The C-Terminal of NaV1.7 Is Ubiquitinated by NEDD4L

Abstract

Na_V1.7, the neuronal voltage-gated sodium channel isoform, plays an important role in the human body's ability to feel pain. Mutations within Na_V1.7 have been linked to pain-related syndromes, such as insensitivity to pain. To date, the regulation and internalization mechanisms of the Na_V1.7 channel are not well known at a biochemical level. In this study, we perform biochemical and biophysical analyses that establish that the HECT-type E3 ligase, NEDD4L, ubiquitinates the cytoplasmic C-terminal (CT) region of Na_V1.7. Through in vitro ubiquitination and mass spectrometry experiments, we identify, for the first time, the lysine residues of Na_V1.7 within the CT region that get ubiquitinated. Furthermore, binding studies with an NEDD4L E3 ligase modulator (ubiquitin variant) highlight the dynamic partnership between NEDD4L and Na_V1.7. These investigations provide a framework for understanding how NEDD4L-dependent regulation of the channel can influence the Na_V1.7 function.

Figure 1

Na_V1.7 gets ubiquitinated in its C-terminal domain. (A) Scheme representation of the Na_V1.7 topology. Each domain and transmembrane helix are labeled accordingly. The cytoplasmic regions are shown as loops. The C-terminal of Na_V1.7 is shown as green cylinders representing alpha-helices as displayed in the crystallographic structures of other isoforms Na_V1.4 and Na_V1.5. The location of CaM is also highlighted in magenta. The canonical NEDD4L PY binding motif is indicated at residues 1955–1958. The two auxiliary beta subunits (β1 and β2) are shown in yellow. (B) Scheme of the GST-fusion with a precision protease cleavage sequence construct of Na_V1.7^CTspanning residues 1761–1988 (green). Coexpression was performed with full-length CaM (magenta). (C) In vitro ubiquitination time course of FL^NEDD4L in the absence and presence of the Na_V1.7^CT-CaM substrate. Equal amounts of sample were taken at the indicated time points and quenched with reducing loading buffer. The purified Na_V1.7^CT-CaM protein is in the last lane. Unmodified FL^NEDD4L and Na_V1.7^CT substrate protein bands were quantified by a densitometry analysis as a function of time. All assays were repeated at least twice with good reproducibility (n = 2). Gel is stained with colloidal Coomassie Blue. (D) Fluorescent Western blot analysis of the in vitro ubiquitination assays as carried out in C using anti-ubiquitin (red) and anti-NEDD4L (green) antibodies. (n = 2.) (E) Na_V1.7^CT sequence coverage after band excision from the in vitro assay. Identified peptides are highlighted in shaded green regions. Bold, red lysine residue with a yellow square above them indicate Ub-modification and the numbers in parentheses indicate total number of PSMs for each lysine identified from MS. Lysine residues detected by MS but not modified with ubiquitin are highlighted in bold, white. The IQ region is highlighted in bold and a purple box; the PY motif is bold and underlined. (F) Scheme of Na_V1.7^CT-CaM with the ubiquitinated lysine sites labeled with yellow squares based on location. (G) Representative MS/MS spectrum and sequence coverage of the peptide containing a Ub-modification on Na_V1.7^CT-CaM Lys1930. Lower case k indicates Ub-modification and lower case m indicates an oxidized methionine.

Figure 2

Lys63-linked ubiquitin chains are assembled on Na_V1.7^CT. (A) LC/MS/MS sequence coverage of ubiquitin after di-Ub-Na_V1.7^CT band excision from the in vitro assay. Identified peptides are highlighted in shaded, boxed gray regions. Bold, red letters with a yellow square above them are the identified Lysine residues with ubiquitin chain formation. (B) Ubiquitin lysine chain linkage of Na_V1.7^CT modified by FL^NEDD4L in vitro ubiquitination assay was analyzed by LC/MS/MS. The peptide spectrum matches of each ubiquitin Lysine residue seen with a Ub-modification is represented as a pie graph. (C) Representative MS/MS spectrum and sequence coverage of the peptide containing a Ub-modified Lys63. Lower case k indicates Ub-modification. (D) In vitro ubiquitination assays of Na_V1.7^CT-CaM in the presence of Ub^WT, Ub^K48R, Ub^K63R, or Ub^K48/63R. Samples were quenched with reducing 2× SDS-PAGE loading buffer at 0, 10, and 30 min, and the gel was stained with a colloidal Coomassie Blue stain. Unmodified Na_V1.7^CT bands were quantified by a densitometry analysis as a function of time. All assays were repeated at least twice with good reproducibility (n = 2).

