Distinct Roles of Inositol 1,4,5-Trisphosphate Receptor Types 1 and 3 in Ca2+ Signaling

Mitsuharu Hattori,Akinobu Z Suzuki,Takayasu Higo,Hiroshi Miyauchi,Takayuki Michikawa,Takeshi Nakamura,Takafumi Inoue,Katsuhiko Mikoshiba

doi:10.1074/jbc.m311456200

Mitsuharu Hattori, Akinobu Z Suzuki + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m311456200

Copy DOI

Abstract

Three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R1, IP(3)R2, and IP(3)R3) Ca(2+) release channel share basic properties but differ in terms of regulation. To what extent they contribute to complex Ca(2+) signaling, such as Ca(2+) oscillations, remains largely unknown. Here we show that HeLa cells express comparable amounts of IP(3)R1 and IP(3)R3, but knockdown by RNA interference of each subtype results in dramatically distinct Ca(2+) signaling patterns. Knockdown of IP(3)R1 significantly decreases total Ca(2+) signals and terminates Ca(2+) oscillations. Conversely, knockdown of IP(3)R3 leads to more robust and long lasting Ca(2+) oscillations than in controls. Effects of IP(3)R3 knockdown are surprisingly similar in COS-7 cells that predominantly (>90% of total IP(3)R) express IP(3)R3, suggesting that IP(3)R3 functions as an anti-Ca(2+)-oscillatory unit without contributing to peak amplitude of Ca(2+) signals, irrespective of its relative expression level. Therefore, differential expression of the IP(3)R subtype is critical for various forms of Ca(2+) signaling, and, particularly, IP(3)R1 and IP(3)R3 have opposite roles in generating Ca(2+) oscillations.

Highlights

Three subtypes of inositol 1,4,5-trisphosphate receptor (IP3R1, IP3R2, and IP3R3) Ca2؉ release channel share basic properties but differ in terms of regulation
Activation of the phospholipase C pathway by hormones, growth factors, and neurotransmitters results in generation of a second messenger inositol 1,4,5-trisphosphate (IP3),1 which diffuses to the cytoplasm and binds to an IP3 receptor (IP3R), a Ca2ϩ release channel on the endoplasmic reticulum (ER) [1]
To confirm that our pan-IP3R antibody recognized all the IP3R subtypes the microsome fraction from Sf9 cells overexpressing each IP3R subtype was separated with SDSPAGE, followed by silver staining (Fig. 1B, left panel) or immunoblotting using the pan-IP3R antibody (Fig. 1B, right panel)

Summary

EXPERIMENTAL PROCEDURES

Reagents—Short interfering RNA (siRNA) duplexes were purchased from Dharmacon (Lafayette, CO) and resolved in Universal buffer (20 mM KCl, 6 mM HEPES-KOH (pH 7.5), 0.2 mM MgCl2) to a final concentration of 20 ␮M. Microsome fractions from Sf9 cells overexpressing murine IP3R2 were kindly provided by Drs Miwako Iwai and Takayuki Michikawa in our department. The entire mixture was added to the cells, resulting in a final concentration of 40 nM for the siRNAs. The cells were incubated for 5– 8 h in a CO2 chamber, washed once, and supplied with 2 ml of the fresh culture medium. After three washes with PBST, the membrane was incubated with horseradish peroxidaseconjugated secondary antibody (Amersham Biosciences; 1:4000) for 1 h at room temperature. The fura-2-loaded cells were placed on the stage of an inverted fluorescence microscope (IX-70; Olympus, Japan) and perfused with balanced salt solution (115 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose, 20 mM HEPES (pH 7.4), and either 2 mM CaCl2 or nominally Ca2ϩ free), at a rate of 2.0 ml/min. With alternate illumination at 340- and 380-nm excitation, pairs of fluorescence images (F340 and F380, respectively) were obtained every 3 s through an objective lens (UApo 20ϫ/340; Olympus) and an emission filter (510 –550 nm), captured with a siliconintensified target video camera (C2400 – 8; Hamamatsu Photonics) and digitized with an image processor

RESULTS

TABLE I Sequence of siRNAs

DISCUSSION