Poon, Wing Yee Louisa; (2003) Identification of sensory-neuron-specific sodium channel Nav1.8 interacting proteins by yeast-two hybrid screen. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Voltage-gated sodium channels initiate and propagate action potentials in excitable cells. Ten distinct pore-forming α-subunits of voltage-gated sodium channels have been identified. The tetrodotoxin-resistant (TTX-r) sodium channel Navl.8 (also known as SNS/PN3) is primarily expressed in small diameter C-fiber-associated sensory neurons. Behavioral studies on Navl.8 null mutant mice have demonstrated a role for Nay 1.8 in the detection of noxious thermal, mechanical and inflammatory stimuli. Unlike other sodium channels, Nayl.8 is poorly expressed in mammalian cell lines even in the presence of accessory β-subunits. Experimental evidence suggests that Nay 1.8 requires accessory neuronal co-factors for high-level expression to produce currents comparable to characteristics exhibited in sensory neurons by endogenous Nayl.8. A yeast two-hybrid screen was used to identify novel regulatory proteins for Nayl.8. Twenty-eight interacting clones were identified by using the intracellular loops of the α-subunit of Nayl.8 as baits in the yeast two-hybrid screen. All clones identified are expressed in small-diameter sensory neurons as indicated by in situ hybridization, and co-immunoprecipitation studies confirmed most of the clones bind strongly to Nayl.8. After initial screening, one of the clones, pi 1, which is a member of the family of SI00 related calcium-binding proteins, was chosen for further studies, pi 1 binds directly to the N-terminus of Nayl.8 α-subunit and promotes the translocation of Nayl.8 to the plasma membrane resulting in channel activity in CHO cell line. The endogenous Nayl.8 current in sensory neurons is inhibited by antisense down- regulation of pi 1 expression. Thus, a pi 1-Nay 1.8 direct interaction is required for functional expression of Nayl.8 in sensory neurons. However, the biophysical properties of the pll-induced Nayl.8 current in Chinese Hamster Ovary (CHO) cells are different from endogenous Nayl.8 current found in neurons. This suggests that other regulatory factors, perhaps extracellularly interacting ones, are involved in the functional expression of this channel. Hence, a second yeast two-hybrid screen was carried out using the extracellular loops as bait.

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