Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl Transport in Retinal Amacrine Cells
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 Ca 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 Ca elevations and cytosolic Cl release. TMEM16A is a Ca-activated Cl channel that is functionally coupled with CFTR in epithelia. Both proteins are also expressed in neurons. Based on this and its Ca 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 GABA 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 () 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.
Publication Source (Journal or Book title)
Frontiers in cellular neuroscience
Rodriguez, T. C., Zhong, L., Simpson, H., & Gleason, E. (2022). Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl Transport in Retinal Amacrine Cells. Frontiers in cellular neuroscience, 16, 937060. https://doi.org/10.3389/fncel.2022.937060