Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl− Transport in Retinal Amacrine Cells

Abstract

Postsynaptic cytosolic Cl− concentration determines whether GABAergic and glycinergic synapses are inhibitory or excitatory. We have shown that nitric oxide (NO) initiates the release of Cl− from acidic internal stores into the cytosol of retinal amacrine cells (ACs) thereby elevating cytosolic Cl−. In addition, we found that cystic fibrosis transmembrane conductance regulator (CFTR) expression and Ca2+ elevations are necessary for the transient effects of NO on cytosolic Cl− levels, but the mechanism remains to be elucidated. Here, we investigated the involvement of TMEM16A as a possible link between Ca2+ elevations and cytosolic Cl− release. TMEM16A is a Ca2+-activated Cl− channel that is functionally coupled with CFTR in epithelia. Both proteins are also expressed in neurons. Based on this and its Ca2+ dependence, we test the hypothesis that TMEM16A participates in the NO-dependent elevation in cytosolic Cl− in ACs. Chick retina ACs express TMEM16A as shown by Western blot analysis, single-cell PCR, and immunocytochemistry. Electrophysiology experiments demonstrate that TMEM16A functions in amacrine cells. Pharmacological inhibition of TMEM16A with T16inh-AO1 reduces the NO-dependent Cl− release as indicated by the diminished shift in the reversal potential of GABAA receptor-mediated currents. We confirmed the involvement of TMEM16A in the NO-dependent Cl− release using CRISPR/Cas9 knockdown of TMEM16A. Two different modalities targeting the gene for TMEM16A (ANO1) were tested in retinal amacrine cells: an all-in-one plasmid vector and crRNA/tracrRNA/Cas9 ribonucleoprotein. The all-in-one CRISPR/Cas9 modality did not change the expression of TMEM16A protein and produced no change in the response to NO. However, TMEM16A-specific crRNA/tracrRNA/Cas9 ribonucleoprotein effectively reduces both TMEM16A protein levels and the NO-dependent shift in the reversal potential of GABA-gated currents. These results show that TMEM16A plays a role in the NO-dependent Cl− release from retinal ACs.