Analysis of Voltage-Gated Sodium Channel Membrane Dynamics in Hippocampal Neurons via a Fluorescent Protein and Biotin Tagged Nav1.6 Channel

Abstract

Voltage-gated sodium channels (Nav) are densely accumulated at the axon initial segment (AIS) of neurons where they are responsible for action potential initiation. The dense clustering of channels at the AIS involves ankyrinG binding, however the details of trafficking these channels to the AIS remains elusive. Furthermore, it is unclear what percentage of AIS channels is actually conducting. Since the large sodium channel cDNAs are difficult to manipulate and suffer from rearrangements in E. coli, the most elegant trafficking work to date has utilized chimeric proteins containing the sodium channel ankyrin-binding motif fused to other membrane proteins. To fully address trafficking in real time, an appropriately tagged full-length and functional channel is required. Therefore, we developed a Nav1.6 tagged with either GFP or Dendra2 fluorescent-proteins on the C-terminus and an extracellular biotin-acceptor-domain (BAD). The BAD allows for visualization and single molecule tracking of quantum-dot-bound sodium channels on the neuronal surface. This modified Nav1.6 demonstrated wild-type activity when expressed in hippocampal neurons. The tagged channel efficiently trafficked to the cell surface and was localized at the AIS as indicated by both confocal and TIRF microscopy. Alexafluor 594-conjugated-streptavidin binding indicated the surface-density of channels at the AIS was approximately 60 times greater than on the soma, comparable to endogenous Nav1.6 channels. Fluorescence recovery after photobleaching (FRAP) and single particle tracking showed that channels at the AIS had recovery time constants of greater than 2 hours and were confined to 60nm +/- 20nm compartments. In summary, we constructed a sodium channel with fluorescent protein and extracellular biotin reporters that has both wild-type trafficking and biophysical properties. This construct will permit the examination of sodium channel turnover, trafficking, diffusion and location-dependent function in neuronal cells.