Persistent Na Channels and the NaCa2 Exchanger

A number of in vitro experiments have shown that intra-axonal influx of calcium via the Na+/Ca2+ exchanger can produce irreversible physiological failure and subsequent degeneration of the white matter axon (Stys et al., 1992a; see also Chapter 19). Stys et al. (1990) performed a series of experiments that have delineated the cascading interaction of persistently activated Na+ channels, the Na+/Ca2+ exchanger (which can import calcium when operating in its "reverse" mode in response to high levels of intracellular Na+ and/or depolarization), and the resultant inflow of calcium into the axon. In these studies, the isolated optic nerve was used as a model and was subjected to a standardized insult consisting of 60 minutes of anoxia. After the anoxic challenge, quantitative analysis of compound action potential (CAP) recovery is used as a functional outcome measure of axonal integrity. When measured after 60 minutes of anoxia, the area of the postanoxic CAP is substantially depressed and remains at only about 30% of preanoxia levels with no additional recovery, indicating that only 30% of the axons are capable of conducting action potentials (Stys et al. 1990, 1991a, 1991b). At the ultrastructural level, the development of permanent structural damage parallels the reduced functional capacity. Anoxia produces injury to the axonal membrane and mitochondria, and severe disruption of cytoskeletal structures, similar to calcium-mediated injury of the cytoskeleton within axons (Waxman et al., 1993).

Notably, even though excitotoxicity is not the trigger for neuronal damage in this model of white matter injury (Ransom et al., 1990), the presence of extracellular Ca2+ is necessary for anoxia-induced axonal degeneration to occur. Removing Ca2+ from the perfusate, and then subjecting optic nerves to the same duration (60 minutes) of anoxia results in complete (100%) recovery of CAP to preanoxia levels (Stys et al., 1990). Ultrastructural examination of anoxia-exposed axons in Ca2+-free conditions reveals the retention of normal axonal cytoarchitecture (Waxman et al., 1993).

In this form of axonal injury, calcium enters the axon via reverse operation of the Na+/Ca2+ exchanger (Fig. 1.) (Stys et al., 1991a; 1992a; see also Chapter 19). In its normal mode, this exchanger functions as an antiporter—extruding one Ca2+

EXTRACELLULAR

Figure 1 Cellular pathophysiological events leading to terminal intracellular influx of calcium. Anoxia leads to ATP depletion with resultant failure of ATPase activity and depolarization (1). Persistent Na+ channels (2) provide a route for Na+ influx. Collapse of the Na+ gradient forces the Na+/Ca2+ exchanger (3) to operate in reverse causing influx of Ca2+ into the axon. Voltage Ca2+ channels (4) provide a route for Ca2+. (Modified from Stys et al., 1992a.)

INTRACELLULAR

Figure 1 Cellular pathophysiological events leading to terminal intracellular influx of calcium. Anoxia leads to ATP depletion with resultant failure of ATPase activity and depolarization (1). Persistent Na+ channels (2) provide a route for Na+ influx. Collapse of the Na+ gradient forces the Na+/Ca2+ exchanger (3) to operate in reverse causing influx of Ca2+ into the axon. Voltage Ca2+ channels (4) provide a route for Ca2+. (Modified from Stys et al., 1992a.)

ion in exchange for three Na+ ions—as Na+ ions run down their gradient from the extracellular compartment to the intra-cellular compartment. However, in situations where the normal Na+ transmembrane gradient has collapsed or has even been reversed because of the intracellular rise of Na+, or when there is membrane depolarization, the Na+/Ca2+ exchanger can be forced to operate backward, now driving Ca2+ ion into the axon (Baker et al., 1969; Cervetto et al., 1989). Anoxia appears to trigger an elevation of intra-axonal Na+ via ATPase run-down because of energy failure and activation of Na+ channels resulting from depolarization (Stys et al., 1992a). High concentrations of intra-axonal Na+ have been correlated with reverse action of the Na+/Ca2+ exchanger; using electron probe microanalysis during membrane depolarization or anoxia of optic nerve, levels of intracellular Na+ are observed to rise concomitant with parallel increases in Ca2+, consistent with reverse function of the exchanger (LoPachin and Stys. 1995).

Of importance, the time-course of sodium influx in the anoxic optic nerve indicates that the increase in intra-axonal Na+ levels is the result of persistent Na+ influx via a nonin-activating Na+ conductance (Stys et al., 1992a). Although the presence of this standing Na+ conductance was not directly demonstrated in the studies of anoxic injury, sucrose gap studies on the normal optic nerve have shown the presence of a noninactivating tetrodotoxin (TTX)-sensitive Na+ conductance in optic nerve axons (Stys et al., 1993). Although the channel(s) responsible for the persistent Na+ influx in injured axons has not been definitively identified, several studies suggest that Nav1.6 sodium channels, which are present in high density in the axonal membrane at the nodes of Ranvier (Caldwell et al., 2000), contribute a significant fraction (if not all) of the noninactivating Na+ conductance. Biophysical studies have demonstrated that the Nav1.6 sodium channel produces a sizable persistent current (Smith et al., 1998;

Burbidge et al., 2002; Herzog et al., 2003). Furthermore, immunochemical studies demonstrate the co-localization of Nav1.6 sodium channels and the Na+/Ca2+ exchanger within degenerating axons in EAE (Craner et al., 2004a) and in MS (Craner et al., 2004b), as described in Chapter 7.

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