TMEM44 is not expressed in sweet, bitter, umami, and sour cells.

<p>Expression of TMEM44 in LE, FG TB, and CV TB (A) as well as top and bottom portions of CV TB (B) by microarray analyses. * p<0.05 compared to LE. Expression units are GC-RMA normalized average intensities of microarray signals. Double label <i>in situ</i> hybridization (ISH) for TMEM44 and TRPM5 (C–E and I–K). TMEM44 (C, I) and TRPM5 (D, J) are expressed in different cells in the merged images (E, K). Identical results were obtained in CV (C–E) or FG taste buds (I–K). Double label ISH for TMEM44 and PKD1L3 (F–H and L–N). TMEM44 (F, L) and PKD1L3 (G, M) are expressed in different cells in the merged images (H, N). Identical results were obtained in primate CV (F–H) or FG taste buds (L–N). Scale bar is 30µm in C and represents scale for C–H. Scale bar is 20µm in K and represents scale for I–N. Images are oblique sections with varying orientations from primate CV papilla. O, Pie chart illustrating fraction of cells expressing TMEM44, TRPM5, or both TMEM44 and TRPM5. P, Pie chart illustrating fraction of cells expressing TMEM44, PKD1L3, or both TMEM44 and PKD1L3.</p

MCTP1 is expressed in TRPM5 cells.

<p>Expression of MCTP1 in LE, FG TB, and CV TB (A) as well as top and bottom portions of CV TB (B) by microarray analyses. * p<0.005 compared to LE (A) or CV TB bottom (B). Expression units are GC-RMA normalized average intensities of microarray signals. Double label <i>in situ</i> hybridization (ISH) for MCTP1 and TRPM5 (C–H). MCTP1 (C, F) and TRPM5 (D, G) are expressed in similar cells in the merged images (E, H). Double label ISH for MCTP1 and PKD1L3 (I–N). MCTP1 (I, L) and PKD1L3 (J, M) are expressed in different cells in the merged images (K, N). Single taste buds are illustrated in F–H and L–N. Scale bar is 30µm in E and represents scale for C–E and I–K. Scale bar is 25µm in H and represents scale for F–H and L–N. Images are from primate CV papilla. O, Pie chart illustrating fraction of cells expressing MCTP1, TRPM5, or both MCTP1 and TRPM5. Cells with only TRPM5 signals may contain MCTP1 transcripts below the detection limit of ISH. P, Pie chart illustrating fraction of cells expressing MCTP1, PKD1L3, or both MCTP1 and PKD1L3.</p

MCTP1 is expressed in TRPM5 cells in mouse.

<p>Double label <i>in situ</i> hybridization (ISH) for MCTP1 and TRPM5 (A–F). MCTP1 (A, D) and TRPM5 (B, E) are expressed in similar cells in the merged images (C, F). Double label ISH for MCTP1 and PKD2L1 (G–L). MCTP1 (G, J) and PKD2L1 (H, K) are expressed in different cells in the merged images (I, L). Images in D–F and J–L depict single taste buds at higher magnification. Scale bar is 40µm in C and represents scale for A–C and G–I. Scale bar is 25µm in F and represents scale for D–F and J–L. Images are from mouse CV papilla. M, Pie chart illustrating fraction of cells expressing MCTP1, TRPM5, or both MCTP1 and TRPM5. N, Pie chart illustrating fraction of cells expressing MCTP1, PKD2L1, or both MCTP1 and PKD2L1.</p

Single Residue Substitutions That Confer Voltage-Gated Sodium Ion Channel Subtype Selectivity in the Na_V1.7 Inhibitory Peptide GpTx‑1

There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit Na_V1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with Na_V1.7 and related Na_V subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on Na_V1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a Na_V1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work

ANO7 is expressed in TRPM5 cells in mouse.

<p>Double label <i>in situ</i> hybridization (ISH) for ANO7 and TRPM5 (A–F). ANO7 (A, D) and TRPM5 (B, E) are expressed in similar cells in the merged images (C, F). Double label ISH for ANO7 and PKD2L1 (G–L). ANO7 (G, J) and PKD2L1 (H, K) are expressed in different cells in the merged images (I, L). Images in D–F and J–L depict single taste buds at higher magnification. Scale bar is 40µm in C and represents scale for A–C and G–I. Scale bar is 10µm in F and represents scale for D–F and J–L. Images are from mouse CV papilla. M, Pie chart illustrating fraction of cells expressing ANO7, TRPM5, or both ANO7 and TRPM5. N, Pie chart illustrating fraction of cells expressing ANO7, PKD2L1, or both ANO7 and PKD2L1.</p

Genes encoding transmembrane proteins are expressed in human CV taste buds.

