Insertion of ncAAs between transmembrane segments 1 and 2 of TRPV1. (A) Missense variants encoded in the TM domains of human TRPV1 (residues 427–680) as annotated in the gnomAD ver. 2.1.1. The blue line is the absolute variant count for the given residue filtered across three residues with a boxcar kernel. TM segments 1–6 are illustrated above the plot (yellow cylinders) and connecting loops are shown as grey regions within the plot. Extracellular (blue) and intracellular (red) loop regions above TM segments are potential sites for ncAA insertion. (B) A reconstructed structural model of one TRPV1 subunit from cryo-EM data (PDB accession no. 7L2H; green) reveals details of the extracellular loop between transmembrane segments 1 and 2 (yellow). (C) Ca2+ imaging experiments to determine the successful expression of full-length TRPV1 constructs containing tag codons in the S1–S2 extracellular loop. RNA-synthetase/tRNA plasmid and type of ncAA used are given at the foot of the plot. The median value of the set is indicated with a purple triangle. Significant differences between Ca2+ imaging responses from non-responding cells (black) and cells with responses to 500 nM capsaicin (colored) within the same imaging experiment were determined by Mann–Whitney U-test. (D) Alignment of rat TRPV1 (rTRPV1) sequence (454–472; S1–S2 extracellular loop) against homologous sequences from several mammals (m: mouse [UniProt Q704Y3]; cl: dog [UniProt Q697L1]; rab: rabbit [UniProt Q6RX08]; cp: guinea pig [UniProt Q6R5A3]; ic: 13-lined ground squirrel [UniProt I3LZN5]; b: bovine [UniProt E1BGM0]; hor: equine [UniProt A0A3Q2H4H5]; gor: gorilla [UniProt G3R334]; h: human [UniProt Q8NER1]). Variant positions identified in the gnomAD 2.1.1. Database are listed below the conservation alignments with P456, V457, G459, P461, K464, T468, and G470 standing out as highly conserved residues (conservation >90%). Our first round of calcium imaging with P456tag, V457tag, K464tag, T468tag, and V469tag returned functional, ncAA-dependent responses with K464tag and T468tag.
Figure 2.

Insertion of ncAAs between transmembrane segments 1 and 2 of TRPV1. (A) Missense variants encoded in the TM domains of human TRPV1 (residues 427–680) as annotated in the gnomAD ver. 2.1.1. The blue line is the absolute variant count for the given residue filtered across three residues with a boxcar kernel. TM segments 1–6 are illustrated above the plot (yellow cylinders) and connecting loops are shown as grey regions within the plot. Extracellular (blue) and intracellular (red) loop regions above TM segments are potential sites for ncAA insertion. (B) A reconstructed structural model of one TRPV1 subunit from cryo-EM data (PDB accession no. 7L2H; green) reveals details of the extracellular loop between transmembrane segments 1 and 2 (yellow). (C) Ca2+ imaging experiments to determine the successful expression of full-length TRPV1 constructs containing tag codons in the S1–S2 extracellular loop. RNA-synthetase/tRNA plasmid and type of ncAA used are given at the foot of the plot. The median value of the set is indicated with a purple triangle. Significant differences between Ca2+ imaging responses from non-responding cells (black) and cells with responses to 500 nM capsaicin (colored) within the same imaging experiment were determined by Mann–Whitney U-test. (D) Alignment of rat TRPV1 (rTRPV1) sequence (454–472; S1–S2 extracellular loop) against homologous sequences from several mammals (m: mouse [UniProt Q704Y3]; cl: dog [UniProt Q697L1]; rab: rabbit [UniProt Q6RX08]; cp: guinea pig [UniProt Q6R5A3]; ic: 13-lined ground squirrel [UniProt I3LZN5]; b: bovine [UniProt E1BGM0]; hor: equine [UniProt A0A3Q2H4H5]; gor: gorilla [UniProt G3R334]; h: human [UniProt Q8NER1]). Variant positions identified in the gnomAD 2.1.1. Database are listed below the conservation alignments with P456, V457, G459, P461, K464, T468, and G470 standing out as highly conserved residues (conservation >90%). Our first round of calcium imaging with P456tag, V457tag, K464tag, T468tag, and V469tag returned functional, ncAA-dependent responses with K464tag and T468tag.

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