Chloride Channels that Mediate a Transient Nitric Oxide-dependent Synaptic Polarity Switch in Retinal Amacrine Cells.

Cytosolic chloride (Cl-) concentration determines whether GABAergic and glycinergic synapses are inhibitory or excitatory. Ionotropic GABA and glycine receptors passively conduct Cl­- leading to membrane depolarization or hyperpolarization depending on the Cl- equilibrium potential. Accordingly, the distribution of Cl- across the plasma membrane has the potential to determine the properties of networks of retinal amacrine cells (ACs) and their postsynaptic partners. Ion channels are responsible for establishing Cl- reversal potential. Nitric oxide (NO) is a signaling molecule that can be generated in the inner retina where ACs form synapses. We have shown that NO initiates a release of Cl- from internal stores into the cytosol in retinal ACs, leading to elevated cytosolic Cl-. Our research implicates a novel role for the cystic fibrosis transmembrane conductance regulator (CFTR) in the 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. Chapter 2 shows CFTR is necessary for the transient effects of NO on cytosolic Cl- levels but the full mechanism remains to be elucidated. Chapter 3 further informs the mechanism with involvement of TMEM16A, a Ca2+ activated Cl- channel demonstrated to be functionally coupled with CFTR in some epithelial cell types. TMEM16A is expressed in the chicken retina as determined by western blot analysis, immunocytochemistry, and single cell RT-PCR. Pharmacological inhibition of TMEM16A with T16inh-AO1 reduced the NO-dependent Cl- release (NOdClr) measured as a reduction in the shift in the reversal potential of GABAA receptor-mediated currents. To confirm the involvement of TMEM16A in the NodClr, we used CRISPR/Cas9 via two different modalities targeting TMEM16A. We delivered either an all-in-one plasmid or functional ribonucleoprotein resulting in different effects on protein levels. The plasmid vector was unsuccessful in reducing the membrane expression of TMEM16A protein within the timeframe of culture viability. However, delivery of TMEM16A-specific crRNA/tracrRNA/Cas9 ribonucleoprotein was effective in reducing both TMEM16A protein levels and the NO-dependent shift in reversal potential of GABA-gated currents. These results demonstrate that ACs express CFTR and TMEM16A with both playing a role in the NodClr. Ultimately, the flexibility of retinal processing may be increased by co-expression of CFTR with different TMEM16 paralogues, thus allowing subsets of amacrine cells to perform specific and unique functions