Functional properties and differential neuromodulation of Nav1.6 channels
Introduction
Voltage-gated sodium channels are widely distributed in neurons of the central nervous system to maintain normal patterns of neuronal electrical activity. Through their role in initiation and propagation of action potentials, sodium channels are essential in defining the input–output relationships of neurons, and they influence integration of dendritic responses, action potential threshold, burst duration, and pattern of firing (Colbert et al., 1997, Jung et al., 1997, Mickus et al., 1999, Johnston et al., 1999, Stuart, 1999, Stuart and Hausser, 2001, Gonzalez-Burgos and Barrioneuvo, 2001). Different central neurons have distinct firing properties, and differential expression, function, and regulation of distinct sodium channel isoforms may contribute to these neuron-specific functions.
Voltage-gated sodium channels are encoded by a family of 10 genes in mammals (Catterall, 2000, Catterall et al., 2005). NaV1.3 channels are primarily expressed in embryonic and neonatal rodent brain, whereas Nav1.1, Nav1.2, and Nav1.6 are highly expressed in adult brain (Catterall et al., 2005). In contrast to rodents, NaV1.3 expression remains high in adult human brain (Chen et al., 2000, Whitaker et al., 2001). Deletion of each of the genes encoding the Nav1.1, Nav1.2, and Nav1.6 channel isoforms expressed in adult rodent brain leads to lethality (Burgess et al., 1995, Planells-Cases et al., 2000, Yu et al., 2006), suggesting that these sodium channels are essential for life and have unique functional roles. Electrophysiological studies have revealed only subtle differences in the properties of these sodium channel subtypes; however, slight changes in sodium channel function can alter action potential firing, as in inherited forms of periodic paralysis, cardiac arrhythmia, epilepsy, chronic pain, and congenital indifference to pain (Balser, 2002, Heron et al., 2007, Keating and Sanguinetti, 2001, Lossin et al., 2002, Meisler et al., 2001, Sugawara et al., 2001, Venance et al., 2006, Dib-Hajj et al., 2007). Although the brain sodium channel isoforms have similar functional properties, distinct subcellular localization and/or regulation of these isoforms may give them unique functional roles. Nav1.1 and Nav1.3 are localized primarily in the soma of CNS neurons, whereas Nav1.2 is primarily in unmyelinated axons (Westenbroek et al., 1989, Westenbroek et al., 1992, Gong et al., 1999). In contrast, Nav1.6 channels are localized in high density in nodes of Ranvier and axon initial segments (Boiko et al., 2001, Caldwell et al., 2000) and in lower density in dendrites and cell bodies of some neurons. They are selectively expressed at high levels in cerebellar Purkinje neurons (Raman et al., 1997).
cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylate brain sodium channels in vitro and in intact neurons (Costa et al., 1982, Costa and Catterall, l984a, Costa and Catterall, l984b, Rossie and Catterall, 1987, Rossie and Catterall, 1989, Rossie et al., 1987) and reduce peak sodium currents in heterologous expression systems (Dascal and Lotan, 1991, Numann et al., 1991) and in neurons (Numann et al., 1991, Surmeier et al., 1992, Li et al., 1992, Cantrell et al., 1996, Cantrell et al., 1997, Cantrell et al., 1999a, Surmeier and Kitai, 1997, Carr et al., 2002). By reducing sodium currents, protein phosphorylation can regulate burst duration and pattern of action potential firing in neurons (Carr et al., 2003). The inhibition of sodium channel activity by PKA and PKC is voltage-dependent and involves enhancement of the intrinsic slow inactivation gating process (Li et al., 1993, Cantrell et al., 1999a, Cantrell et al., 2002, Carr et al., 2003, Chen et al., 2006). Regulation by PKC requires anchored PKCε (Chen et al., 2005). Regulation by PKA requires interaction with A Kinase Anchoring Protein 15 (AKAP15), which binds to the intracellular loop between domains I and II (LI–II; Cantrell et al., 1999b, Cantrell et al., 2002, Few et al., 2007). The key sites of phosphorylation by PKA and PKC in NaV1.1 and NaV1.2 channels are located in the inactivation gate (West et al., 1991) and in LI–II (Murphy et al., 1993, Smith and Goldin, 1996, Smith and Goldin, 1997, Cantrell et al., 1997, Cantrell et al., 2002).
The Nav1.6 channel has been extensively studied in Purkinje neurons, where it conducts substantial peak, persistent, and resurgent sodium currents (Raman and Bean, 1997, Raman et al., 1997). The NaV1.6 channel contributes to peak sodium currents and repetitive firing of neurons in cortical pyramidal cells (Maurice et al., 2001), subthalamic neurons (Do and Bean, 2004), dorsal root ganglion neurons (Cummins et al., 2005), retinal ganglion cells (Van Wart and Matthews, 2006), globus pallidus neurons (Mercer et al., 2007), and trigeminal neurons (Enomoto et al., 2007). Nav1.6 has also been expressed in Xenopus oocytes and in dorsal root ganglion neurons, and its functional properties have been compared with Nav1.1 and Nav1.2 channels in those cell types (Smith et al., 1998, Rush et al., 2005, Rush et al., 2007). In this study, we transfected Nav1.6 channels into tsA-201 cells in order to analyze this channel expressed alone in mammalian non-neuronal cells, compare its functional properties and neuromodulation with NaV1.2 channels, and identify functional and regulatory properties that may be important for its role in action potential generation and repetitive firing.
Section snippets
Activation and fast inactivation of Nav1.6 channels
The properties of voltage-gated sodium channels vary depending on the cell background in which they are expressed. This is reflected in the variable findings in previous reports of the properties of NaV1.6 channels (Burbidge et al., 2002, Rush et al., 2005, Smith et al., 1998). To allow comparison with previous reports on the functional properties and neuromodulation of Nav1.2a channels from our laboratory, we examined the functional properties of Nav1.6 α subunits with clearly defined primary
Similar functional properties of NaV1.2 and NaV1.6 channels
Voltage-gated sodium channels are critical for neuronal excitation because they initiate and propagate action potentials. Three isoforms of sodium channels, Nav1.1, Nav1.2 and Nav1.6, are highly expressed in adult central nervous system (Westenbroek et al., 1989, Trimmer and Rhodes, 2004). The kinetics and voltage dependence of activation and fast inactivation of Nav1.1 and Nav1.2 channels are essentially identical when expressed in tsA-201 cells (Chen et al., unpublished results). Sodium
Molecular biology, transfection, and cell culture
Plasmid pCDM8-rIIA containing the cDNA encoding the full-length rat Nav1.2a α subunit has been described previously (Auld et al., 1990, Linford et al., 1998). Wild-type mouse Nav1.6 cDNA was a kind gift of Dr. Alan Goldin (UC Irvine). It was expanded and subcloned it into pCDM8 for expression in mammalian cells. Following subcloning into pCDM8, we fully sequenced the cDNA and corrected its sequence to correspond with the RefSeq database (NM_011323). The full-length human β4 cDNA was subcloned
Acknowledgments
This work was supported by National Institutes of Health Research Grant NS15751 to W. A. C. and NIH NRSA NS43065 to Y. C.
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