THEMES
Pathobiology of Visceral Pain: Molecular Mechanisms and Theraputic Implications
I. Cellular and molecular biology of sodium channel beta -subunits: therapeutic implications for pain?*

Lori L. Isom

Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109-0632


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Voltage-gated sodium channel alpha -subunits have been shown to be key mediators of the pathophysiology of pain. The present review considers the role of sodium channel auxiliary beta -subunits in channel modulation, channel protein expression levels, and interactions with extracellular matrix and cytoskeletal signaling molecules. Although beta -subunits have not yet been directly implicated in pain mechanisms, their intimate association with and ability to regulate alpha -subunits predicts that they may be a viable target for therapeutic intervention in the future. It is proposed that multifunctional sodium channel beta -subunits provide a critical link between extracellular and intracellular signaling molecules and thus have the ability to fine tune channel activity and electrical excitability.

cell adhesion molecules; extracellular matrix


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ABNORMAL EXPRESSION of voltage-gated sodium channels in primary sensory neurons [dorsal root ganglion (DRG) neurons and trigeminal neurons] is thought to contribute to the molecular pathophysiology of pain. A number of review articles have appeared recently that describe the role of sodium channel alpha -subunits, especially the TTX-insensitive channels PN3/SNS (SCN10A) and NaN/SNS2 (SCN11A), in the molecular basis of pain (4, 8, 19, 23, 25, 36, 37). It is important to remember that sodium channels are heterotrimeric structures composed of a central, pore-containing alpha -subunit and two auxiliary subunits: a noncovalently associated beta 1-subunit (or its splice-variant isoform beta 1A) and a disulfide-linked beta 2-subunit, which do not form the pore but play critical roles in channel gating, voltage dependence of activation and inactivation, channel protein expression levels, and interaction with other signaling molecules such as extracellular matrix and the cytoskeleton (3, 10, 16). This article examines recent advances in our understanding of sodium channel auxiliary beta -subunit function. In the future, specific modulation of beta -subunit expression may result in the ability to fine tune sodium channel activity in localized tissues, much like a rheostat, such that pain sensations can be managed without serious side effects.


    MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF SODIUM CHANNEL beta -SUBUNITS
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beta 1 (36 kDa), beta 1A (45 kDa), and beta 2 (33 kDa) are transmembrane proteins with type I topology (11, 12, 16). All three molecules contain extracellular immunoglobulin domains that are structurally homologous to the V-set of the immunoglobulin superfamily that includes cell adhesion molecules (CAMs) (9). Sequence analysis of beta 2 revealed that its extracellular domain contains an immunoglobulin fold and an extended region with similarity to the CAM contactin (12). Two distinct regions of the extracellular domain of contactin have >40% amino acid sequence identity with sodium channel beta 2-subunits. Subsequent analysis of the extracellular domains of beta 1 and beta 1A showed a similar homology to the CAM myelin Po (9). It was proposed that beta -subunits may function as CAMs as well as modulators of channel kinetics and plasma membrane expression levels.

Coexpression of beta 1-subunits with brain [SCN1A (32), SCN2A (11), SCN3A (24), and SCN8A (31)] or skeletal muscle [SCN4A (30)] alpha -subunits in Xenopus oocytes increased the size of the peak sodium current, accelerated its inactivation, and shifted the voltage dependence of inactivation to more negative membrane potentials, indicating that beta 1 is crucial in the assembly, expression, and functional modulation of the rat brain sodium channel heterotrimeric complex. Coexpression of SCN2A alpha - and beta 1-subunits in mammalian cells increased the level of sodium channels at the plasma membrane two- to fourfold as determined from [3H]saxitoxin binding but did not affect the dissociation constant for saxitoxin (13). Coexpression of beta 1-subunits in these cells also shifted the voltage dependence of sodium channel inactivation to more negative membrane potentials by 10-12 mV and shifted the voltage dependence of channel activation to more negative membrane potentials by 2-11 mV (13). Interestingly, beta 1 does not appear to modulate all sodium channel alpha -subunits. Most important for this discussion is the observation that SNS/PN3 alpha -subunits are not affected by coexpression of beta 1 (26, 33). These results suggest that other beta 1-like subunits may be present in sensory neurons. To begin to answer this question, a splice variant of beta 1, beta 1A, was recently identified in DRG, as described below. Its functional effects on the TTX-resistant sodium channels have not yet been determined.

