ReviewRegulation of sodium channel activity by phosphorylation
Introduction
Voltage-gated sodium channels are responsible for the initial depolarization of the action potential in most excitable cells. In addition, non-inactivating persistent sodium current (INa) supports maintained depolarization during and between action potentials. Finally, a component of INa termed resurgent is triggered upon repolarization and supports repetitive firing in some types of neurons. In the 30 years since sodium channels were first isolated, it has become recognized that they are regulated in a variety of ways including by phosphorylation. Here we review current information on effects of phosphorylation and the specific molecular events that are responsible. Voltage-gated sodium channels include an α subunit that contains the ion conducting pore and the gating machinery (Fig. 1) [1]. Each α subunit consists of 4 homologous domains arranged pseudosymmetrically around the central axis. Each domain consists of a pore forming module that contributes to the ion conducting pore and a voltage gating module. Sodium channels also contain one or two β subunits, a β2 or β4 subunits that is disulfide linked to the α subunit as well as a β1 or β3 subunit. There are 9 α subunits in the genome that have been functionally expressed. The transmembrane portions of different isoforms are extremely similar to each other. Instead, the N and distal C-termini as well as the first two intracellular loops connecting the homologous domains are divergent. The intracellular loop connecting homologous domains I and II (loop I–II) contains many of the functionally relevant phosphorylation sites in most isoforms (Fig. 1, red). Other intracellular regions contain additional targets for isoform-specific phosphorylation and protein–protein interactions [2].
This review highlights phosphorylation events that are reflected in sodium channel function or expression and for which specific phosphorylation sites have been identified. Although the α subunits are structurally similar, regulation is different and thus each is considered separately.
Section snippets
Modulation of Nav1.1, Nav1.2, Nav1.3 α subunits by phosphorylation
Nav1.1, Nav1.2 and Nav1.3 sodium channel α subunits are highly expressed in the brain and may be similarly regulated by a variety of kinases. Nav1.2 has been studied in the greatest detail and is emphasized here. Physiological regulation of brain sodium channels by protein kinase A (PKA, cAMP-dependent protein kinase) in a variety of brain neurons is well-established. In rat striatal neurons the dopamine D1 receptor agonist, SKF 81297, raises threshold for firing and this change is associated
Nav1.4 α subunit
Skeletal muscle sodium channels were phosphorylated by PKA despite lacking the large loop I–II that is the target of PKA in other sodium channels [36]. Nav1.4 expressed in HEK293 cells is phosphorylated by activation of PKA or PKC pathways on overlapping and/or interacting sites [37]. However, activation of PKA had little effect on the current [14], [38]. Activation of PKC caused a large decrease in current that was blocked by inhibitors of PKC [39], [40]. This effect was not blocked by
Nav1.5 α subunit
Nav1.5 is the major sodium channel expressed in the heart. In cardiac myocytes activation of the β adrenergic system using isoproterenol produced variable effects on INa, some of which were attributed to activation of PKA while others were attributed to direct actions of G proteins on the channel. Most reports suggested an increase in current, at least partially due to a hyperpolarizing shift in the voltage dependence of activation and inactivation [42], [43].
Activation of PKA increased INa due
Nav1.6 α subunit
Current due to Nav1.6 is reduced by activation of PKA or PKC [7]. However, the degree of reduction is far less in neurons [6] and in tissue culture cells [7] relative to modulation of Nav1.2 after analogous stimulation. Conversely, Nav1.6 is potently modulated by p38 MAP kinase [59]. This kinase is activated after a variety of insults including injury and hypoxia. P38 MAP kinase colocalizes and coimmunoprecipitates with Nav1.6, and kinase activation strongly decreases INa. Only loop I–II of Nav
Nav1.7 α subunit
Effects of PKA regulation of Nav1.7 depend on expression system and whether long (11L) or short (11S) splice variants affecting loop I–II are expressed. PKA decreased currents due to Nav1.7 11L expressed in Xenopus oocytes [61] but had no effect after expression in mammalian cells. PKA caused currents due to Nav1.7 11S expressed in mammalian cells to activate at more negative potentials [62]. Conversely, PKC causes a large depolarizing shift in activation of currents due to Nav1.7 11L expressed
Nav1.8 α subunit
Hyperalgesia mediated by DRG neurons due to adenosine, PGE2 and serotonin effects have been linked to activation of PKA [68], [69], [70]. PGE2 increases the excitability of neonatal dorsal root ganglion (DRG) neurons acting at least partially through Nav1.8 (SNS, PN3) TTX-resistant sodium channels. TTX-resistant INa was increased by shifting the voltage-dependence of activation and inactivation to more negative potentials. This effect is mimicked by activation of PKA and blocked by a peptide
Na channel β subunits
Sodium channel β1 subunits are substrates for tyrosine phosphorylation which inhibits interaction of the intracellular C-terminal tail of β1 with ankyrin [79]. In cardiac myocytes, tyrosine phosphorylated β1 subunits were localized to intercalated disks whereas unphosphorylated β1 subunits were found in the transverse tubules [80]. Thus, β1 subunit phosphorylation controls its interactions and localization in a variety of cells.
Conclusions
Phosphorylation acts to link a broad range physiologic stimuli to an equally broad range of sodium channel responses. Phosphorylation results in changes in gating, acute increases and decreases in current as well as longer term changes in current that are mediated by channel trafficking onto and off of the membrane. A surprisingly common theme is the involvement of loop I–II in the majority of these diverse effects, regardless of sodium channel isoform. Future work will certainly add new
Acknowledgement
This work was supported by National Institutes of Health grant NS64428.
References (80)
From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels
Neuron
(2000)- et al.
Protein–protein interactions involving voltage-gated sodium channels: post-translational regulation, intracellular trafficking and functional expression
Int J Biochem Cell Biol
(2009) - et al.
Functional properties and differential neuromodulation of Nav1.6 channels
Mol Cell Neurosci
(2008) - et al.
Cyclic-AMP-dependent phosphorylation of voltage-sensitive sodium channels in primary cultures of rat brain neurons
J Biol Chem
(1987) - et al.
Identification of an intracellular domain of the sodium channel having multiple cAMP-dependent phosphorylation sites
J Biol Chem
(1987) - et al.
Phosphorylation of the α subunit of rat brain sodium channels by cAMP-dependent protein kinase at a new site containing Ser686 and Ser687
J Biol Chem
(1989) - et al.
Functional modulation of brain sodium channels by cAMP-dependent phosphorylation
Neuron
(1992) - et al.
Transmitter modulation of slow, activity-dependent alterations in sodium channel availability endows neurons with a novel form of cellular plasticity
Neuron
(2003) - et al.
Neuromodulation of Na+ channel slow inactivation via cAMP-dependent protein kinase and protein kinase C
Neuron
(2006) - et al.
AKAP15 anchors cAMP-dependent protein kinase to brain sodium channels
J Biol Chem
(1998)