A Ubiquitous Splice Variant and a Common Polymorphism Affect Heterologous Expression of Recombinant Human SCN5A Heart Sodium Channels

Genotyping at Positions 558, 559, 618, and 1027

Allele frequencies for the SCN5A variants were established by direct genomic DNA sequencing of 400 reference alleles derived from the 100 Caucasian human variation panel (Coriell Cell Repositories) and the 100 African-American human variation panel (National Institute of General Medical Sciences). Protein-encoding sequences harboring these variants were amplified by polymerase chain reaction (PCR). PCR products were enzymatically treated to remove unincorporated dNTP and primers and subsequently sequenced. The resulting chromatograms were analyzed for the specific variant.

Identification, Characterization, and Quantification of Alternatively Spliced SCN5A at Position 1077

Direct DNA sequencing was performed on exon 17/18–targeted RT-PCR–generated products of messenger RNA extracted from myocardial tissue. The tissue came from autopsy specimens from sudden infant death syndrome (n=5), nonaccidental infant death (n=5), or accidental adult death victim (n=5). Additional tissue came from surgical myectomy specimens in adults with hypertrophic cardiomyopathy (n=5). The total test group contained 9 males and 11 females, and the infant group contained 5 white and 5 black subjects. Total RNA from heart tissue was isolated and first-strand cDNA synthesis was performed in triplicate on total RNA. PCR was performed, and products were purified and sequenced. The relative quantity of the two alternatively spliced transcripts were quantified. Triplicate cDNA samples from each case were subject to PCR amplification using the same PCR primers and conditions. Control templates representing homozygous Q1077 and Q1077del transcripts were synthesized and PAGE purified. The resulting radionucleotide incorporated PCR products representing (1) the transcript (135 base pairs) encoding the inclusion of glutamine at position 1077 (Q1077) or (2) the transcript (132 base pairs) that does not encode for glutamine at position 1077 (Q1077del) were separated by denaturing gel electrophoresis on a polyacrylamide/urea gel. Autoradiography was used for signal quantification of the two alternatively spliced transcripts.

Gene Expression, Mutagenesis, and Nomenclature

The SCN5A clone hH1 (GenBank M77235)² was kindly provided by Dr Al George in prcCMV (Invitrogen). The hH1c construct (GenBank AY148488) was made from the hH1b clone (GenBank AF482988)⁴ by mutating the arginine (R) at 558 to histidine (H), and the isoleucine (I) at positions 618 to leucine (L). The consensus reference sequence at this writing (GenBank NM_000335 August 2003) has 2015 amino acids. In this article, we used the full-length 2016 amino acid deduced sequence from the IHGSC (GenBank AC137587, deposited April 2003) as the reference sequence for SCN5A in order to preserve the standard numbering system. Single amino acid variations are referred to by the amino acid substitution without brackets. Channel variants of the full sequence are denoted by the amino acid substitutions from the reference sequence separated by semicolons and contained in brackets as recommended by den Dunnen and Antonarakis.⁵ The channels SCN5A, [H558R], [H558R;Q1077del], [H558R;Q1077del;M1766L], and [Q1077del;M1766L] were made by mutagenesis at appropriate positions using a PCR-based site-directed mutagenesis kit. Transformed bacteria were grown and the DNA isolated and purified with spin-column preps. All constructs were sequenced to verify incorporation of the intended amino acid change and to confirm that no unwanted changes were introduced. These constructs were placed in an expression vector and transfected into HEK-293 cells. Cells 24 hours after transfection were used to measure macroscopic I_Na. Experiments were done with transient transfection unless otherwise noted. In some instances, stable colonies were selected for by addition of the G418 antibiotic. After stabilization, RNA was isolated (RNAisol from LPS) and screened by RT-PCR analysis. Colonies that tested RT-PCR–positive were then analyzed by voltage clamp.

Voltage-Clamp Techniques

The whole-cell patch-clamp technique was utilized to measure macroscopic I_Na.⁶ The voltage-clamp protocols are described briefly with the data and have been published previously in detail.⁷ Peak I_Na and late I_Na were obtained after passive leak subtraction and saxitoxin subtraction as described previously.^6,7⇓ Parameter fits were obtained and one-way ANOVA with Bonferroni correction was performed to determine statistical significance among 3 or more groups of mean data. Statistical significance was determined by a value P<0.05.

