Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin

Frontiers in Cell and Developmental Biology

Janna Blechman¹  Gil Levkowitz¹  Savani Anbalagan¹  Gary G. Matthews² 

[1] Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel;Department of Neurobiology and Behavior, Stony Brook University, New York, NY, United States;

关键词: neuropeptide;    cGMP-gated ion channel;    neurohypophysis;    monoclonal antibody;    oxytocin;   

DOI  :  10.3389/fcell.2018.00117

来源: DOAJ

【 摘 要 】

Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools.

【 授权许可】

Unknown