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Purpose: A common genetic variant (rs3812718) in a splice donor consensus sequence within the neuronal sodium channel gene SCN1A (encoding NaV1.1) modulates the proportion of transcripts incorporating either the canonical (5A) or alternative (5N) exon 5. A pharmacogenetic association has been reported whereby increased expression of exon 5N containing NaV1.1 transcripts correlated with lower required doses of phenytoin in epileptics. We tested the hypothesis that SCN1A alternative splicing affects the pharmacology of NaV1.1 channels.
Methods: To directly examine biophysical and pharmacologic differences between the exon 5 splice variants, we performed whole-cell patch clamp recording of tsA201 cells transiently coexpressing either NaV1.1-5A or NaV1.1-5N with the β1 and β2 accessory subunits. We examined tonic inhibition and use-dependent inhibition of NaV1.1 splice isoforms by phenytoin, carbamazepine, and lamotrigine. We also examined the effects of phenytoin and lamotrigine on channel biophysical properties and determined concentration–response relationships for both splice variants.
Key Findings: We observed no significant differences in voltage dependence of activation, steady-state inactivation, and recovery from inactivation between splice variants. However, NaV1.1-5N channels exhibited enhanced tonic block by phenytoin and lamotrigine compared to NaV1.1-5A. In addition, NaV1.1-5N exhibited enhanced use-dependent block by phenytoin and lamotrigine across a range of stimulation frequencies and concentrations. Phenytoin and lamotrigine induced shifts in steady-state inactivation and recovery from fast inactivation for both splice isoforms. No splice isoform differences were observed for channel inhibition by carbamazepine.
Significance: These results suggest NaV1.1 channels containing exon 5N are more sensitive to the commonly used antiepileptic drugs phenytoin and lamotrigine.
Voltage-gated sodium (NaV) channels are critically important for initiating and propagating action potentials in most excitable cells (Hille, 1993). Sodium channels exist as heteromultimeric complexes comprising the large (approximately 260 kDa) pore-forming α-subunit and smaller accessory β-subunits (Yu & Catterall, 2003; George, 2005). Nine genes (SCN1A, SCN2A, and so on) encode distinct sodium channel α-subunits (NaV1.1, NaV1.2, and so on) and four genes encode the accessory β-subunits (Isom, 2001; George, 2005). Mutations in NaV channel genes are associated with various genetic epilepsies, and >500 mutations have been identified in SCN1A (encoding NaV1.1; Claes et al., 2009; Lossin, 2009). The central role of NaV channels in neuronal excitability is further highlighted by the numerous clinically utilized antiepileptic drugs (AEDs) including phenytoin, carbamazepine, and lamotrigine, which exhibit anticonvulsant effects by inhibiting NaV channel activity (Ragsdale et al., 1991; Kuo & Lu, 1997; Kuo, 1998).
Sodium channel transcripts undergo alternative mRNA splicing. One splice variation in SCN1A leads to the incorporation of an alternate exon 5 encoding a portion of the domain 1 S3/S4 helices (Copley, 2004). A corresponding splicing event is conserved among other neuronal (NaV1.2, NaV1.3, NaV1.6 and NaV1.7) as well as the cardiac (NaV1.5) NaV channel isoforms (Copley, 2004; Diss et al., 2004; Onkal et al., 2008). The resultant splice isoforms differ by 1–6 amino acids within the voltage-sensing portion of domain 1, and functional studies have revealed splice isoform specific channel gating (Diss et al., 2004). Interestingly, the biophysical effects of disease-causing NaV channel mutations can be modified by the splice isoform background, as demonstrated for NaV1.7 (Jarecki et al., 2009).
Previous pharmacogenetic studies have demonstrated that a common SCN1A genetic variant (rs3812718) located within an intron splice donor site alters the proportion of human brain SCN1A transcripts incorporating the canonical (exon 5A) or alternative (exon 5N) exon 5 (Tate et al., 2005). The 5N/5A terminology arose due to initial reports suggesting that exon 5N was expressed primarily during the neonatal period, whereas the exon 5A was the adult exon (Sarao et al., 1991; Gustafson et al., 1993). However, strict developmental regulation of this splicing event may be relaxed, as evidenced by the detection of NaV1.1 transcripts (up to 50% of transcripts) incorporating exon 5N in adult human brain (Tate et al., 2005; Heinzen et al., 2007). In population studies, the two alleles generated by rs3812718 (A or G) occur with approximately equal frequency, producing genotype frequencies of 25% AA, 50% AG, and 25% GG. Importantly, the A allele disrupts the consensus splice donor sequence immediately following exon 5N, thereby completely blocking Nova2-mediated incorporation of exon 5N into mature SCN1A mRNA transcripts. Heinzen et al. (2007) reported that inclusion of exon 5N ranged between 1 and 50% for the AA and GG haplotypes, respectively.
Genetic variation in SCN1A or other NaV channel genes might also influence susceptibility to or treatment responses in non-Mendelian forms of epilepsy. The aforementioned pharmacogenetic study demonstrated that epileptic patients expressing the GG genotype (expressing NaV1.1-5N) required lower doses of phenytoin and carbamazepine for treatment (Tate et al., 2006, 2005), but subsequent analyses indicated that this common variant was not associated with carbamazepine dosage levels in other populations, suggesting that other genetic factors may contribute to carbamazepine sensitivity, and illustrating the variability associated with pharmacogenetic studies (Abe et al., 2008; Zimprich et al., 2008). Nonetheless, these findings prompted the hypothesis that NaV1.1 channels translated from transcripts containing exon 5N may respond differently to AEDs.
