Identification and localisation of the sodium channel alpha subunits carrying the Ttx-sensitive sodium current (INa) in the enteric nervous system of the guinea PIG intestine

Abstract

The sodium channel protein consists of an alpha subuint in association with auxiliary beta subunits. The 5 neuronal alpha subnnit isoforms that carry the TTX-sensittve current are differentially expressed according to the neuronal types and have different functional properties, which result in distinct conductances in specific cells. Our work was designed in order to identify which alpha subunits carry the TTX-sensitive Na current in the enteric nervous system. Experiments were performed on fixed whole mount preparations of the myenteric and submucous plexuses of guinea pig (GP) small and large intestine using polydonal antibodies (ABs) raised against rat and/or human epitopes of the following alpha subunits : Navl.1, Nay1.2, Navl.3, Navl.6 and Nay1.7. Positive controls on non enteric nervous system were obtained for all ABs except that against Nay1.7. AB selectivity was verified using Western blots performed on rat and GP brain membrane preparations. Only NaV1.2 and Navl.3 alpha subunits were expressed in enteric neurons. NaV1.2 was expressed in the majority of the enteric neurons and immunoreactivity was located in the soma. Interestingly, it was highly expressed in a single axon hillock of the enteric sensory neurons. Some of the myenteric NaV1.2 immunoreactive non-sensory neurons are likely to be motor neurons that innervate the longitudinal muscle. Navl.3-fike immunoreactivity was observed only in descending filamentous neurons It was located in the soma, the long ramifying dendrites and the axon. Numerous smooth nerve fibers were also labeled. These fibres are extrinsic in origin since they enter the gut wall in large bundles running from the mesentery. Using Western blots performed on adult and new born rat and GP brain membrane preparations, the Navl.2 AB, raised against epitopes located in the I-I1 intracellular loop (M1 IL), stained a band at 230 kDa. Using rat brain membrane preparations, the Navl.3 AB, also raised against an epitope located in 1-II 1L, stained a band at 130 kDa. A band of similar low molecular weight was also stained using a Pan AB raised against the loop llI-IV. The expression of both the 230 and 130 kDa protein was age-dependent as previously described for Navl.2 and Navl 3 (Felts eta;. 1997). Our data strongly suggests that in enteric sensory neurons and in some motor neurons of the GP the TTX-sensitive INa is exclusively car'ned by the Navl.2 alpha subunit. This work is supported by GSK and CNRS.