Cellular neuroscienceHeterologous expression of the invertebrate FMRFamide–gated sodium channel as a mechanism to selectively activate mammalian neurons
Section snippets
cDNA constructs
HaFaNaC constructs were contracted out to sequencing companies for subcloning into the mammalian expression vector pCMVTnT (Promega, Madison, WI, USA). HaFaNaC cDNA (EMBL accession number: X92113) was donated by Drs. Eric Lingueglia and Michel Lazdunski (CNRS-Université de Nice-Sophia, Antipolis, France). To generate pCMVTnT/HaFaNaC, the entire coding sequence of HaFaNaC was polymerase chain reaction (PCR) amplified and ligated between XbaI and SalI sites (Epoch Biolabs, Sugar Land, TX, USA).
Results
In this study we have examined the efficacy with which the invertebrate ligand-gated ion channel HaFaNaC can be expressed in mammalian neurons. In addition to its functional expression, we have investigated whether the expression of HaFaNaC or the application of FMRFamide would have any secondary confounding effects on mammalian neuronal function. Finally, we examined the subcellular localization of HaFaNaC in the somata, dendrites and axons of HaFaNaC-expressing mammalian neurons. We used
Discussion
We have shown that the invertebrate HaFaNaC can be functionally expressed in mammalian neurons in vitro. When HaFaNaC was expressed in CA3 pyramidal neurons of organotypic slice cultures, somatic application of FMRFamide was able to produce large depolarizations sufficient to elicit a burst of APs. The responses to FMRFamide were inhibited by amiloride and showed similar current–voltage relationships to those previously observed in cell lines and oocytes (Lingueglia et al., 1995). Importantly,
Conclusion
In conclusion, we have described the utility of heterologously expressing HaFaNaC in mammalian neurons as a potential genetically targeted method to excite specific subsets of neurons in intact mammalian CNS tissue. FMRFamide activation of heterologous expressed HaFaNaC could be used to study the effect of the excitation of specific subsets of neurons that are tonically active in the mammalian CNS. Strategies to improve the kinetics of activation of HaFaNaC and modify its subcellular
Acknowledgments
The authors would like to thank Drs. E. Lingueglia and M. Lazdunski for the donation of their FMRFamide-gated sodium channel, Dr. R. Y. Tsien for the tdTomato, Dr. E. Ruthazer for donating the GFP-tagged synaptophysin, and Dr. J. Dempster for the gift of his electrophysiological software. Microscopy was performed at the VCU-Dept. of Neurobiology and Anatomy Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant 5P30NS047463. A. Rory McQuiston is supported by a
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