Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Regulation of cytosolic Cl- is an important determinant in synaptic function. Our lab has discovered that cytosolic Cl- is regulated not only by transport across the plasma membrane but that Cl- can also be released from internal Cl- stores (Hoffpauir et al., 2006) in amacrine cells (ACs). Nitric oxide (NO) is a small molecule neurotransmitter that has been demonstrated to be involved in cytosolic Ca2+ elevations and Cl- regulation via activation of the cystic fibrosis transmembrane regulator (CFTR) (Krishnan et al., 2017). Here, I test the hypothesis that intracellular Ca2+ signaling mediates the NO-dependent Cl- regulation in retinal ACs. To determine this, I investigated the role of Ca2+- sensitive adenylate cyclase 1 (AdC1) in the NO-dependent release of Cl- by applying pharmacological inhibitors and activators of cAMP pathway. Voltage-clamp recordings reveal that AdC1 plays a specific role in CFTR activation. The role of intracellular Ca2+ was evaluated by suppressing Ca2+ elevations and by artificially inducing them in zero external Ca2+. The results demonstrate that Ca2+ from internal stores is required for NO-dependent release of Cl-. Simultaneous activation of the cAMP pathway and stimulated store release replicated the effects of NO indicating that Ca2+ elevations and cAMP activity are sufficient to explain the effects of NO. Insights into the mechanisms and functions of NO-dependent Ca2+ in cytosolic Cl- regulation were provided by pre-applied TMEM16A inhibitor T16Ainh-A01, inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor 2-APB and ryanodine receptor (RyR) inhibitor ryanodine, respectively in Ca2+ imaging and voltage- ramp recordings. T16Ainh-A01, 2-APB, and ryanodine were efficient in suppressing the NO-dependent Ca2+ responses and NO-dependent release of Cl-. Co-application of T16Ainh-A01 and 2-APB produced the same inhibition in release of Cl- as 2-APB alone, similar results were found in T16Ainh-A01 and ryanodine, indicating TMEM16A may regulate IP3R-and RyR-related signaling upstream to the Ca2+ elevations in the NO-dependent Cl- release. Taken together, these results suggest that AdC1 provides a link between NO-dependent cytosolic Ca2+ signaling and cytosolic Cl- regulation, TMEM16A, IP3R, and RyR are involved in the NO-dependent Ca2+ elevations and this signaling is necessary for cytosolic Cl- regulation in retinal ACs.

Date

7-19-2022

Committee Chair

Gleason, Evanna

Available for download on Saturday, July 19, 2025

Included in

Biology Commons

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