Immunocytochemistry

The FLAG epitope was introduced between S1 and S2 in domain I for channels used in the immunocytochemistry experiments with confocal microscopy as previously described.⁴ The rabbit anti-Calnexin IgG was used as an endoplasmic reticulum (ER) marker to test for colocalization and was applied to transfected cells immediately after incubation with fluorescein-conjugated goat anti-mouse secondary antibody.

Sequence Comparisons for SCN5A Clones and Genotyping of Control Panels

The sequences of hH1,² hH1a,³ and hH1b⁴ were compared and the amino acid differences at 5 positions are shown in Table 1, along with the GenBank Accession number where available. In a previous study of the hH1b clone,⁴ hH1 and hH1a were resequenced and the differences between these two clones were found to be only the 3 positions included in Table 1 rather than at the 9 positions reported previously.³

We investigated allelic frequencies from genomic DNA at the 5 positions in question from a panel of 200 human controls (100 white subjects and 100 black subjects). The most common amino acids at these 5 positions are reported as hH1c in Table 1. For positions 559, 618, and 1027, 100% of the 400 reference alleles possessed T559, L618, and R1027, indicating that each of the existing clones contains a rare variant and that none represents the common sequence (Table 1). A search of the Celera human genome database showed that the deduced amino acid sequence agreed with hH1c. The deduced amino acid sequence in the NIH International Human Genome Sequencing Consortium (IHGSC) database also agreed with the hH1c sequence except for the additional presence of glutamine (Q) at position 1077. This glutamine is present in the hH1 clone but not hH1a or hH1b.

A Common Polymorphism, H558R

The residue at position 558 hosts the common polymorphism involving a substitution of histidine (H) with arginine (R) H558R⁸ with a reported frequency in the population of 19% to 24%.⁹ We confirm that H558R is a common polymorphism among both blacks (53% homozygous for H, 40% heterozygous, and 7% homozygous for R) and whites (65%/30%/5%). The apparent higher frequency of the R-encoding allele in blacks (27% versus 20%) is not statistically significant (paired student t test, Fischer’s exact). Other less common variations also exist in the human population. One of the more common variants is the S1103Y (also annotated Y1102) variant with a reported heterozygous frequency of 13.2% among blacks.¹⁰ We found approximately the same frequency among blacks in our panel. Otherwise, no variations exceeding 5% were found in our panels.

A Common Alternatively Spliced Variant, Q1077del

The hH1 clone contained a glutamine (Q) present at both amino acid positions 1076 (the final codon of exon 17) and also at 1077 (the first codon of exon 18).² The acceptor site sequence for exon 18 was annotated as ggggtcttttcagCAGGAATCC, where the lowercase letters represent the intronic sequences and the uppercase letters represent the 5′ exonic sequences of exon 18 (see Figure 1). The bold lowercase letters indicate the predicted “ag” acceptor site rule for splice site recognition. However, splice-site analytical tools indicate that the CAG following the ag shown above could also comprise the terminal intronic sequence and may be the preferred acceptor site for splicing, resulting in a deletion of glutamine at position 1077 (Q1077del). Does SCN5A most commonly have a glutamine (Q) at both amino acid positions 1076 and 1077, as in hH1, or only at 1076 as in hH1a and hH1b?

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Figure 1. Alternative splicing of SCN5A at exon 18. Top panel shows an example of sequencing data for RT-PCR products from mRNA isolated from human ventricle (see Materials and Methods). Note the single sequence present in the exon 17 coding region, but a dual sequence in the exon 18 coding region. Splice acceptor site in the genomic DNA has two “cag” repeats leading to the alternative splicing of a glutamine at position 1077. This results in two splice variants, one of 2015 amino acids (mRNA top) and one with 2016 amino acids (mRNA bottom). The two alternative numbering systems after position 1076 may cause confusion; we use and recommend numbering as in the bottom full-length sequence.

We sequenced RT-PCR products derived from mRNA isolated from human left ventricular myocardial specimens and generated with primers targeting exons 17 and 18. The specimens came from infants with sudden infant death syndrome (SIDS), infants with a structurally normal heart, adults with hypertrophic cardiomyopathy, and adults with a structurally normal heart. All subjects contained transcript with a “cag” in-frame insertion indicating the universal presence of alternative splicing involving this acceptor site (Figure 1). The relative abundance for each alternatively spliced transcript was quantified by autoradiography and phosphoimaging (Figure 2). Examples of RT-PCR products generated from myocardial RNA (Figure 2A) show the expected size products for control experiments with the 135-bp template containing the extra cag codon (Q1077) and for the 132-bp template lacking the extra cag codon (Q1077del). Summary data (Figure 2B) show that Q1077del was the preferred alternatively spliced variant being significantly more abundant in every group tested. The proportions were not affected by age or by the presence of SIDS or ventricular hypertrophy (Figure 2) or by sex or race (data not shown). Overall, the proportion of alternatively spliced variant containing Q1077 was 35±2.0% (range 31% to 38%, n=20) and the Q1077del transcript was 65±2.0% (range 62% to 69%, n=20). In addition, the proportion of Q1077del was the same whether RNA was obtained from right atrium, left atrium, right ventricle, or left ventricle (data not shown).

