ, 30 μm BD1063, and 100 nm PTZ + 30 μm BD1063, respectively, relative to the control.

Immunoprecipitation of IP₃R-3 and ankyrin B in the five cell lines and analysis of complex content. A, identification of IP₃R-3 and ankyrin B in MCF-7 cells. MCF-7 cells (untransfected) were extracted and subjected to SDS-PAGE and Western blotting as described under “Experimental Procedures,” using mouse anti-ankyrin B or mouse anti-IP₃R-3 antibodies. Visualization is by chemiluminescence. IP₃R-1 (IP₃R type 1) was not detected in separate experiments using anti-IP₃R-1 antibody (data not shown). B, immunoprecipitation of IP₃R-3 and ankyrin B complexes. Untransfected MCF-7 cells and the lines expressing either wild-type or truncated sigma-1 receptors were cultured to about 90% confluency, lysed, and immunoprecipitated with anti-IP₃R-3 antibody as described under “Experimental Procedures.” The immunoprecipitate was then subjected to SDS-PAGE and Western blotting using anti-ankyrin B and anti-IP₃R-3 antibodies. Actin was used as internal standard for normalization of loading (not shown), and bands were visualized by chemiluminescence. C, immunoblots (shown in B) were digitally scanned and densitometrically analyzed using a Kodak Image Station 2000R, to obtain relative optical density units. Data were then expressed as a bar graph of ratio of ankyrin B220 over IP₃R-3. Data shown are mean ± S.E. from three separate determinations. Numbering corresponds to cell line order in B. Lines sg101, 11, and 41 all showed significantly lower ratios when compared with untransfected MCF-7 cells (M7), with p = 0.0096, 0.01, 0.0067, respectively. Line K3 showed a somewhat lower ratio, but was not significantly different from untransfected MCF-7 cells (p = 0.0858). Asterisks indicate a significant difference: * = p < 0.05; ** = p < 0.01; *** = p < 0.0001. D, extracts from the five cell lines were immunoprecipitated using anti-ankyrin B antibody and subjected to SDS-PAGE and Western blotting as described under “Experimental Procedures.” The blots were probed with C-terminal directed anti-sigma-1 antibody (1:100 dilution) to detect intact sigma-1 receptor and the C-terminal fragment and the N-terminal directed anti-sigma-1 antibody (1:200 dilution) to detect the N-terminal fragment.

Effect of vasopressin and ATP on [Ca²⁺]_i in cell lines expressing the sigma-1 receptor and its truncated forms. The various cell lines were loaded with Fura-2 as described under “Experimental Procedures.” After recording base line, vasopressin (final 800 nm) or ATP (final 50 μm) was rapidly injected, and [Ca²⁺]_i levels recorded and analyzed as described under “Experimental Procedures.” For each panel, the graph on the left shows representative time versus [Ca²⁺]_i traces for each cell line, with all values normalized to base line. Each point is the averaged value from 6 wells. For clarity, the error bars have been removed. Standard error across the 6 wells was within 4.0–5.7%. Experiment was repeated six times. The graph on the right is a comparison of peak values from the time versus [Ca²⁺]_i traces shown as a bar graph, with each value representing the mean of peak [Ca²⁺]_i values from six separate experiments, ± S.E. Student's t test was used to analyze differences across cell lines relative to the untransfected control. A, vasopressin, 800 nm. ***, p < 0.001 for lines 41, 11, sg101, and K3; p = 0.9816 for empty vector transfected. B, ATP, 50 μm. ***, p < 0.0001 for lines 41 and sg101; *, p = 0.0317 and 0.0349 for lines 11 and K3, respectively; p = 1.021 for empty vector transfected.