The Spatial Distribution of Inositol 1,4,5-Trisphosphate Receptor Isoforms Shapes Ca2+ Waves

Erick Hernandez,M Fatima Leite,Mateus T Guerra,Emma A Kruglov,Oscar Bruna-Romero,Michele A Rodrigues,Dawidson A Gomes,Frank J Giordano,Jonathan A Dranoff,Michael H Nathanson

doi:10.1074/jbc.m700746200

Erick Hernandez, M Fatima Leite + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m700746200

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Abstract

Cytosolic Ca(2+) is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca(2+) waves and other spatial patterns of Ca(2+) signals. To investigate the mechanism responsible for the formation of Ca(2+) waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca(2+) wave formation. Ca(2+) signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca(2+) signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca(2+) waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca(2+) waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca(2+) waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca(2+) signals.

Highlights

Electrolyte secretion, exocytosis, cell-cell communication, and morphogenesis (3–7)
Because the type II InsP3R is the isoform with the highest affinity for InsP3 (17), it has been suggested that Ca2ϩ waves begin in the apical region of hepatocytes because the type II isoform is concentrated there (8)
AR4-2J cells were used as a tool to examine the efficacy of the adenoviral antisense constructs because these cells, like hepatocytes, are epithelia that almost exclusively express the types I and II InsP3R (20)

Summary

Introduction

Electrolyte secretion, exocytosis, cell-cell communication, and morphogenesis (3–7). Ca2ϩ wave formation is due entirely to the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R), because that is the only intracellular Ca2ϩ release channel in this cell type (8) and because Ca2ϩ waves in hepatocytes do not rely on influx of extracellular Ca2ϩ (9). The type I InsP3R is distributed relatively uniformly throughout the hepatocyte, whereas the type II isoform is concentrated in the apical region (8). Because the type II InsP3R is the isoform with the highest affinity for InsP3 (17), it has been suggested that Ca2ϩ waves begin in the apical region of hepatocytes because the type II isoform is concentrated there (8). The purpose of this study was to determine whether the subcellular distribution of InsP3R isoforms is important for organizing the pattern of Ca2ϩ waves

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