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Figure 2. Quantitative analysis of alternatively spliced variant SCN5A transcripts that either encode Q1077 or exclude Q1077 (Q1077del). A, Examples of RT-PCR products from 3 control experiments and 4 subjects. A sample without DNA (NEG) served as a negative control, and synthesized template of 135 bp containing the “cag” repeat (Q1077) and template of 132 bp without the “cag” repeat (Q1077del) served as positive controls. RT-PCR from samples of mRNA taken at autopsy from a subject with sudden infant death syndrome (SIDS), an infant without structural heart disease (Infant), an adult with no structural heart disease (Adult), and from a myomectomy specimen from a patient with hypertrophic cardiomyopathy (HCM) all show the presence of both transcripts. B, Summary data from 20 subjects show that the transcript coding for Q1077del was consistently more abundant relative to Q1077. Bars represent the mean and standard deviation of the relative abundance of each transcript from 5 subjects determined by autoradiography and phosphoimaging. See text and Materials and Methods for details.

Four Common SCN5A Variants in the Human Population

Based on the predicted amino acid frequency estimates from our genomic sequencing in controls and our measurement of the frequency of the splice variant Q1077del, we estimated the population frequency of the existing clones and other full-length sequences (Table 1). These estimates assume independence between the probability of the Q1077del splice variant (65%) and the genomic variant containing H558R (30%). Note that the three clones hH1, hH1a, and hH1b used in previous studies are estimated to have a 0% frequency in the population because of the presence of a mutation in each that was not seen in our panel. The most common variant, [Q1077del] at 45%, is identical to the hH1c sequence, the Celera sequence, and also to the NCBI Reference Sequence for SCN5A (Accession no. NM_000335). The full-length sequence SCN5A is actually less frequent than [Q1077del] at 25%. Only slightly less common are the variants with the H558R polymorphism designated as [H558R;Q1077del] at 20% and [H558R] at 10% (not in Table 1).

Functional Characterization of SCN5A Variants

Constructs for expressing SCN5A and the other common variants, [H558R], [Q1077del], and [H558R;Q1077del], were made and transfected individually into HEK-293 cells for voltage-clamp study. For comparison, we also expressed hH1 contemporaneously under the same conditions with the four variants because most previous studies in the literature have used this clone. Representative currents for the four variants and hH1 are shown in Figure 3A. For those variants that expressed robust current, no significant differences were identified in the parameters of activation, current decay, or the late currents (Table 2). The midpoint of inactivation for hH1, however, was significantly negative by 7 mV to the variants lacking Q1077 ([Q1077del] and [H558R;Q1077del], Table 2). Consistent with this negative shift in inactivation, the recovery for hH1 was also significantly slower (Table 2).

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Figure 3. Voltage-clamp data for SCN5A variants. A, Current traces for hH1 and four common SCN5A channel variant sequences were elicited by step depolarizations from a holding potential of −140 mV to various test potentials of 24-ms duration from −140 to +60 mV. No obvious differences in current time course were noted. B, Summary data for peak I_Na densities for the variants shown. Currents were elicited by a step depolarization to −20 mV from a holding potential of −140 mV and normalized to membrane capacitance. Data for the hH1 clone is included as a historical control. Results for [H558R] coexpressed with the β₁ subunit are shown in the rightmost bar. Bars depict the mean and standard error of the mean derived from n measurements with the n for each construct shown within the bar. I_Na density for [H558R] was significantly lower (P<0.05) than [Q1077del] and [H558R;Q1077del] but not the Q1077 containing variants hH1 or SCN5A.

