Trauma-Induced Changes in Recombinant and Native Nav1.2 Channel Kinetics

Nav1.2, the Nav isoform of neuronal cell bodies and proximal axon initial segments (AIS), is a “high threshold” channel compared to Nav1.6, the distal AIS and node of Ranvier isoform. For recombinant Nav1.6, we showed that bleb formation (mechanically-induced by aspiration of cell-attached oocyte patches) irreversibly left-shifts availability and activation (Wang et al 2009 Am J Physiol 297:C823); in situ, the resultant window current shift would constitute a pathological Na-leak. Here we show that, like human Nav1.6, recombinant rat Nav1.2 expressed in oocytes (without/with rat β1 subunit) and monitored in cell-attached patches, current availability and activation left-shifted progressively and irreversibly with progressively stronger pipette aspiration. Sometimes, just the act of patch formation was mechanically traumatic enough to left-shift channel operation (as we found previously with Nav1.6). In computations, left-shifting even a fraction of an axon's Nav channel population yields multiple deleterious effects for excitability and ion homeostasis (Boucher, Joos, Morris; BPSoc2011 abstracts) and this makes it important to determine if native Nav channels misbehave like recombinants when subjected to membrane trauma. To test native Nav1.2, we did cell-attached patching of entorhinal cortex pyramidal neurons in rat brain slices. Well-clamped whole-cell recordings from these neurons (Milescu et al 2010 J Neurosci 30:7740) show the threshold for Nav1.2 activation to be −45 mV. Seals were difficult to maintain (spontaneous break-in was frequent within a minute of sealing), but assuming Vrest=0 or =-65 mV (i.e. hiK bath; normal bath, respectively), we observed Nav current starting at Vm= −60 mV or −55 mV, i.e. close to threshold activation values for maximally traumatized recombinant Nav1.2 current in oocyte patches. Establishing the cell-attached configuration in these neurons, we conclude, unavoidably subjected patches to mechanical trauma that put the native Nav1.2 channels in a permanently left-shifted condition.