Coexpression of beta 2 with alpha -subunits in Xenopus oocytes caused an increase in functional expression of sodium channels, an increase in the fraction of alpha -subunits gating in a fast mode, and a small negative shift in the voltage dependence of channel inactivation, similar to the effects observed for beta 1 (12). Expression of higher levels of beta 2 also caused a fourfold increase in the capacitance of the Xenopus oocyte, which resulted primarily from an increase in the number and surface area of the plasma membrane microvilli. Interestingly, this beta 2-subunit-mediated increase in membrane capacitance did not depend on coexpression of alpha . The sequence similarity of beta 2-subunits to contactin, their ability to expand the cell surface membrane, and their appearance in developing neurons and axons suggested that they may modulate cell surface expression and function of sodium channels during neurogenesis and synaptogenesis (12).

Association of neuronal alpha - and beta 2-subunits is a late event in sodium channel biosynthesis (29). In primary cultures of rat embryonic brain neurons, a large, metabolically stable, intracellular pool of newly synthesized "free" alpha -subunits (not disulfide-linked to beta 2-subunits) could be detected. alpha -Subunits disulfide-linked to beta 2-subunits were found preferentially at the cell surface. This intracellular pool of alpha -subunits was found to be available to serve as a source of precursors to form functional cell surface sodium channels. It was proposed that association with beta 2 could be a rate-limiting step in regulation of the cell surface density and localization of sodium channels in developing neurons. Because high-affinity beta -subunit antibodies were not available at the time of these studies, the association of alpha  and beta 1 was not described. However, more recent data have shown that beta 1-subunit expression is a critical regulator of sodium channel density in the plasma membrane of transfected cells (13). Thus an important function of beta -subunits may be to direct, promote, and/or stabilize sodium channel alpha -subunit localization in the plasma membrane. In the future, tight regulation of beta -subunit expression might serve as a therapeutic mechanism to control sodium channel density and thus electrical excitability of targeted neurons in pain pathways.

beta 1 is expressed only after birth in the developing brain (24, 27). However, the developmental time course of beta 1 expression in rat forebrain showed multiple size bands at earlier time points (20). These immunoreactive bands were also present in adrenal gland, heart, and skeletal muscle. In an attempt to identify beta 1-subunit isoforms, a rat adrenal cDNA library was screened with a probe specific to the coding region of beta 1. A cDNA clone was isolated that contained identity to beta 1 at the 5' end followed by a novel 3' region. This beta 1 isoform, beta 1A, was found to be a splice variant of beta 1 that is the result of the retention of intron 3 containing an in-frame stop codon (16). This alternate splicing event produces a novel carboxy terminus that includes a transmembrane segment and short intracellular domain. The developmental time course of beta 1A vs. beta 1 mRNA expression in rat brain showed that beta 1A is expressed early in embryonic development. Its expression declines to undetectable levels after birth, concurrent with the expression of beta 1. Thus alternate splicing of beta 1 mRNA appears to be developmentally regulated. Immunohistochemical analysis of beta 1A expression showed that it is expressed in adult DRG, spinal cord, and heart. Functional coexpression of SCN2A with beta 1A in transfected Chinese hamster lung fibroblasts resulted in a 2.5-fold increase in current density compared with cells expressing the alpha -subunit alone. [3H]saxitoxin binding analysis confirmed these results. This increase in current density reflected two distinct effects of beta 1A: 1) an increase in the proportion of cells expressing detectable sodium currents and 2) an increase in the levels of functional sodium channels in expressing cells. Increases in sodium channel expression with beta 1A are similar to previous results obtained with the adult brain beta 1 isoform in both mammalian cells (13) and Xenopus oocytes (11). These observations are consistent with the hypothesis that beta 1- and beta 1A-subunits facilitate the expression of sodium channels and/or stabilize channels in the plasma membrane. Sodium currents in beta 1A-expressing cell lines also exhibited subtle functional differences compared with the parent alpha -subunit-expressing cell line. Inactivation curves in alpha beta 1A-expressing cell lines were shifted to slightly more positive potentials than inactivation curves for alpha  alone. This finding differs from previous results showing that coexpression of alpha  and beta 1 in Chinese hamster lung cells shifts inactivation to potentials ~10 mV more negative than for cells expressing alpha  alone. Apparently, differences between these two beta -subunit isoforms in the putative transmembrane and intracellular segments are responsible for these differences in functional effects.


