In this study, we have demonstrated the presence of the NCX2 isoform of the Na+/Ca2+ exchanger (NCX), together with sodium channel isoforms NaV1.6, NaV1.7, NaV1.8, and NaV1.9 within intra-epidermal free nerve terminals which include nociceptive endings. Our results are novel in demonstrating the presence of NCX2 in virtually all of the small-diameter axons and their terminals in the epidermis, and show that NaV1.6, NaV1.7, NaV1.8, and NaV1.9 are detectable in a majority of epidermal nerve endings. Consistent with our observation of NCX2 in these terminals, Verdru et al.  reported sodium-calcium exchange, and expression of mRNAs for multiple NCX isoforms including NCX2, in rat DRG neurons, and our studies demonstrate NCX2 within DRG neuronal cell bodies. Our observations in epidermal nerve endings extend earlier observations by Toledo-Aral et al.  who observed NaV1.7 within the growth cones of DRG neurons in culture, Black et al.  who observed NaV1.6 in axons within the skin, and Zhao et al.  who described NaV1.8 and NaV1.9 in nerve endings in epidermis. Notably, our results demonstrate that NCX2 is present within virtually all epidermal nerve terminals and thus indicate that NCX is present in terminals that express NaV1.6, NaV1.7, NaV1.8 and NaV1.9. Our results also demonstrate the co-localization of NaV1.7 and NaV1.8 within intra-epidermal nerve fibers, a result that is functionally important since NaV1.7 and NaV1.8 play different and interdependent roles in electrogenesis [6, 21].
Electrophysiological recordings indicate that a tetrodotoxin (TTX)-resistant sodium channel isoform, presumably NaV1.8 on the basis of kinetics, is expressed at levels that can support action potential electrogenesis, together with unspecified TTX-sensitive sodium channels that contribute to excitability, within terminal branches of C- and Aδ-type dural afferent axons . Similarly, TTX-resistant sodium channels have been shown to support action potentials within polymodal, mechanosensory, and cold-sensitive nerve terminals in the cornea, where action potential electrogenesis is also partially supported by TTX-sensitive sodium channels . There is also evidence indicating that TTX-resistant sodium channels (probably NaV1.8) allow the superficial endings of slowly conducting nociceptive fibers to conduct impulses at low temperatures . A role of sodium channels in transduction in sensory axon terminals is also suggested by the observation that 6.0 μM TTX attenuates receptor potentials in Pacinian corpuscles , although this concentration does not differentiate between TTX-sensitive and TTX-resistant channels and thus provides no information about the molecular identity of the channels. There is also electrophysiological evidence indicating that, along C-fiber trunks, NaV1.6  and sodium channels with physiological properties suggestive of NaV1.7 and NaV1.8  contribute to action potential propagation.
It is possible that some intra-epidermal nerve fibers may express low densities of channels that are below the threshold for detection by immunocytochemical methods, so the percentages of fibers expressing sodium channel isoforms that we report may represent underestimates. Moreover, our results do not provide information about the density of functional sodium channels within the membrane of the epidermal nerve endings. Low sodium channel densities (as low as 1-3/μm2) can support action potential electrogenesis in neuronal compartments such as small-diameter axons with high input impedances [27, 28]. There may in fact be functional benefit to limiting sodium channel density within sensory terminals since even moderate sodium channel densities (30/μm2 in a 1.0 μm diameter axon) can produce spontaneous firing as a result of channel noise or spontaneous channel opening in the context of a high input resistance, low capacitance per unit length, and shorter length constant [29–31].
While the presence of sodium channels along axons is not unexpected, the precise functional roles of each of the four sodium channel isoforms within intra-epidermal nerve terminals remain to be determined. The sodium channel isoforms that we have observed in intra-epidermal nerve terminals display a spectrum of biophysical and physiological properties which tune them so that they support sodium influx over a range of voltage and time domains. NaV1.6, the major channel at nodes of Ranvier , is also present along the trunks of central non-myelinated axons  and peripheral C-fibers  and, as a result of rapid recovery from inactivation , produces sodium influx that contributes to high-frequency firing. NaV1.7 displays slow closed-state inactivation [34, 35] and generates an inward sodium current in response to small slow depolarizing inputs such as generator potentials in the subthreshold range. NaV1.8 displays rapid recovery from inactivation  and depolarized activation and inactivation voltage-dependence  which permit it to generate a large inward sodium current during the rising phase of the action potential [38, 39] including high-frequency firing in response to sustained depolarization . NaV1.9 displays broad overlap between activation and inactivation together with extremely slow inactivation , and can generate a persistent sodium current at subthreshold potentials so as to amplify and prolong depolarizing inputs, decrease action potential threshold, and depolarize resting potential [41, 42]. Although the majority of NaV1.9  and possibly other sodium channels may be inactivated at the resting potential of sensory terminals , expression and current density of NaV1.7 [43, 44], NaV1.8 [45–47] and NaV1.9 [6, 41] are known to be up-regulated by pro-inflammatory molecules, so that these channels may contribute to sensory axon sensitization under conditions of inflammation.
Small caliber C- and Aδ-fibers that terminate as free nerve endings in the epidermis each represent spatially and molecularly heterogeneous populations, expressing non-uniform combinations of ligand-mediated ionotropic and metabotropic receptors, voltage-gated ion channels, heat/cold receptors and neuropeptides (see e.g. ). For instance, nerve endings expressing the G-protein coupled receptor Mrgprd terminate in the stratum granulosum , where they are suggested to play an important role in noxious mechanical nociception . Conversely, the non-overlapping peptidergic, TRPV1+ nerve endings terminate in stratum spinosum and appear to participate in noxious heat sensitivity [4, 48]. Most intra-cutaneous nerve terminals express NaV1.7, NaV1.8 and NaV1.9, while NaV1.6 is detectable in 70% of the nerve endings. It is not clear whether the different subsets of free nerve endings exhibit different patterns of sodium channel expression.
The expression of NCX2 along intra-epidermal nerve terminals provides a molecular substrate for sodium-calcium exchange in these fibers. NCX is known to be present along the trunks of myelinated axons  where, under normal (non-pathological) conditions, sodium-calcium exchange is coupled to sodium influx, and contributes to calcium extrusion following physiological activity . However, the presence of NCX2 together with sodium channels within epidermal nociceptive terminals may also have pathophysiological implications. Even a small ongoing sodium influx may increase intracellular sodium levels within small-diameter axons, which have a large surface-volume ratio, thereby imposing a substantial energetic load . Persistent currents and ramp responses to small, slow depolarizations are produced over multiple overlapping voltage domains extending from the resting potential of DRG neuron somata to nearly 0 mV by the sodium channels present within these sensory terminals, NaV1.6 [50, 51], NaV1.7 , NaV1.8  and NaV1.9 . Persistent sodium influx via sodium channels has been shown to drive injurious, calcium-importing reverse sodium-calcium exchange in myelinated axons under conditions of energy deprivation such as anoxia or ischemia [53–55]. In addition, repetitive action potential activity at physiological frequencies can also render axons vulnerable to metabolic insults so that a combination of energy deprivation and electrical activity can lead to axonal degeneration . Expression of NCX2 together with sodium channels in intra-epidermal axon terminals may thus make these fine-diameter nerve fibers especially sensitive to injury when energetically challenged.