A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl⁻ from acidic organelles in amacrine cells

Abstract

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.

Inhibition of endocytosis suppresses the nitric oxide-dependent release of Cl- in retinal amacrine cells

“…We have demonstrated that CFTR expression and function is required for the NOdrCl [ 7 ]. We began here by testing whether the CFTR protein was localized to the same regions of the cell as SV2, a synaptic vesicle marker protein.…”

“…ab131553; Abcam). We have previously characterized this antibody with a western blot showing a single band at the appropriate molecular weight [ 7 ]. Antibodies were diluted at 1:1000 in dilution solution and applied to cells at 4°C overnight then washed with PBS.…”

“…Cells were incubated for 1 hour at room temperature in secondary antibodies then washed in PBS. Secondary antibody-only controls were prepared in the same solutions and produced no labeling [ 7 ]. The primary monoclonal antibody raised against SV2 developed by K.M.…”

“…The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl - transporter that can be found in internal membranes of vertebrate neurons [ 5 , 6 ]. Recently our lab has shown that pharmacological inhibition and knockdown of CFTR expression in ACs blocks the NO-dependent shift in E rev - GABA [ 7 ].…”

CRISPR-Cas9-Mediated Correction of the 1.02 kb Common Deletion in CLN3 in Induced Pluripotent Stem Cells from Patients with Batten Disease

“…These isogenic controls could be differentiated into neuronal cells and compared side-by-side with affected (i.e., uncorrected) cells for rigorous disease modeling or drug selection experiments. A similar approach has been utilized to investigate the pathophysiological mechanisms of disease in cells with mutations that cause Duchenne muscular dystrophy, 33 cystic fibrosis, 34 or beta thalassemia. 35,36 Although mutations in CLN3 cause neuronal cell death throughout the CNS, the light-sensing photoreceptor cells of the neural retina are the first to succumb, suggesting they are especially sensitive to CLN3 dysfunction and that using them as a disease model may provide critical insight into how CLN3 mutations specifically alter neuronal function, leading to cell death.…”