Abstract title: Examination of the role of Nav1.7 and Nav1.8 in the generation of action potentials both in-vivo and in-vitro
Poster number: PTH114
Presentation time: Thursday, September 13, 9:30 – 10:30 AM
Authors: Lunbin Deng1, Victory Joseph1, Susanne Lardell2, Justin Elstrott1, Paul Karila2 and David Hackos1
Affiliations: 1Department of Neuroscience, Genentech, South San Francisco, CA, USA; 2Cellectricon AB, Mölndal, Sweden
Aim of Investigation: Loss-of-function mutations in Nav1.7 lead to congenital insensitivity to pain (CIP) in humans and a CIP-like phenotype in Nav1.7 KO mice. On the other hand, CIP patients have not been identified with loss-of-function mutations in Nav1.8 and Nav1.8 KO mice show a minimal pain phenotype, suggesting that Nav1.8 plays little if any role in sensory neuron action potential generation or propagation in vivo. Using laser speckle imaging, we established an in vivo assay sensitive to peripheral action potential generation in the skin, allowing us to examine the role that Nav1.7 and Nav1.8 play in action potential generation under resting and inflammatory conditions
Methods: Laser speckle imaging allows real-time detection of blood flow in the glabrous skin of the mouse hind-paw. In this method, AITC (mustard oil) is applied to the skin, which activates TRPA1 channels, allowing calcium influx and depolarization of sensory fibers. Such depolarization is sufficient to generate action potentials, allowing opening of voltage-gated calcium channels that allows more calcium influx, which results in release of peptide neurotransmitters and increased blood flow. Recordings of DRG excitability (using electrophysiology and calcium influx) were also used to examine the interplay between Nav1.7 and Nav1.8 channels in cultured sensory neurons under different conditions.
Results: We observed that AITC-induced increase in blood flow in the mouse hind-paw is dependent on TRPA1, as expected (since AITC is a direct activator of TRPA1 channels). We further observed that the action potential component of this response is Nav1.7-dependent and examined the role that Nav1.8 plays in action potential generation using the Nav1.8 KO mouse. Recordings of DRG neurons in culture show that the dependence of Nav1.7 vs Nav1.8 can be modified depending on the condition of the experiment (resting membrane voltage and inflammation state).
Conclusions: Here we demonstrate a novel assay that allows indirect examination of action potential generation within the peripheral sensory fibers in the skin. We use this assay to determine the role that Nav1.7 and Nav1.8 play in this process and demonstrate that action potential generation is fully dependent on Nav1.7.
Acknowledgments/Disclosures: LD, VJ, JE and DH are employees of Genentech, Inc. SL and PK are employees of Cellectricon AB.