    SODIUM CHANNEL beta -SUBUNITS INTERACT WITH THE EXTRACELLULAR MATRIX PROTEINS TENASCIN-R AND TENASCIN-C
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Glial-derived extracellular matrix molecules, for example tenascin-C (TN-C) and tenascin-R (TN-R), play important roles in cell interactions in developing or injured neuronal cells, such as neuronal migration, neuritogenesis, and neuronal regeneration (6, 28). The tenascins are multifunctional molecules that can promote neurite outgrowth, inhibit growth cone advance, and induce axonal defasciculation in vitro. Two research groups, in separate experiments, showed that sodium channel beta -subunits interact with TN-C (34) and TN-R (34, 38). The tenascins were chosen as candidate molecules because they bind contactin, a CAM with homology to sodium channel beta 2-subunits (34). Incubation of purified sodium channels on microtiter plates coated with TN-C showed saturable and specific binding. Glutathione-S-transferase (GST) fusion proteins containing various domains of TN-C and TN-R were tested for their ability to bind purified sodium channels or the recombinant beta 2-subunit extracellular domain. Both sodium channels and beta 2 bound specifically to the fibronectin (FN) type III repeats 1-2, A, B, and 6-8 of TN-C and FN type III repeats 1-2 and 6-8 of TN-R. No binding was observed to the epidermal growth factor (EGF)-like repeats or the fibrinogen (FG)-like domain of either molecule. Transfected cells expressing beta 1 or beta 2 were repelled from TN-R plated on a nitrocellulose substrate (38). The same TN-R GST fusion proteins used in the first study were used to determine which domains were responsible for the observed repulsion. Both the beta 1- and beta 2-expressing cell lines were strongly repelled by EGF-L (cysteine-rich amino terminus of TN-R plus the EGF-like domains) but adhered well to EGF-S (EGF-like domains only), FN 6-8, FG, and GST, suggesting that the cysteine-rich amino-terminal domain of TN-R may be involved in repulsion of beta 1- or beta 2-expressing cells. Cells expressing beta 1-subunits alone initially adhered to the TN-R recombinant domains FN 6-8 (as found in the first study for beta 2) and EGF-S before repulsion. A mixture of EGF-L, EGF-S, and FN 6-8 fusion proteins added to the cell culture medium blocked the adhesion of beta 1-expressing cells to the EGF-like or FN-like domains of TN-R in a concentration-dependent manner. beta -Subunit-mediated effects in response to TN-R occurred in the absence of alpha -subunits, suggesting that beta -subunits can function as CAMs independently of the ion channel complex.

In two-electrode recordings in Xenopus oocytes, EGF-L fusion protein produced a rapid increase in the amplitude of sodium currents. EGF-L-mediated potentiation was observed in oocytes expressing the SCN2A alpha -subunit alone and in oocytes coexpressing SCN2A, beta 1-, and beta 2-subunits. In contrast, neither FN 6-8 fusion protein nor GST affected sodium currents in oocytes, suggesting that potentiation is a specific effect of the EGF-L domain of TN-R. EGF-L-mediated potentiation was not accompanied by any detectable changes in the voltage dependence of current activation or inactivation or in any obvious effects on current time course.


