Introduction

TMEM16A Ca²⁺-gated Cl^– channels (CaCCs) provide a key depolarising conductance in in contractile vascular cells such as arterial smooth muscle cells and contractile pericytes (Al-Hosni et al., 2024). Single-point mutations in TMEM16A (ANO1) have recently been linked to moyamoya disease (MMD), a cerebrovascular disorder characterised by progressive carotid artery occlusion and an increased risk of ischaemic stroke (Pinard et al., 2023). Several MMD-associated TMEM16A variants have been identified, most exhibiting gain-of-function effects through enhanced Ca²⁺ sensitivity. In contrast, a variant with substitution of arginine (R) 890 to glutamine (Q) appears to cause channel loss of function. However, the mechanisms linking altered TMEM16A activity to MMD remain poorly understood.  

Aims:

We aim to investigate the functional consequences of the MMD-associated hTMEM16A-R890Q mutation and assess its role in the pathological neovascularisation observed in the patient carrying this variant (Pinard et al., 2023). Here, we study the effects of this mutation on TMEM16A channel function.

Methods

Whole-cell and inside-out patch-clamp recordings of heterologously expressed wild-type and mutant TMEM16A currents were used to investigate how TMEM16A currents are affected by the R890Q mutation. Site-directed mutagenesis and stationary noise analysis were used to assess the impact of the mutation on channel gating, open probability (Pₒ), and single-channel current amplitude (i). Immunocytochemistry was employed to evaluate surface expression of HA-tagged hTMEM16A and hTMEM16A-R890Q channels.

Results

Heterologous hTMEM16A-R890Q currents were reduced compared to wild-type in a range of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) (e.g. by 84±20% at 100 mV in the presence of 100 nM Ca²⁺_I, n=13 in each case, p<0.001). Inside-out patch-clamp recordings revealed a decrease in maximal current amplitude from 0.90±0.13 nA for hTMEM16A-WT channels to 0.32±0.06 nA for hTMEM16A-R890Q channels (n=6 in each case, p<0.01). This reduction in channel activity was accompanied by a significant rightward shift in the Ca²⁺ concentration–response relationship, with the concentration causing half maximal activation (EC₅₀) for intracellular Ca²⁺ increasing from 386 ± 12 nM in hTMEM16A-WT channels to 528 ± 26 nM in hTMEM16A-R890Q channels (n = 6 for each group; p < 0.001). Stationary noise analysis demonstrated that hTMEM16A-R890Q has reduced plasmalemmal expression by 72±26% (n=6, p<0.01), and altered channel gating. These findings were further supported by immunocytochemical analyses, which demonstrated reduced plasma membrane expression of hTMEM16A-R890Q channels. In addition, the R890Q mutation altered both anion selectivity and conductance across a range of permeant anions.

Conclusions

Collectively, our findings reveal a complex biophysical alteration of hTMEM16A-R890Q function, involving impaired plasma membrane trafficking and altered channel gating that together reduce macroscopic currents. This provides new insights into MMD pathogenesis and into how TMEM16A channel dysfunction may contribute to cerebrovascular disease.