Abstract
Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity.
Highlights
One of the primary functions of intracellular Ca2+ signaling in polarized epithelia is the regulation of fluid and electrolyte secretion [1,2,3]
To determine which isoforms are expressed in the liver, we performed Real Time quantitaive PCR in whole rat liver extracts and primary rat hepatocytes in collagen sandwich culture
E-Syt1 and E-Syt2 were detected, whereas E-Syt3 was absent from both whole rat liver and rat hepatocytes
Summary
One of the primary functions of intracellular Ca2+ signaling in polarized epithelia is the regulation of fluid and electrolyte secretion [1,2,3]. Ca2+ signals in these cells are organized as polarized Ca2+ waves that are initiated apically due to local clustering of the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel [4, 5]. This apical targeting of InsP3Rs creates a ‘‘trigger zone’’ that allows local increases in Ca2+ concentration [4, 6,7,8], which are important for exocytosis [9], the insertion of key membrane transporters into the apical membrane [10, 11] and their function [12, 13], which together drive the secretory activity of these cells. InsP3R deficiency is a common feature in patients with different types of secretory diseases [18]
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