    SODIUM CHANNEL beta -SUBUNITS INTERACT HOMOPHILICALLY AND RECRUIT ANKYRIN TO THE PLASMA MEMBRANE
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Cell adhesion molecules of the immunoglobulin superfamily interact homophilically and heterophilically to transduce signals between adjacent cells or adjacent axons, where they participate in axonal fasciculation. For example, certain CAMs interact homophilically in a trans mechanism to produce cellular aggregation (14). After homophilic binding, CAMs of the L1 family that have intracellular carboxy-terminal domains transduce signals resulting in the recruitment of ankyrin and spectrin to points of cell-cell contact. Drosophila S2 cells are a classic model system in which potential CAMs have been tested for these properties (1). S2 cells do not express endogenous CAMs and grow as a suspension culture. cDNAs of interest are then cloned into the S2 cell expression vector under the control of an inducible Drosophila metallothionine promoter. On induction of protein expression, S2 cells transfected with CAMs aggregate. Immunocytochemical localization of endogenously expressed ankyrin can then be performed to determine whether trans-homophilic binding results in ankyrin recruitment to the plasma membrane. The S2 cell model system has been used to investigate whether sodium channel beta 1- and beta 2-subunits behave in a similar manner. S2 cells transfected with beta 1 or beta 2 display homophilic interactions (18). Immunocytochemical analysis of the cell aggregates revealed recruitment of ankyrin to sites of cell-cell contact. Coimmunoprecipitation experiments from rat brain membranes showed that ankyrinG, which is expressed at mammalian nodes of Ranvier and axon initial segments (17), and beta 1- or beta 2-subunits are physically associated in mammalian neurons (J. D. Malhotra and L. L. Isom, unpublished observations). Thus sodium channel beta -subunits appear to behave like classic CAMs: they bind homophilically, recruit ankyrin to points of cell-cell contact, and interact with extracellular matrix molecules.

The intracellular domains of beta 1 and beta 2 are critical for ankyrin recruitment. Truncation mutants lacking the intracellular carboxy-terminal domains of beta 1 and beta 2, respectively, were transfected into S2 cells and the transfected cells were induced to aggregate. Although both cell lines aggregated, ankyrin staining was diffuse and not concentrated to points of cell-cell contact as in cells expressing full-length beta -subunits (18). It was concluded that the intracellular carboxy-terminal domains of beta 1 and beta 2 are necessary for trans-homophilic-mediated signal transduction to the cytoskeleton.


    SODIUM CHANNELS ARE MODULATED BY CONTACTIN
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Because of its homology to beta 2-subunits, contactin was also investigated as a possible modulator of sodium channels (15). Stable coexpression of SCN2A and contactin in Chinese hamster lung cells had no obvious effects on channel gating, voltage dependence, or expression levels. However, coexpression of SCN2A, contactin, and beta 1 resulted in a fourfold increase in [3H]saxitoxin binding and current amplitude compared with coexpression of SCN2A and beta 1. This effect was limited to sodium channel expression levels, since no changes were observed in channel-gating kinetics or voltage dependence. Contactin-mediated effects on sodium channel expression appeared to require the presence of beta 1, perhaps through heterophilic cell adhesive interactions. The distribution of sodium channels and contactin was investigated in sciatic nerve using immunocytochemical techniques. Although contactin and sodium channels were clearly separated into paranodal and nodal regions, respectively, in adult animals, it was found that during embryonic development as well as during remyelination these molecules were colocalized. After nerve injury, the interaction of sodium channel beta -subunits and contactin may play a key role in reformation of nodes of Ranvier and reestablishment of saltatory conduction.


    SODIUM CHANNEL beta -SUBUNIT MRNAS ARE DIFFERENTIALLY REGULATED IN A RAT MODEL OF NEUROPATHIC PAIN
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In rat models of neuropathic pain, it has been shown that chronic constrictive injury results in dynamic changes in the relative expression of sodium channel alpha -subunits in the DRG as well as in the spinal cord (36). In the Bennett and Xie model of neuropathic pain, levels of beta 1 and beta 2 mRNA in the dorsal horn of the spinal cord are differentially regulated as well (2). At 12-15 days after neuropathy, beta 1 mRNA levels increased, whereas beta 2 mRNA levels decreased significantly within laminae I-II on the ipsilateral side of the spinal cord relative to the contralateral side. In laminae II-IV, beta 1 mRNA levels remained constant and beta 2 levels again showed a small but significant decrease. It was proposed that a functional downregulation of beta 2-subunits may decrease the interaction of beta -subunits with tenascins, thereby promoting axonal growth and projection of neurons into the superficial laminae in which new synaptic contacts could be initiated. It remains to be seen whether beta -subunit protein levels correspond to the reported changes in mRNA after neuronal injury. Future research must also include a correlation of neurons in which beta -subunit levels change with localized changes in alpha -subunit expression as well as with specific pain pathways.


