An expanded biological repertoire for Ins(3,4,5,6)P4 through its modulation of ClC-3 function

Abstract

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.

Figure 1. Electrophysiological Analysis of the Inhibition of CaMKII-Activated Cl⁻ Current by Ins(3,4,5,6)P₄ in tsA^ClC-3 Cells

(A) Representative whole-cell currents from tsA^ClC-3 cells in which autonomous CaMKII (20 μg/ml) was included in the pipette solution. Upper left panel: Representative time-course of current elicited during depolarizing voltage pulses to 110 mV from a holding potential of −40 mV every 10 s. Upper right panel: Families of currents recorded at the initiation (“initial”) and peak of CaMKII-mediated current activation, during hyper-polarizing and depolarizing voltage pulses from a holding potential of −40 mV to potentials between −110 and 110 mV at 10 mV intervals. Lower panels: Average relationship between current and voltage from seven cells. (B–E) Analysis of whole-cell current responses to 100 μM Ins(1,3,4,5,6)P₅, 100 μM Ins(1,4,5,6)P₄, or 10 μM (unless otherwise indicated) Ins(3,4,5,6)P₄. (B) Average relationship between current and voltage for current families elicited as in (A). (C) Mean current densities determined at 110 mV and −110 mV, with cell numbers indicated above each bar. (D) Effect of Ins(3,4,5,6)P4 concentration upon CaMKII-activated current (n = 3-6). Data are fitted to a single-site binding curve. (E) Representative time course of current activation by CaMKII (peak current = 3.18 ± 0.34 nA, n = 7) plus (in the pipette solution) either 10 μM Ins(3,4,5,6)P₄ + 100 nM okadaic acid (“OKA”) (peak current = 2.64 ± 0.68 nA, n = 3) or 10 μM Ins(3,4,5,6)P₄ alone. In panels that contain error bars, these represent standard errors of the mean.

Figure 2. Ins(3,4,5,6)P₄ Regulates Endosomal Acidification by Inhibiting ClC-3

(A) Lysates of BHK cells (control) or BHK^ClC-3 cells were separated by 7% SDS-PAGE and transferred to nitrocellulose, and ClC-3 was detected with a murine monoclonal antibody. The same antibody was used for detection of ClC-3 in methanol-fixed cells via immunofluorescence microscopy (see Experimental Procedures). (B) WT BHK cells or BHK^ClC-3 cells were each allowed to endocytose FITC-transferrin at 37°C. For calibration purposes, cells were equilibrated in buffers of known pH containing ionophores, followed by fluorescence-ratio imaging. An example (for BHK^ClC-3 cells at pH 7.3) is shown in (B), and for clarity of presentation (but not during data analysis), the brightness and contrast were increased by 30% with Microsoft Powerpoint. One cell is magnified and annotated with yellow arrows to highlight the regions that were defined as vesicular early endosomes and selected for pH analysis [32]. (C) A representative calibration plot is shown. (D) Endosomal pH in cells that were treated either for 30 min with 0.5 μM bafilomycin or for 1 hr with 100 μM Bt₂⁻Ins(3,4,5,6)P₄/PM, 100 μM Bt₂-Ins(1,4,5,6)P₄/PM, or 200 μM butyrate (Bt). Data are from 3–6 experiments. Asterisks indicate p < 0.001 versus control. (E) Cells were treated for 1 hr with 0, 10, 20, or 100 μM Bt₂-Ins(3,4,5,6)P₄/PM. The data (n = 3–4) show the relationship between ΔpH and the estimated (EST) intracellular concentration of Ins(3,4,5,6)P₄, based upon a delivery efficiency ([Ins(3,4,5,6)P₄]_IN / [Bt₂-Ins(3,4,5,6)P₄/PM]_OUT) of 16% [13, 34] and assuming 1 μM Ins(3,4,5,6)P₄ in resting cells [13]. In panels that contain error bars, these represent standard errors of the mean.

Figure 3. CaMKII-Activated ClC-3 Current Is Inhibited by Ins(3,4,5,6)P₄ in Cultured Rat P5 Hippocampal Neurons

Whole-cell currents were obtained, as described in Figure 1, from cultured hippocampal neurons in which autonomous CaMKII (20 μg/ml) was included in the pipette solution for 25 min plus, where indicated, either 10 μM Ins(3,4,5,6)P₄ or 100 μM Ins(1,3,4,5,6)P₅. (A) Families of currents recorded during hyperpolarizing and depolarizing voltage pulses. (B) Relationship between current and voltage. (C) Average current densities at 110 mV and −110mV, with cell numbers indicated below each bar. In panels that contain error bars, these represent standard errors of the mean.