<p>Section of human CV papilla before (A) and after (B) laser capture microdissection of taste buds. Collected taste bud regions (C), were isolated from CV papilla and used for molecular analysis of gene expression. A laser beam was used to cut the perimeter of taste buds and physically separate them from surrounding lingual epithelium. Taste buds were next lifted away from the tissue section with an adhesive cap. Panel C is an image of six isolated taste bud regions, devoid of surrounding lingual epithelium and connective tissue, on the adhesive cap. Scale bar is 40µm. Semi-quantitative PCR (D) for known taste genes (TRPM5 and PKD2L1), genes predicted or known to encode transmembrane proteins, and the housekeeping gene GAPDH in isolated CV taste buds (black bars) or non-gustatory lingual epithelium (white bars) collected by laser capture microdissection. Relative expression is shown on a logarithmic scale.</p

ANO7 is expressed in TRPM5 cells.

<p>Expression of ANO7 in LE, FG TB, and CV TB (A) as well as top and bottom portions of CV TB (B) by microarray analyses. * p<0.005 compared to LE (A). Expression units are GC-RMA normalized average intensities of microarray signals. Double label <i>in situ</i> hybridization (ISH) for ANO7 and TRPM5 (C–E). ANO7 (C) and TRPM5 (D) are expressed in similar cells in the merged image (E). Double label ISH for ANO7 and PKD1L3 (F–H). ANO7 (F) and PKD1L3 (G) are expressed in different cells in the merged image (H). Images are from primate CV taste buds. Scale bar is 15µm in E and represents scale for C–H. I, Pie chart illustrating fraction of cells expressing ANO7, TRPM5, or both ANO7 and TRPM5. J, Pie chart illustrating fraction of cells expressing ANO7, PKD1L3, or both ANO7 and PKD1L3.</p

TMEM44 cells localize to the bottom and sides of taste buds.

<p>Double label <i>in situ</i> hybridization (ISH) for TMEM44 and TRPM5/PKD1L3 in CV taste bud; longitudinal section (A–C). TMEM44 cells (A) are enriched towards the base and sides of taste buds whereas TRPM5/PKD1L3 cells (B) are enriched toward the center and top of taste buds. A merged image with nuclei stained blue (DAPI, C) highlights these signals in a longitudinal section. Double label ISH for TMEM44 and TRPM5 in FG taste bud; tangential section in middle of taste bud (D–F). TMEM44 cells (D) surround TRPM5 cells (E) in merged image (F). Some TMEM44 cells extend processes toward the taste pore (G–I). Double label IHC-ISH for Keratin-19 (G, IHC), TMEM44 (H, ISH) and merge (I) in CV taste bud (longitudinal section). Pink arrows track a TMEM44 cell process towards the taste pore. Double label ISH for TMEM44 (blue) with TRPM5 (red) (J), SHH (blue) with TRPM5 (red) (K), and SHH (blue) with TMEM44 (red) (L) in primate CV taste buds (longitudinal sections). Small white arrows denote cells that express both TMEM44 and SHH transcripts, whereas small red arrows denote cells that express only TMEM44 transcripts. Large arrows denote taste pore region. Note that TMEM44 stain in panel A is a colorimetric signal (DIG-labeled ISH probe) that is pseudocolored green, whereas the TMEM44 stain in panel H is a fluorescent signal (FITC-labeled ISH probe). Colorimetric signals highlight the nuclear envelope and cytoplasm whereas fluorescent signals highlight intranuclear regions. Scale bar is 20µm in C and represents scale for A–L.</p

Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the Na_V1.7 Sodium Channel

Na_V1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on Na_V1.7 (IC₅₀ = 10 nM) and promising selectivity against key Na_V subtypes (20× and 1000× over Na_V1.4 and Na_V1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp²⁹, Lys³¹, and Phe³⁴ near the C-terminus are critical for potent Na_V1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against Na_V1.4. A structure-guided campaign afforded additive improvements in potency and Na_V subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]­GpTx-1 with a Na_V1.7 IC₅₀ value of 1.6 nM and >1000× selectivity against Na_V1.4 and Na_V1.5

Baseline movement and tactile sensitivity of Nav1.7 KOs are not different from WT/HET.

<p>Overall locomotion was evaluated in Nav1.7 WT/HET and KO animals by scoring both total rearing behavior and basic movement using beam breaks in an automated open-field box. <b>A.</b> There was no statistically significant difference in rearing behavior between WT/HET (11187±492, n = 20) and KO (11036±781, n = 17) littermates (pairwise t-test, p = 0.2438). <b>B.</b> There was no statistically significant difference in basic movement between WT/HET (935±85, n = 20) and KO (835±75, n = 17) littermates (mean ± S.E.M.; pairwise t-test, p = 0.528). <b>C.</b> Tactile sensitivity, as assayed by measuring threshold of paw withdrawal to von Frey fibers of increasing force, was not significantly different between WT/HET (1.225 g ±0.05 g, n = 11) and KO littermates (1.18 g ±0.08 g, n = 7) (mean ± S.E.M.; Wilcoxon Two-Sample Exact Test, p = 0.6434). Dashed line represents the level at which the animal’s paw was physically lifted by the von Frey monofilament and is included to show that the measured responses are due to true behavioral tactile response.</p