γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligand-gated ion channels support the influx of Cl However, the electrochemical gradient for Cl across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl levels support inhibition, whereas higher levels of cytosolic Cl can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl from acidic organelles and transiently elevates cytosolic Cl, making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage-clamp recordings of currents through GABA-gated Cl channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl from acidic organelles. Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission.
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.
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