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. 2022 Jul 27:16:937060.
doi: 10.3389/fncel.2022.937060. eCollection 2022.

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

Tyler Christopher Rodriguez  1 , Li Zhong  1 , Hailey Simpson  1 , Evanna Gleason  1
Affiliations

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

Tyler Christopher Rodriguez et al. Front Cell Neurosci. .

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.

Keywords: CRISPR/Cas9; TMEM16/anoctamin; amacrine cell; intracellular Cl−; nitric oxide (NO); retina.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
TMEM16A transcript and protein are expressed in retinal amacrine cells. (A) RT-PCR of RNA extracted from E16 retinal tissue for TMEM16 paralogues. (B) Single-cell PCR amplifying TMEM16A from individual AC. Each cell was DNase-treated prior to reverse transcription. RT (–) and template (–) controls showed no amplification. (C) Western blot of subcellular fractionated protein samples (i) and Triton x-114 cloud point extraction samples (ii) show immunodetection of TMEM16A (Abcam ab190803) and CFTR (Abcam ab131553) in the same fraction with both types of membrane preparation. (D) Immunocytochemistry for TMEM16A reveals a punctate labeling along with the soma. The image is displayed with a magenta-hot look-up table that shows puncta with high-fluorescence intensity as white.
Figure 2
Figure 2
Pharmacological inhibition of TMEM16A reduces the transient shift in ErevGABA. (A) Current–voltage relationship for leak subtracted GABA-gated currents recorded before and after NO injection in control and TMEM16A inhibitor (T16inh-AO1) conditions. (B) Truncated violin plots for the mean shift in the reversal potential of GABA-gated currents (ErevGABA). The shape of the violin shows frequency distribution of the data, top and bottom of the violin are data extrema, red lines are quartiles, and the dashed line is the median of the data. The mean shift in ErevGABA was reduced, and frequency distribution becomes bimodal when TMEM16A inhibitor (n = 11) was in the external solution compared to control conditions (n = 12). Recordings were from E16 cultures (Welch's t-test, ** denotes p < 0.005).
Figure 3
Figure 3
CRISPR/Cas9 edits TMEM16A of retinal amacrine cells when delivered by an all-in-one plasmid. (A) (top) Graphical representation of target genetic loci showing the cut site for each sgRNA. Primers are denoted by the arrows, and sgRNA target regions are indicated by the dashed lines. (Bottom) Agarose gel electrophoresis of in vitro Cas9 cleaved DNA (asterisks) for each sgRNA of a 606 bp PCR product containing exon 3 of TMEM16A. The pair of faint bands below the digestion fragments correspond to sgRNA secondary structures (arrowheads). (B) Simplified plasmid map of pSpHiFiCas9-T2A-tdTomato which was electroporated into dissociated chick retinal cells. (C) AC in culture expressing tdTomato is aspirated into a glass pipette for single-cell genomic PCR. (D) List of observed mutations detected in p222 transfected cells. (E) Percent indels detected from 4 days and 9–11 days post-transfection. (F) Representative sequence traces from genomic PCR of a wild-type cell (wt/wt) and an edited cell with ~30% WT and 70% +1 insertion alleles (wt/c221_222insA) according to TIDE. (G) TIDE output from cell in (F) shows substantial aberrant sequence starting at the expected cut site due to a mixture of WT and +1 amplicons in the edited cells PCR. * indicates cleavage products.
Figure 4
Figure 4
Transfection with P222 has no effect on TMEM16A protein expression (A–D). Representative images of TMEM16A antibody immunofluorescence and tdTomato fluorescence from e18 cells transfected with control plasmid (A,B) or p222 plasmid (C,D) targeting TMEM16A. (E) The mean integrated density for all objects identified within the TMEM16A mask; (F) the mean fluorescent intensity of the objects; (G) the mean objects size; (H) the log-transformed integrated density for tdTomato. There is no statistical difference between groups in (E–H) (two-tailed Welch's t-tests).
Figure 5
Figure 5
Mean shift in the reversal potential of GABAARs for control and cells targeted with p222 do not significantly differ. Current–voltage relationship for leak subtracted GABA-gated currents in a representative control AC (A) and in a representative p222 transfected cell (B). Paired data points for ErevGABA before and after NO injection shift positively for all cells tested in control (C) and p222 transfected (D) groups. Violin plots of the mean shift in ErevGABA (E) show no difference and similar frequency distribution between control and p222 transfected groups (control n = 17, p222 n = 20, p = 0.75 two-tailed Welch's t-tests).
Figure 6
Figure 6
Targeting TMEM16A exon 3 with dual gRNA Cas9 ribonucleoprotein produces deletions within 3 days. (A) The target loci in TMEM16A showing cleavage sites. (B,C) Graphical depictions of observed mutations from single amacrine cells that were dual gRNA-transfected showing an inversion c.222_424inv201 (B) and deletion c.222_424del202 (C) between the two target sites. (D) Edits were present as a smaller PCR amplicon from a separate amplification of mixed cultures 3 days post-transfection (white arrowhead).
Figure 7
Figure 7
Dual gRNA Cas9 ribonucleoprotein reduces TMEM16A expression (A–D). Representative immunofluorescence images of GFP expressing amacrine cells labeled with TMEM16A antibodies in control-transfected (A,B) and dual-guide RNA-transfected (C,D) conditions. (E) The mean integrated density for all objects identified within the TMEM16A mask is reduced in the dual-guide condition. (F) The mean fluorescent intensity of TMEM16A objects and the mean TMEM16A object size (G) are significantly reduced in dual-guide RNA-transfected cells. (H) Log-transformed integrated density of the GFP signal is lower in dual gRNA group. * denotes p < 0.05, ** denotes p < 0.005, **** denotes p < 0.00005.
Figure 8
Figure 8
Dual gRNA Cas9 ribonucleoproteins targeting TMEM16A reduce the NO-dependent shift in ErevGABA. Recording traces from GABA-gated currents before and after injecting NO from control-transfected (A) (n = 23) and dual gRNA/RNP-transfected cells (B) (n = 25). Paired data showing before and after NO injection for control (C) and dual-guide RNA targeting TMEM16A (D). Delivery of the Cas9 ribonucleoprotein led to a population of cells exhibiting significantly reduced NO-dependent shift [(D), brace] (E) Violin plots for the mean shift in ErevGABA show a reduction under dual gRNA/RNP condition and a lower quartile [(E), brace] that is far lower than control (control n = 25, dual gRNA/RNP n = 28, p = 0.003, ** denotes p < 0.005).
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