An Expanded Biological Repertoire for Ins(3,4,5,6)P4 through its Modulation of ClC-3 Function

Ins(3,4,5,6)P(4) inhibits plasma membrane Cl(-) flux in secretory epithelia [1]. However, in most other mammalian cells, receptor-dependent elevation of Ins(3,4,5,6)P(4) levels is an "orphan" response that lacks biological significance [2]. We set out to identify Cl(-) channel(s) and/or transporter(s) that are regulated by Ins(3,4,5,6)P4 in vivo. Several candidates [3-5] were excluded through biophysical criteria, electrophysiological analysis, and confocal immunofluorescence microscopy. Then, we heterologously expressed ClC-3 in the plasma membrane of HEK293-tsA201 cells; whole-cell patch-clamp analysis showed Ins(3,4,5,6)P4 to inhibit Cl(-) conductance through ClC-3. Next, we heterologously expressed ClC-3 in the early endosomal compartment of BHK cells; by fluorescence ratio imaging of endocytosed FITC-transferrin, we recorded intra-endosomal pH, an in situ biosensor for Cl(-) flux across endosomal membranes [6]. A cell-permeant, bioactivatable Ins(3,4,5,6)P4 analog elevated endosomal pH from 6.1 to 6.6, reflecting inhibition of ClC-3. Finally, Ins(3,4,5,6)P(4) inhibited endogenous ClC-3 conductance in postsynaptic membranes of neonatal hippocampal neurones. Among other ClC-3 functions that could be regulated by Ins(3,4,5,6)P4 are tumor cell migration [7], apoptosis [8], and inflammatory responses [9]. Ins(3,4,5,6)P4 is a ubiquitous cellular signal with diverse biological actions.