    SODIUM CHANNEL beta -SUBUNIT MRNA EXPRESSION IS REGULATED BY PHOSPHORYLATION AND NEUROTROPHINS
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Protein kinase A (PKA) modulation of TTX-resistant sodium channels may underlie the hyperalgesic responses of sensory neurons to agents such as serotonin and PGE2 (5, 8). The action of these agents can be mimicked by drugs that upregulate PKA phosphorylation of sodium channels, such as forskolin and 8-bromo-cAMP (5, 8). SNS/PN3 channels are phosphorylated by PKA, resulting in a time-dependent increase in sodium current amplitude as well as a marked hyperpolarizing shift in the current-voltage relationship, reducing the threshold for activation (7). Interestingly, sodium channel beta 1- and beta 2-subunit mRNA levels are upregulated in spinal cord astrocytes, B50 neuroblastoma cells, optic nerve astrocytes, sciatic nerve astrocytes, and Schwann cells after exposure to increased levels of cAMP (21, 22). Perhaps increases in cAMP mediated by hyperalgesic agents can result in hyperexcitability via two mechanisms: phosphorylation of alpha -subunits and upregulation of beta -subunit expression. Upregulation of beta 1 and beta 2 levels would be expected to result in increased sodium current amplitude as well as hyperpolarizing shifts in the voltage dependence of channel activation and inactivation.

Neuronal sodium channel expression has been shown to be influenced by nerve growth factor (NGF) (35). Changes in sodium channel expression in DRG neurons after axotomy within the sciatic nerve have been shown to be, in part, due to loss of access to peripheral pools of NGF (36). Direct delivery of NGF to DRG cell bodies in vitro resulted in downregulation of type III alpha -subunits (SCN3A) and upregulation of SNS/PN3 expression, thus partially preventing the redistribution of sodium channels normally seen after axotomy (36). Sodium channel beta 1-subunit mRNA is upregulated in DRG neurons in NGF-containing medium (39). Again, like the situation with cAMP, neurotrophins may modulate sodium channel expression in DRG neurons via differential effects on alpha - and beta -subunits. Thus future research focusing on methods of modulating beta -subunit expression with neurotrophins in selective neuronal cell bodies in the DRG may yield effective therapies for pain.


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In conclusion, sodium channel beta -subunits present a unique and exciting experimental problem. beta -Subunits participate in modulation of the voltage dependence of channel activation and inactivation, channel gating mode, and channel expression levels at the plasma membrane. Now we see that the beta -subunits are members of the immunoglobulin superfamily and play roles in cellular adhesion and repulsion as well. Thus sodium channel beta -subunits are multifunctional proteins that present a number of possibilities for future therapeutics. It may be possible to target beta -subunit expression in selective DRG or spinal cord neurons. Tight control of beta -subunit expression levels in certain classes of neurons may be one way of regulating sodium current density and therefore hyperexcitability after neuronal injury. Alternatively, site-directed mutagenesis of specific residues in the immunoglobulin fold or the intracellular domains, or rational drug design directed toward these regions, may make it possible to regulate beta -subunit-mediated interactions with extracellular matrix, other CAMs, or cytoskeletal molecules. In this way, one might gain control over sodium channel clustering, axonal sprouting, or axonal fasciculation during remodeling events after nerve injury. Sodium channel beta -subunits may indeed be critical links between the extra- and intracellular neuronal environments and may now provide insights into future therapeutic interventions for pain.


    FOOTNOTES

*  First in a series of invited articles on Pathobiology of Visceral Pain: Molecular Mechanisms and Therapeutic Implications.

Address for reprint requests and other correspondence: L. L. Isom, Dept. of Pharmacology, The Univ. of Michigan, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632 (E-mail: lisom{at}umich.edu).


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