Figure 3

Binding of Na_V1.7^CT-CaM to FL^NEDD4L is enhanced by a ubiquitin variant. (A) Scheme of the SPR binding kinetics between Na_V1.7^CT-CaM and FL^NEDD4L. The ligand Na_V1.7^CT-CaM was covalently attached to a CM5 chip. The analyte, FL^NEDD4L, recognizes the PY motif of the Na_V1.7^CT unstructured tail (black line). (B) FL^NEDD4L binding to Na_V1.7^CT-CaM was evaluated by parallel kinetics. Each binding sensorgram at 6.25, 100, and 400 nM of FL^NEDD4L were fit with a one-to-one binding model. N = 2. (C) Size exclusion chromatogram profile for Na_V1.7^CT-CaM + FL^NEDD4L–UbvNL.1 (solid gold line) showing a ∼ 0.5 mL shift to the left of the FL^NEDD4L–UbvNL.1 peak (dashed maroon line) indicating interaction and larger molecular weight. Molecular weight standard (Biorad) is shown in gray. Bold numbers indicated each protein within the molecular weight standard: 1. thyroglobulin, 2. γ-globulin, 3. ovalbumin, 4. myoglobin, 5. vitamin B12. (D) SDS-PAGE gel showing the elution fractions from (B). The FL^NEDD4L–UbvNL.1-Na_V1.7^CT-CaM complex elutes are ∼12 mL and excess Na_V1.7^CT-CaM elutes at 14 mL. (E) Scheme of the SPR binding kinetics as carried out in (A) with the analyte being FL^NEDD4L + 100 nM of ubiquitin variant, UbvNL.1. (F) FL^NEDD4L + UbvNL.1 binding to Na_V1.7^CT-CaM was evaluated by parallel kinetics SPR. Each binding sensorgram at 6.25, 100, and 400 nM of FL^NEDD4L + UbvNL.1 was fit with a one-to-one binding model. N = 2. (G) In vitro ubiquitination time course of Na_V1.7^CT-CaM in the absence and presence of UbvNL.1. Equal amounts of samples were taken at the indicated time points and quenched with reducing loading buffer. Unmodified Na_V1.7^CT substrate band was quantified by a densitometry analysis as a function of time. N = 2.

Figure 4

Mapping the locations of the ubiquitinated lysine residues of Na_V1.7^CT–CaM by sequence and structural analysis. (A) CryoEM structure of full-length human Na_V1.7 (PDB ID 7W9K) with a zoomed in view of the C-terminal domain (CTD). Na_V1.7 is shown in forest green, the β1 subunit in cyan, and the β2 subunit in orange. The position of the identified lysine residues of Na_V1.7^CT-CaM are shown in cyan sticks. (B) Structure of Na_V1.5^CT with apo-CaM (PDB ID 4OVN). Na_V1.5^CT is shown in green and apo-CaM in hot pink. The corresponding position of the identified lysine residues of Na_V1.7^CT-CaM are shown in cyan sticks. (C) Structure of Na_V1.5^CT with calcium-bound CaM (PDB ID 6MUD) structurally aligned to (A). Similarly to (A), Na_V1.5^CT is shown in split pea and calcium-CaM in red. The corresponding positions of the identified lysine residues of Na_V1.7^CT-CaM are shown in cyan sticks. (D) Structure of Na_V1.5^CT with apo-CaM and an FGF13 (PDB ID 4DCK) structurally aligned to (A). Similarly to (A), Na_V1.5^CT is shown in lime green, apo-CaM in dark red, and FGF13 in purple. The corresponding positions of the identified lysine residues of Na_V1.7^CT-CaM are shown in cyan sticks.