Contrasting effects of phosphatidylinositol 4,5‐bisphosphate on cloned TMEM16A and TMEM16B channels

Abstract

Ca2+ -activated Cl À channels (CaCCs) are gated open by a rise in intracellular Ca 2+ concentration ([Ca 2+ ] i ), typically provoked by activation of G q -protein coupled receptors (G q PCR). G q PCR activation initiates depletion of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Here, we determined whether PIP 2 acts as a signalling lipid for CaCCs coded by the TMEM16A and TMEM16B genes. EXPERIMENTAL APPROACHPatch-clamp electrophysiology, in conjunction with genetically encoded systems to control cellular PIP 2 content, was used to define the mechanism of action of PIP 2 on TMEM16A and TMEM16B channels. KEY RESULTSA water-soluble PIP 2 analogue (diC8-PIP 2 ) activated TMEM16A channels by up to fivefold and inhibited TMEM16B by~0.2-fold. The effects of diC8-PIP 2 on TMEM16A currents were especially pronounced at low [Ca 2+ ] i . In contrast, diC8-PIP 2 modulation of TMEM16B channels did not vary over a broad [Ca 2+ ] i range but was only detectable at highly depolarized membrane potentials. Modulation of TMEM16A and TMEM16B currents was due to changes in channel gating, while single channel conductance was unaltered. Co-expression of TMEM16A or TMEM16B with a Danio rerio voltage-sensitive phosphatase (DrVSP), which degrades PIP 2 , led to reduction and enhancement of TMEM16A and TMEM16B currents respectively. These effects were abolished by an inactivating mutation in DrVSP and antagonized by simultaneous co-expression of a phosphatidylinositol-4-phosphate 5-kinase that catalyses PIP 2 formation. CONCLUSIONS AND IMPLICATIONSPIP 2 acts as a modifier of TMEM16A and TMEM16B channel gating. Drugs interacting with PIP 2 signalling may affect TMEM16A and TMEM16B channel gating and have potential uses in basic science and implications for therapy. AbbreviationsCaCC, calcium-activated chloride channel; DrVSP, Danio rerio voltage-sensitive phosphatase; E rev , reversal potential; G q PCR, G q -protein coupled receptors; IP 3 , inositol triphosphate; PIP 2 , phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PIPK, PIP 5-kinase type Iγ; V m , membrane potential

Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1)

“…Figure 1B summarizes this finding and shows ATP was critical for channel recovery. Our findings revealed that the PI(4,5)P2 dephosphorylation caused by Dr-VSP decreased TMEM16A activity in agreement with a recent report showing that exogenous PI(4,5)P2 increases TMEM16A activity whereas PI(4,5)P2 depletion has the opposite effect (Ta et al, 2017). …”

“…PI(4,5)P2 stabilizes the current by preventing rundown but if PI(4,5)P2 is dephosphorylated then TMEM16A activity decreases. Our results are in agreement with those of Ta and co-workers showing that exogenous PI(4,5)P2 increases the activity of TMEM16A, but not TMEM16B in a Ca 2+ -dependent manner and activation of the Dr-VSP phosphatase inhibited TMEM16A (Ta et al, 2017). A previous report showed that adding diC8-PIP2 decreased the current through Ca 2+ -dependent Cl − channels activated by 500 nM Ca 2+ in inside-out patches excised from rat pulmonary artery cells (Pritchard et al, 2014); however, we do not have an explanation for this discrepancy.…”

“…A dephosphorylated PI(4,5)P2 could inactivate ERM proteins that subsequently down regulate TMEM16A or impair actin-channel interaction (Perez-Cornejo et al, 2012; Fehon et al, 2010; Zhang et al, 2012; Sasaki et al, 2014) or become less effective at attracting Ca 2+ ions to bind TMEM16A. However, PI(4,5)P2 can activate and prevent rundown of TMEM16A in inside out patches that lack cytoskeleton (Ta et al, 2017; present data). In addition, our data show that the channel’s activity decreases after PI(4,5)P2 dephosphorylation in cells treated with latrunculin A that disrupt actin filaments or with CK-666 that inhibits nucleation of branching acting filaments (Hetrick et al, 2013).…”

“…Two reports showed opposite effects of PI(4,5)P2 on TMEM16A; PI(4,5)P2 inhibited the Ca 2+ -activated Cl − current (I Ca,Cl ) in pulmonary artery cells (Pritchard et al, 2014) but increased TMEM16A I Ca,Cl in HEK 293 cells (Ta et al, 2017). Here we study the regulation of TMEM16A I Ca,Cl by PI(4,5)P2 using the Danio rerio voltage-sensitive phosphatase (Dr-VSP).…”

“…One of them demonstrates that exogenous PI(4,5)P2 down regulates the Ca 2+ -dependent Cl − current (I Ca,Cl ) in inside-out patches from pulmonary artery cells (Pritchard et al, 2014). But the other shows that exogenous PI(4,5)P2 increases TMEM16A activity in inside out patches isolated from HEK 293 cells and that PI(4,5)P2 dephosphorylation induces the opposite effect (Ta et al, 2017). …”