The most dramatic finding was that [H558R], which contains Q1077, expressed very low current density (summary data in Figure 3B). Other constructs that contained Q1077 (hH1 and SCN5A) also tended to have reduced currents, but the difference did not reach statistical significance. RT-PCR of mRNA from cells that were stably transfected with [H558R] showed abundant SCN5A transcript, but just as with the transient transfections little or no current was observed. The β₁ subunit has been shown to increase wild-type current density when coexpressed with the hH1 α subunit¹¹ and also to increase expression of a trafficking defective SCN5A mutant.¹² When [H558R] was coexpressed with the β₁ subunit, increased currents were observed, although not at the levels of the other α subunits expressed alone (Figure 3). To determine if the small currents seen with [H558R] could be endogenous currents, we voltage-clamped nontransfected HEK-293 cells. In 8 cells, only 1 cell had current (100 pA) for an average density of <0.5 pA/pF compared with 7.5 pA/pF in [H558R] alone and 14 pA/pF in [H558R] cotransfected with the β₁ subunit. We were unable to significantly increase endogenous currents in untransfected cells by transfection with β₁ (1.0±0.5 pA/pF n=9), incubation with 100 μmol/L mexiletine for 24 hours (1.1±0.4 pA/pF n=10), or incubation at 27°C (0.9±0.4 pA/pF n=8). We conclude that [H558R] did indeed generate small currents. As SCN5A is a monomeric channel, a dominant-negative suppression mechanism would not be expected, but to test of the possibility that the two variants might interact both variants: [H558R] and [H558R;Q1077del] were coexpressed (plasmid DNA 0.75 μg each) and normal current densities were observed (data not shown). Hyperpolarization of the holding potential to −200 mV for 1 second did not elicit current, suggesting the decreased density is not caused by a large shift in the voltage dependence of fast inactivation.

Cell Trafficking of the [H558R] Variant

Some SCN5A variants with decreased current density have been shown by immunocytochemistry to have defective trafficking^4,13⇓ and do not make it to the cell surface. To determine if [H558R] trafficked to the cell surface, we labeled the channel variants with a FLAG epitope and localized the channels by immunofluorescence and confocal microscopy. In each panel of Figure 4, a light microscopy image is shown allowing for identification of the nucleus and the cell surface followed by the confocal immunofluorescence image(s). A nontransfected cell gave no fluorescent signal, as expected (Figure 4A). The [Q1077del] variant that gave robust current densities showed a rim of fluorescence at the cell surface, as expected for a normally trafficking channel (Figure 4B). The [H558R] variant expressed almost no current, but also showed fluorescence at the cell surface (Figure 4C), suggesting that the lack of current was not caused by a trafficking defect.

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Figure 4. [H558R] has normal trafficking to the cell surface. Standard light photography in the leftmost image of each panel is compared with confocal micrographs of HEK-293 cells with immunostaining of a FLAG epitope inserted into the Na⁺ channel. A, Nontransfected HEK-293 cell shows no immunostaining. B, [Q1077del] channel shows normal immunostaining pattern in the periphery and around the nucleus. C, [H558R] also shows normal staining pattern, indicating that it trafficks to the cell surface. D, With the mutation M1766L in the [Q1077del] background, the second image shows immunostaining is confined to a perinuclear location. Third image shows immunostaining with an endoplasmic reticulum marker (see Materials and Methods) and the fourth image (where the previous two images are superimposed) shows colocalization of the nontrafficking channel with the endoplasmic reticulum marker. E, When the M1766L mutation was engineered into the [H558R;Q1077] background, normal trafficking to the cell periphery was seen. All results represented here were seen in at least 7 additional experiments.

As a positive nontrafficking control for our experiment with the [H558R] variant, we overlaid the mutation M1766L into the variants and show data from two of these experiments (Figures 4D and 4E). We had previously shown M1766L to be trafficking defective in hH1a [T559A;Q1077del] but to be normally trafficking in hH1b [H558R;L618I;Q1077del].⁴ When the M1766L mutation was put into [Q1077del] to make [Q1077del;M1766L] (Figure 4D), the fluorescence was restricted to the area around the nucleus without any labeling in the periphery, consistent with a trafficking defect. This eliminates the possibility that the rare variant T559A in hH1a was responsible for the trafficking defect previously described.⁴ An image with a marker for the endoplasmic reticulum is shown in the third panel of Figure 4D, and the fourth image in Figure 4D superimposes the second and third image to show colocalization of the channel with the endoplasmic reticulum marker. When M1766L was placed in the [H558R;Q1077del] background, the channel localizes to the cell periphery (Figure 4E), consistent with previous data and also showing that the rare variant L618I in hH1b was not responsible for “rescuing” the trafficking defect.