A Role for Phosphorylation in the Maintenance of Resurgent Sodium Current in Cerebellar Purkinje Neurons

Abstract

Cerebellar Purkinje neurons express voltage-gated, tetrodotoxin (TTX)-sensitive sodium channels that not only open and inactivate rapidly during depolarization but also reopen during repolarization, carrying an unusual "resurgent" sodium current. Expression of Na(V)1.6 alpha subunits appears necessary but not sufficient to generate this component of current; Purkinje cells without Na(V)1.6 lack resurgent current, but resurgent current is absent from many other Na(V)1.6-expressing neurons. These observations raise the question of how modulation or modification of the Na(V)1.6 subunit may lead to production of resurgent current. Previous studies have suggested that sodium channels of Purkinje neurons are subject to a rapid, voltage-dependent, open channel block by an endogenous particle whose unbinding allows resurgent current to flow. To investigate the nature of this block, we recorded TTX-sensitive sodium currents in outside-out patches from Purkinje cells acutely isolated from mice. In all patches, step depolarizations evoked transient current, and step repolarizations evoked resurgent current. The amplitudes of the transient and resurgent currents were highly correlated across patches (R(2) > 0.99), suggesting that the blocking agent is closely associated with the channel. Intracellular protease eliminated fast inactivation, indicating that the blocking element, like the fast inactivation gate, may be proteinaceous. Intracellular application of alkaline phosphatase abolished resurgent current and significantly slowed inactivation of transient current. The phosphatase inhibitor vanadate reduced these effects. Together, the results suggest that constitutive phosphorylation of the sodium channel complex of Purkinje neurons is necessary to maintain a functional blocking element and produce resurgent sodium current.

Production of Resurgent Current in NaV1.6-Null Purkinje Neurons by Slowing Sodium Channel Inactivation with β-Pompilidotoxin

“…To verify that the resurgent component of sodium current could be selectively abolished by dephosphorylation, we first recorded sodium currents from inside-out patches from wild-type Purkinje cells. In control solutions, the kinetics of transient and resurgent currents in inside-out patches were similar to those measured in whole-cell recordings and outside-out patches (Grieco et al, 2002). Application of alkaline phosphatase (3 mg/ml) to the intracellular face of the patch consistently slowed inactivation of transient currents at 0 mV (from ϭ 0.42 Ϯ 02 to 0.68 Ϯ 12 msec; p ϭ 0.04) and abolished resurgent currents (n ϭ 5 of 5 patches) (Fig.…”

“…In wild-type Purkinje cells, constitutive phosphorylation is necessary to maintain a functional open-channel blocker, because outsideout patches exposed to intracellular alkaline phosphatase, a broad-spectrum phosphatase, lack resurgent current (Grieco et al, 2002). To verify that the resurgent component of sodium current could be selectively abolished by dephosphorylation, we first recorded sodium currents from inside-out patches from wild-type Purkinje cells.…”

“…Na V 1.6 alone is not sufficient to generate resurgent kinetics, however, because several cell types that express Na V 1.6 have no resurgent component to their sodium currents Pan and Beam, 1999). Our working hypothesis is that, although these cells express Na V 1.6, they lack other factors that are required to produce sodium channels with resurgent kinetics, including the presence of an endogenous open-channel blocker and/or constitutive phosphorylation of an unknown substrate (Grieco et al, 2002).…”

Persistent Changes in Spontaneous Firing of Purkinje Neurons Triggered by the Nitric Oxide Signaling Cascade

“…Recent evidence suggests that phosphorylation state tightly controls a resurgent Na ϩ current in Purkinje neurons (Grieco et al, 2002). Our results imply that PKG may be responsible for maintaining the phosphorylation state of resurgent current.…”

Axonal Propagation of Simple and Complex Spikes in Cerebellar Purkinje Neurons

“…For example, somatic voltage-gated Na channels have "resurgent" kinetic properties that promote rapid recovery from inactivation even at potentials that are only moderately hyperpolarized (Raman and Bean, 2001). This rapid recovery appears to be facilitated primarily by Na V 1.6 ␣ subunits associated with a putative protein that limits inactivation Grieco et al, 2002;Khaliq et al, 2003;Grieco and Raman, 2004). Although Na V 1.6 is the primary Na channel ␣ subunit in nodes in most parts of the nervous system Krzemien et al, 2000), it is unknown whether the nodal Purkinje Na channels share the distinctive kinetics of the somatic channels.…”