Background The TMEM16A Ca²⁺-activated Cl^– channel plays a key role in the control of vascular tone and blood flow. TMEM16A has a pore with sections exposed to plasmalemmal lipids (1-3). This structural arrangement may confer the channel sensitivity to plasmalemmal lipids, including phosphatidylinositol 4,5-bisphospate (PIP₂) (4). The lysosomal NPC1 protein regulates cellular distribution of lipids (5). Loss-of-function mutations in NPC1 lead to Niemann-Pick disease Type C (NPC) a prematurely fatal neurodegenerative disorder with a range of systemic alterations including vascular (5). Here, we ask whether TMEM16A is modulated by NPC1 and examine the impact of this modulation on the tone of isolated systemic arteries, where TMEM16A is highly expressed.

Methods Whole-cell patch-clamp recordings of native and cloned TMEM16A currents, isometric tension recordings, confocal imaging and Förster Resonance Energy Transfer (FRET) were used in this study. For patch-clamp recordings, the external solution contained (mM): 150 NaCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, 10 D-mannitol, and 10 HEPES (pH 7.4); the pipette solution contained (mM): 130 CsCl, 10 EGTA, 1 MgCl₂, 10 HEPES and 8 CaCl₂ (pH 7.3). The tone of isolated artery rings obtained from mice carrying Npc1 deletion (Npc1^-/-) before and after 5-week treatment (4g/kg/week) with 2-hydroxypropryl-β-cyclodextrin (bCD), was assessed via wire myography. Expression of mRNA for phospholipase C (PLC) isoforms was conducted via quantitative RT-PCR (qRT-PCR).  Data are given as mean±SEM alongside the number of independent experiments.

Results Heterologously expressed TMEM16A currents were enhanced by 2.3±0.3 fold (n=14) during pharmacological inhibition of NPC1 or by 2.6±1.0 fold (n=15) as a consequence of genetic deletion of the Npc1 gene (knockout). These increases were prevented by treatment with b-cyclodextrin or re-introduction of Npc1 gene in the knockout cells. The activation of cloned TMEM16A currents by NPC1 inhibition was independent on KCNE1, a proposed TMEM16A auxiliary subunit. Depletion of plasmalemmal PIP₂ or an inactivating mutation in the channel PIP₂ binding site (TMEM16A-R482A), prevented TMEM16A activation during NPC1 inhibition. Artery (aorta and mesenteric) rings obtained from Npc1 null mice showed increased contractility in response to phenylephrine, which was prevented by Ani9, a selective TMEM16A inhibitor and enhanced by increasing the depolarising Cl^– gradient. The underlying mechanism involves augmented plasmalemmal PIP₂ levels during NPC1 inhibition, assed via genetically-encoded PH-PLCd domains and FRET imaging. The plasmalemmal PIP₂ level was increased by 1.2 ±0.6 fold (n=67) during pharmacological inhibition of NPC1 and was rescued by treatment with b-cyclodextrin. This change in PIP₂ homeostasis was presumably caused by reduction in the expression of phospholipase C during NPC1 inhibition, assessed using qRT-PCR.

Conclusions PIP₂-dependent changes in TMEM16A activity may form the basis of vascular overactivity during pathology caused by loss of NPC1 function and establish a role for the lysosome in the control of cell excitability and vascular tone.