A novel cyclic nucleotide-gated ion channel enriched in synaptic terminals of isotocin neurons in zebrafish brain and pituitary

Abstract

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels opened by binding of intracellular cyclic GMP or cyclic AMP. CNG channels mediate sensory transduction in the rods and cones of the retina and in olfactory sensory neurons, but in addition, CNG channels are also expressed elsewhere in the central nervous system, where their physiological roles have not yet been well defined. Besides the CNG channel subtypes that mediate vision and olfaction, zebrafish has an additional subtype, CNGA5, which is expressed almost exclusively in the brain. We have generated CNGA5-specific monoclonal antibodies, which we use here to show that immunoreactivity for CNGA5 channels is highly enriched in synaptic terminals of a discrete set of neurons that project to a subregion of the pituitary, as well as diffusely in the brain and spinal cord. Double labeling with a variety of antibodies against pituitary hormones revealed that CNGA5 is located in the terminals of neuroendocrine cells that secrete the nonapeptide hormone/transmitter isotocin in the neurohypophysis, brain, and spinal cord. Furthermore, we show that CNGA5 channels expressed in Xenopus oocytes are highly permeable to Ca 2+ , which suggests that the channels are capable of modulating isotocin release in the zebrafish brain and pituitary. Isotocin is the teleost homolog of the mammalian hormone oxytocin, and like oxytocin, it regulates reproductive and social behavior. Therefore, the high calcium permeability of CNGA5 channels and their strategic location in isotocinsecreting synaptic terminals suggest that activation of CNGA5 channels in response to cyclic nucleotide signaling may have wide-ranging neuroendocrine and behavioral effects. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2011 January 13. Barnstable et al., 2004). In keeping with this idea, CNG channels have been reported to modulate transmitter release at synaptic terminals of retinal photoreceptors (Rieke and Schwartz, 1994;Savchenko et al., 1997), in addition to their role in phototransduction. Also, in the mammalian olfactory bulb, Murphy and Isaacson (2003) have shown that in response to moderate elevations in cyclic nucleotides, presynaptic CNG channels increase Ca 2+ entry and thereby enhance spontaneous transmitter release. However, in response to large elevations in cyclic nucleotides, CNG channel opening decreases evoked transmitter release, most likely by depolarizing the presynaptic terminal and thus inducing Na + channel inactivation.CNG channel...

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

“…To exclude the possibility that the retained anti-CNGA2a is due to cross-reactivity with CNGA2b paralog we also generated cnga2a/b double homozygous mutant (CNGA2a-del/CNGA2b-stop) and demonstrated that it still retained mAb L55/54 immunoreactivity ( Figure 4A ). These results suggested that mAb L55/54 immunoreactivity, which was detected in zebrafish OXT neurons by Khan et al (2010) , is due to an antigenic moiety that is different from the CNGA2a protein.…”

“… Khan et al (2010) generated a monoclonal antibody (mAb L55/54) which was raised against CNGA5/CNGA2a carboxy terminal tail. Based on the immunoreactivity of this antibody they concluded that CNGA2a is enriched in synaptic terminals of zebrafish OXT neurons.…”

“…Because the mAb L55/54 we used is directed to a 106 amino acids peptide that corresponds to the cytoplasmic C-terminal tail of CNGA2a ( Khan et al, 2010 ) we expected reduced or no immunoreactivity in cnga2a mutants. Surprisingly, both cnga2a-stop and cnga2a-del mutant alleles retained mAb L55/5 immunoreactivity ( Figure 4A ).…”

“…Surprisingly, it was reported that a zebrafish CNG subunit, denoted CNGA5, exhibits restricted brain-specific expression pattern with only weak expression in the olfactory bulb ( Tetreault et al, 2006 ). A subsequent study by Khan et al (2010) employed a monoclonal anti-CNGA5 antibody and concluded that CNGA5 protein is enriched in synaptic terminals of zebrafish OXT neurons. This inspired us to examine the role CNGA5, which was recently renamed CNGA2a, in the modulation of OXT presynaptic activity.…”

Dependence of the Excitability of Pituitary Cells on Cyclic Nucleotides

“…The mRNA transcripts for the rod CNG were also detected in rat pituitary cells by reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis (41) and RNA blot hybridisation (75). The zebrafish‐specific CNGA5 mRNA and protein transcripts are also expressed in the pituitary cells (76). Further studies are required to clarify their expression at the protein level and their potential role in electrical activity and Ca 2+ signalling in pituitary cells.…”