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Exploring functional roles of TRPV1 intracellular domains with unstructured peptide-insertion screening

View Article: PubMed Central - PubMed

ABSTRACT

TRPV1 is a polymodal nociceptor for diverse physical and chemical stimuli that interact with different parts of the channel protein. Recent cryo-EM studies revealed detailed channel structures, opening the door for mapping structural elements mediating activation by each stimulus. Towards this goal, here we have combined unstructured peptide-insertion screening (UPS) with electrophysiological and fluorescence recordings to explore structural and functional roles of the intracellular regions of TRPV1 in mediating various activation stimuli. We found that most of the tightly packed protein regions did not tolerate structural perturbation by UPS when tested, indicating that structural integrity of the intracellular region is critical. In agreement with previous reports, Ca2+-dependent desensitization is strongly dependent on both intracellular N- and C-terminal domains; insertions of an unstructured peptide between these domains and the transmembrane core domain nearly eliminated Ca2+-dependent desensitization. In contrast, channel activations by capsaicin, low pH, divalent cations, and even heat are mostly intact in mutant channels containing the same insertions. These observations suggest that the transmembrane core domain of TRPV1, but not the intracellular domains, is responsible for sensing these stimuli.

No MeSH data available.


Unstructured peptide-insertion screening.(A) TRPV1 residues bracketing the insertion sites are highlighted in green (functional) and red (non-functional). The linker between the ankyrin-like repeat domain and S1 is shown in blue. (B) A schematic diagram showing the membrane topology of a TRPV1 subunit. Insertion sites that yield functional and non-functional mutants are marked by green and red triangles, respectively. (C–F) Zoom-in view of the channel structures containing peptide insertions. Insertions are named in the format of Xnnn_maa, in which X represents the single-letter amino acid preceding the insertion, nnn represents the residue number, m represents the number of amino acids in the inserted peptide.
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f1: Unstructured peptide-insertion screening.(A) TRPV1 residues bracketing the insertion sites are highlighted in green (functional) and red (non-functional). The linker between the ankyrin-like repeat domain and S1 is shown in blue. (B) A schematic diagram showing the membrane topology of a TRPV1 subunit. Insertion sites that yield functional and non-functional mutants are marked by green and red triangles, respectively. (C–F) Zoom-in view of the channel structures containing peptide insertions. Insertions are named in the format of Xnnn_maa, in which X represents the single-letter amino acid preceding the insertion, nnn represents the residue number, m represents the number of amino acids in the inserted peptide.

Mentions: Guided by the TRPV1 cryo-EM structures2223, we have identified a series of intracellular sites for UPS tests (Fig. 1A,B). Our strategy was to focus on key structural domains as well as junctions between domains. After expressing the sixteen insertion mutants in HEK293 cells, we first conducted confocal imaging to examine the distribution of eYFP-tagged channel proteins. While it is well-known that transient over-expression often leads to a large amount of protein trapped inside the cell, we observed in most cases clear plasma membrane distribution of fluorescence (Fig. 2). To confirm the location of fluorescence signal, a plasma membrane marker, di-8-ANEPPS (a gift from the Santana lab), was used to label the same cells (Fig. 2A). Overlap of channel-eYFP and di-8-ANEPPS signals was seen only at the plasma membrane, suggesting proper trafficking of the mutant channels. Two exceptions, H365_3aa and Q561_4aa, were identified, for which the fluorescence signal was evenly distributed across the cell (Fig. 2B).


Exploring functional roles of TRPV1 intracellular domains with unstructured peptide-insertion screening
Unstructured peptide-insertion screening.(A) TRPV1 residues bracketing the insertion sites are highlighted in green (functional) and red (non-functional). The linker between the ankyrin-like repeat domain and S1 is shown in blue. (B) A schematic diagram showing the membrane topology of a TRPV1 subunit. Insertion sites that yield functional and non-functional mutants are marked by green and red triangles, respectively. (C–F) Zoom-in view of the channel structures containing peptide insertions. Insertions are named in the format of Xnnn_maa, in which X represents the single-letter amino acid preceding the insertion, nnn represents the residue number, m represents the number of amino acids in the inserted peptide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035920&req=5

f1: Unstructured peptide-insertion screening.(A) TRPV1 residues bracketing the insertion sites are highlighted in green (functional) and red (non-functional). The linker between the ankyrin-like repeat domain and S1 is shown in blue. (B) A schematic diagram showing the membrane topology of a TRPV1 subunit. Insertion sites that yield functional and non-functional mutants are marked by green and red triangles, respectively. (C–F) Zoom-in view of the channel structures containing peptide insertions. Insertions are named in the format of Xnnn_maa, in which X represents the single-letter amino acid preceding the insertion, nnn represents the residue number, m represents the number of amino acids in the inserted peptide.
Mentions: Guided by the TRPV1 cryo-EM structures2223, we have identified a series of intracellular sites for UPS tests (Fig. 1A,B). Our strategy was to focus on key structural domains as well as junctions between domains. After expressing the sixteen insertion mutants in HEK293 cells, we first conducted confocal imaging to examine the distribution of eYFP-tagged channel proteins. While it is well-known that transient over-expression often leads to a large amount of protein trapped inside the cell, we observed in most cases clear plasma membrane distribution of fluorescence (Fig. 2). To confirm the location of fluorescence signal, a plasma membrane marker, di-8-ANEPPS (a gift from the Santana lab), was used to label the same cells (Fig. 2A). Overlap of channel-eYFP and di-8-ANEPPS signals was seen only at the plasma membrane, suggesting proper trafficking of the mutant channels. Two exceptions, H365_3aa and Q561_4aa, were identified, for which the fluorescence signal was evenly distributed across the cell (Fig. 2B).

View Article: PubMed Central - PubMed

ABSTRACT

TRPV1 is a polymodal nociceptor for diverse physical and chemical stimuli that interact with different parts of the channel protein. Recent cryo-EM studies revealed detailed channel structures, opening the door for mapping structural elements mediating activation by each stimulus. Towards this goal, here we have combined unstructured peptide-insertion screening (UPS) with electrophysiological and fluorescence recordings to explore structural and functional roles of the intracellular regions of TRPV1 in mediating various activation stimuli. We found that most of the tightly packed protein regions did not tolerate structural perturbation by UPS when tested, indicating that structural integrity of the intracellular region is critical. In agreement with previous reports, Ca2+-dependent desensitization is strongly dependent on both intracellular N- and C-terminal domains; insertions of an unstructured peptide between these domains and the transmembrane core domain nearly eliminated Ca2+-dependent desensitization. In contrast, channel activations by capsaicin, low pH, divalent cations, and even heat are mostly intact in mutant channels containing the same insertions. These observations suggest that the transmembrane core domain of TRPV1, but not the intracellular domains, is responsible for sensing these stimuli.

No MeSH data available.