Alternative splicing of Na(V)1.7 exon 5 increases the impact of the painful PEPD mutant channel I1461T

Abstract

Alternative splicing is known to alter pharmacological sensitivities, kinetics, channel distribution under pathological conditions, and developmental regulation of VGSCs. Mutations that alter channel properties in Na(V)1.7 have been genetically implicated in patients with bouts of extreme pain classified as inherited erythromelalgia (IEM) or paroxysmal extreme pain disorder (PEPD). Furthermore, patients with IEM or PEPD report differential age onsets. A recent study reported that alternative splicing of Na(V)1.7 exon 5 affects ramp current properties. Since IEM and PEPD mutations also alter Na(V)1.7 ramp current properties we speculated that alternative splicing might impact the functional consequences of IEM or PEPD mutations. We compared the effects alternative splicing has on the biophysical properties of Na(V)1.7 wild-type, IEM (I136V) and PEPD (I1461T) channels. Our major findings demonstrate that although the 5A splice variant of the IEM channel had no functional impact, the 5A splice variant of the PEPD channel significantly hyperpolarized the activation curve, slowed deactivation and closed-state inactivation, shifted the ramp current activation to more hyperpolarized potentials, and increased ramp current amplitude. We hypothesize a D1/S3-S4 charged residue difference between the 5N (Asn) and the 5A (Asp) variants within the coding region of exon 5 may contribute to shifts in channel activation and deactivation. Taken together, the additive effects observed on ramp currents from exon 5 splicing and the PEPD mutation (I1461T) are likely to impact the disease phenotype and may offer insight into how alternative splicing may affect specific intramolecular interactions critical for voltage-dependent gating.

Fig. 1. Location of painful NaV1.7 single-point mutations implicated in either PEPD or IEM

Depicted above is a linear representation of the VGSC α-subunit. The coding region of NaV1.7 exon 5, located in the D1/S3–S4 region, is noted with a star (*). Numerical position, amino acid mutation, and locations of the tested mutant channels in this study are designated with a filled (■, IEM) or unfilled (□, PEPD) square. Tested IEM mutant construct was D1/S1 I136V and the tested PEPD mutant construct was D3–D4 I1461T located within the putative inactivation gate of NaV1.7.

Fig. 2. Effects of exon 5 alternative splicing on wild-type (WT) NaV1.7 channels

(A) Representative sodium current traces for WT exon 5N versus 5A channels elicited using an incremental depolarizing step protocol. (B) Normalized fraction of channels available (G / V) during steady-state activating (m_∞ / V) and fast-inactivating (h_∞ / V) protocols. (C) Deactivation kinetics are compared using single-exponential fits to tail currents. (D) Time course for the development of closed-state inactivation (Csi) at −60 mV. The bar graph inset represents the averaged time constant (τ₋₆₀) values for the recordings ± S.E.M. (E) Channel ramp current generation was assayed using a slow depolarizing ramp (0.27 mV/ms) stimulus from −120 mV to +40 mV at a holding potential of −120 mV. Illustrated are the averaged (n = 5–6) ramp current traces for each splice variant. Averaged data for the WT 5N variant is colored in black and the 5A splice variant is colored in red.

Fig. 3. Alternative splicing modifies the voltage-dependent properties of the NaV1.7- PEPD mutation (I1461T) located in the putative inactivation gate

(A) Shown above are representative whole-cell sodium current traces for the exon 5N and 5A forms of the I1461T mutant channel. (B) Comparison of conductance voltage (G / V) properties and steady-state fast-inactivation in response to changes in membrane potential. (C) Changes in channel deactivation for the PEPD were determined at a range of membrane potentials by observing tail current properties. Inset figure shows representative traces elicited during a deactivating protocol. (D) Development plot for the I1461T splice variants during transition to a closed-inactivated state at −60 mV. (E) Averaged ramp current elicited in response to a slow depolarizing stimulus for I1461T mutant channels. Traces were compiled from 5–10 individual recordings and then plotted versus membrane potential.

Fig. 4. Alternative splicing of exon 5 in NaV1.7 does not affect gating properties of the D1/S1 IEM mutant channels

(A) Characteristic inward sodium traces for the I136V-IEM splice forms. (B) Steady-state activating and inactivating properties of I136V are displayed and plotted with respect to membrane potential to determine the fraction available at the range potentials depicted. (C) Voltage-dependent deactivation properties of I136V 5N and 5A splice variants are illustrated in the graph and representative traces are shown within the inset. (D) Development of closed-state inactivation at −60 mV is unchanged when comparing the I136V splice forms. No significant difference in the time constants was observed at −60 mV. (E) Changes in ramp current elicited during a slow-depolarizing stimulus for the I136V splice variants were not observed.

Fig. 5. Summary of changes observed in ramp current and potential mechanistic implications for the charge substitution difference between the NaV1.7 5N and the 5A splice variants in the D1/S3–S4 linker

(A) Summary bar graph of the percent of peak current determined for each channel. Denoted on the y-axis are the channel types (wild-type and numbered IEM or PEPD mutants). The x-axis represents the percentage of peak ramp current elicited for the splice variants in each group, where the 5N forms are highlighted in blackened or filled horizontal bars, and the 5A forms are highlighted with unfilled bars. Raw numerical data were determined by selecting the peak ramp current elicited from each trace, filtered at 250 Hz to reduce stochastic noise, and then divided by the peak transient sodium current elicited during an activating protocol, then multiplied by 100 to yield the percent of peak current for individual recordings. Raw data was then compiled and averaged to determine group mean and standard error. Statistical significance was set at p < 0.05 and noted with a star (*). (Ba) Sequence alignment of NaV1.7 D1/S3 through the region D1/S4. Exon 5 amino acid differences, located within the D1/S3–S4 linker, between the 5N versus the 5A splice forms are highlighted with grayed letters. Conserved native charged residues are illustrated with an encompassing rectangle in the sequence alignment. A substituted charged residue in the D1/S3–S4 of the NaV1.7 5A form is emphasized with a star and a dashed line leading to the amino acid structural differences at physiological pH. In the 5N form there is an uncharged asparagine (N) residue characterized by the grayed amide moiety, whereas the 5A residue is a charged aspartic acid (D), with the charged moiety also noted in gray. (Bb) Structural diagram of the NaV1.7 D1/S3–S4 transmembrane segments and the adjoining extracellular linker predicted for the 5A splice variant. Single-letter residues from the above sequence alignment are inserted along predicted exon 5 coding region chain from the upper portions of the segments and the linker region. Native charged residues conserved between the splicing forms are highlighted with the corresponding charge (+ or −) within the segments and linker regions. Residue substitutions between the 5N and the 5A variants are grayed and highlighted with an arrow. Note that the charge substitution within the linker between the splice variants occurs near the highly charged S4 segment of D1.

Fig. 6. State occupancy diagram illustrates increased open-state probability for 5A PEPD mutant channels

(A) Simplified three-state diagram of transitions between closed (C), open (O), and inactivated (I) where movement between the states is dependent on membrane potential and time. Illustrated in the model on the left of the reaction arrow are theoretical transitions between each state for a wild-type 5N splice form. To the right of the reaction arrow are the effects the PEPD mutation (I1461T) has on transition between the simplified states, primarily on the stability of the inactivated configuration highlighted with dotted lines. Also noted to the right of the reaction arrow are the effects the 5A alternative splicing substitutions have (highlighted by the grayed arrow) on the simplified transitions.