Malik-Hall, Misbah; (2002) Identification of accessory subunits that interact with the sensory neurone-specific sodium channel, SNS (Nav 1.8). 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. Nav1.8/SNS is a tetrodotoxin resistant (TTX-r) voltage-gated sodium channel expressed predominantly in the small diameter sensory neurones in the dorsal root ganglia. This channel is involved in transmission of nociceptive information from sensory neurones to the central nervous system. Recent evidence suggests that in addition to accessory P subunits, Nav1.8 requires some distinct subunit to help produce flinctional expression on the plasma membrane in sensory neurones. Applying Yeast two-hybrid analysis, 27 clones have been identified as interactors for Nav1.8 from a rat DRG cDNA library. Co-immunoprecipitation studies with Nav1.8 have confirmed most of the clones bind strongly to Nav1.8. All the clones were expressed in small diameter sensory neurones as indicated by in situ hybridisation. One clone was chosen for further analysis, I-1 or p11 protein. The 11 kDa protein p11 is a light chain in the annexin II (also known as calpactin I or lipocortin II) complex which is a member of the calcium and phospholipid-binding family of proteins known as annexins. Functional experiments were performed to investigate the tissue distribution and subcellular localization in sensory neurones of this clone by Northern blot analysis. Northern blot analysis revealed high level of expression of p11 mRNA in the DRG, a modest level in the heart and low level in the brain. This confirms the coexpression role of p11 with Nav1.8 as these TTX-r channels are expressed in DRG where there is a high level of p11 expression. Introduction of antisense cDNA of the clones into primary culture of sensory neurones by microinjection methods revealed one clone in particular, p11 protein, caused a significant decrease in the sodium current hence showing that p11 is essential for the expression of a fianctional Nav1.8 channel in DRG. When p11 is transfected into stably transfected Nav1.8 expressing CHO cells (which do not express a functional Nav1 8 channel despite the existence of a considerable amount of Nav1.8 mRNA and protein in the cytosol), Nav1.8 is translocated to the plasma membrane, p11 elicits the functional expression via a direct interaction to the N-terminus of Nav1.8. This expressed channel displayed a Nav1.8 like current thus indicating that p11 is acting as regulatory protein that is necessary for the expression of a fianctional channel. Regulation of Nav1.8 expression by distinct subunits is a good target for possible pain management.

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