We investigated the biophysical and pharmacologic properties of NaV1.1 splice variants containing the different exon 5 sequences. Although these NaV1.1 splice variants differ by three amino acids near the domain 1 voltage sensor, no significant biophysical differences were apparent. In addition, the two splice variants exhibited similar levels of inhibition by carbamazepine. However, NaV1.1-5N channels were more sensitive to phenytoin compared to NaV1.1-5A, which correlates with findings from previous pharmacogenetic studies. Furthermore, NaV1.1-5N channels were also more sensitive to inhibition by lamotrigine, a commonly used AED that has not yet been investigated for pharmacogenetic association with SCN1A.
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- Materials and Methods
- Supporting Information
Alternative mRNA splicing occurs in up to 60% of human genes and is an important mechanism providing protein diversity (Copley, 2004; Matlin et al., 2005). Alternative splicing of NaV genes has been shown to generate channels with unique functional and pharmacologic properties (Tan et al., 2002; Copley, 2004; Diss et al., 2004; Onkal et al., 2008). Our study examined the biophysical and pharmacologic properties of NaV1.1 splice variants incorporating either exon 5A or 5N. This work was motivated by pharmacogenetic studies that identified a common genetic variant in an SCN1A intron splice donor site that influences the proportion of transcripts incorporating either exon 5A or exon 5N (Tate et al., 2005, 2006). Control and epileptic patients carrying the G allele had a larger proportion of NaV1.1-5N transcript in brain (approximately 50% of channels) and required lower therapeutic doses of phenytoin, respectively. We found that NaV1.1-5N channels were more sensitive to both phenytoin and lamotrigine.
The appropriate therapeutic dosage for phenytoin, carbamazepine, and lamotrigine must be empirically determined to account for the unique pharmacodynamic and pharmacokinetic profile of each patient. Our study provides the first direct evidence that the variation in therapeutic phenytoin dosage observed by Tate et al. (2005) arises in part due to differences in how these drugs interact with an alternatively spliced brain sodium channel. Previous work has demonstrated that phenytoin, carbamazepine, and lamotrigine preferentially interact with the inactivated states (Ragsdale et al., 1991; Kuo & Bean, 1994; Kuo et al., 1997; Kuo, 1998; Yang & Kuo, 2002), and this interaction can be described by the modulated receptor model (Hille, 1977). Therefore, altered binding site presentation and differences in drug block could result from isoform specific activation and/or inactivation gating. However, although NaV1.1-5N trended toward activation at more hyperpolarized voltages, no statistically significant differences in biophysical properties between the splice variants were observed (Fig. 1). Only frequency dependent channel rundown differed between splice variants. Despite the absence of major biophysical differences between splice variants, even in the presence of AEDs (Figs 4 and 5), NaV1.1-5N channels exhibited greater tonic and use-dependent inhibition by phenytoin and lamotrigine than NaV1.1-5A (Figs. 2 and 6), suggesting that binding sites for these drugs may be altered, and that the pharmacologic differences may arise from slower inactivation processes. Because the binding sites for the studied drugs are predicted to lie near the DIV-S6 and exon 5 encodes D1-S3/S4, we predict that the divergent pharmacologic properties observed for NaV1.1-5A and NaV1.1-5N result from allosteric modification of the binding sites through subtle structural rearrangements.
Our data suggest that phenytoin, carbamazepine, and lamotrigine interact with NaV1.1 with low blocking efficacy (IC50 > 100 μm) that at first appears to conflict with the free serum concentration for these drugs at therapeutic levels (approximately 10 μm; Rambeck et al., 1987; Johannessen & Tomson, 2006; Dasgupta, 2007). However, the degree of channel inhibition is strongly dependent on the holding potential, which in turn determines binding site presentation (Ragsdale et al., 1991; Kuo, 1998). Therefore, measurements of tonic block by these drugs reveals only binding to a specific set of states that are determined by the holding potential used during the experiment, and the available states change as the membrane potential of the cell changes. Therefore, relative tonic inhibition measurements made in vitro cannot be directly compared with values expected from therapeutic concentrations of these drugs. However, using experiments with a therapeutic concentration of these drugs using repetitive stimulation over a range of frequencies, we demonstrated conclusively that phenytoin and lamotrigine both evoke greater use-dependent block of NaV1.1-5N (Fig. 6).
Our data demonstrate that NaV1.1 splice variants have divergent pharmacologic properties, with NaV1.1-5N exhibiting increased sensitivity to the commonly prescribed AED phenytoin. An important additional finding was that lamotrigine, another widely prescribed drug for the treatment of epilepsy that was not considered in the previous pharmacogenetic studies, more effectively inhibits NaV1.1-5N than NaV1.1-5A. Additional studies are needed to determine whether genetic testing of patients for the SCN1A polymorphism enables more precise dosing of phenytoin, lamotrigine, and possibly other AEDs that target this channel.