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Subunit modification and association in VR1 ion channels.

Rosenbaum T, Awaya M, Gordon SE - BMC Neurosci (2002)

Bottom Line: This dimer persisted under strongly reducing conditions, was not affected by capsaicin or calcium, and was refractory to treatment with transglutaminase inhibitors.The persistence of this dimer even under harsh denaturing and reducing conditions indicates a strong interaction among pairs of subunits.This biochemical dimerization is particularly intriguing given that functional channels are almost certainly tetramers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology, Department of Physiology and Biophysics, University of Washington, Box 356485, Seattle, WA 98195-6485, USA. tronsenba@u.washington.edu

ABSTRACT

Background: The capsaicin (vanilloid) receptor, VR1, is an agonist-activated ion channel expressed by sensory neurons that serves as a detector of chemical and thermal noxious stimuli.

Results: In the present study we investigated the properties of VR1 ion channels expressed in Xenopus oocytes. A VR1 subunit with a FLAG epitope tag at the C-terminus was constructed. When examined for size on an SDS gel, VR1-expressing oocytes produced a doublet corresponding to the size of the monomer and a band at about twice the molecular weight of the monomer. A consensus site for N-linked glycosylation was identified in the primary sequence at position 604. In channels in which the putative glycosylation site was mutated from asparagine to serine (N604S), the larger of the two monomer bands could no longer be detected on the gel. Electrophysiological experiments showed these unglycosylated channels to be functional. The high molecular weight band observed on the gel could represent either a dimer or a monomer conjugated to an unknown factor. To distinguish between these possibilities, we coexpressed a truncated VR1 subunit with full-length VR1. A band of intermediate molecular weight (composed of one full-length and one truncated subunit) was observed. This dimer persisted under strongly reducing conditions, was not affected by capsaicin or calcium, and was refractory to treatment with transglutaminase inhibitors.

Conclusions: The persistence of this dimer even under harsh denaturing and reducing conditions indicates a strong interaction among pairs of subunits. This biochemical dimerization is particularly intriguing given that functional channels are almost certainly tetramers.

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VR1 is glycosylated at N604. (A) Cartoon (not to scale) of proposed subunit topology for a single VR1 subunit. Shown are the epitopes for the N-terminal and FLAG antibodies used in Western blot experiments, and the consensus sequence for glycosylation, located just distal to the fifth transmembrane domain at position 604. The red circle depicts the approximate localization of this consensus sequence. The line parting from the circle points to the sequence of this N-glycosylation site and the yellow box shows the asparagine which was substituted for a serine in order to produce the glycosylation mutant (N604S). (B) Western blot of oocytes expressing VR1 probed with the N-terminal antibody. Uninjected oocytes and oocytes expressing VR1 were prepared as described in Experimental Procedures. The monomer was observed as a doublet at 80 kDa and at 84 kDa in VR1-injected oocytes and a third band of 200 kDa was also observed in these oocytes. No bands were observed in the uninjected oocytes. (C) Western blot of oocytes expressing VR1 probed with the FLAG antibody. Bands are as in (B). (D) Western blot of oocytes expressing VR1 or N604S. Uninjected oocytes and oocytes expressing VR1 or N604S were prepared as described in Experimental Procedures. The monomer doublet in the VR1 is not present in the N604S mutant; only the 80 kDa band is observed. No bands were observed in the uninjected oocytes. This blot was probed with the FLAG antibody.
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Figure 2: VR1 is glycosylated at N604. (A) Cartoon (not to scale) of proposed subunit topology for a single VR1 subunit. Shown are the epitopes for the N-terminal and FLAG antibodies used in Western blot experiments, and the consensus sequence for glycosylation, located just distal to the fifth transmembrane domain at position 604. The red circle depicts the approximate localization of this consensus sequence. The line parting from the circle points to the sequence of this N-glycosylation site and the yellow box shows the asparagine which was substituted for a serine in order to produce the glycosylation mutant (N604S). (B) Western blot of oocytes expressing VR1 probed with the N-terminal antibody. Uninjected oocytes and oocytes expressing VR1 were prepared as described in Experimental Procedures. The monomer was observed as a doublet at 80 kDa and at 84 kDa in VR1-injected oocytes and a third band of 200 kDa was also observed in these oocytes. No bands were observed in the uninjected oocytes. (C) Western blot of oocytes expressing VR1 probed with the FLAG antibody. Bands are as in (B). (D) Western blot of oocytes expressing VR1 or N604S. Uninjected oocytes and oocytes expressing VR1 or N604S were prepared as described in Experimental Procedures. The monomer doublet in the VR1 is not present in the N604S mutant; only the 80 kDa band is observed. No bands were observed in the uninjected oocytes. This blot was probed with the FLAG antibody.

Mentions: To examine the size of VR1 with SDS/PAGE, we constructed a VR1 subunit with a FLAG epitope tag on its extreme C-terminal end (Figure 2A). By using both this C-terminal FLAG epitope and an N-terminal epitope for which a commercial antibody is available, we could determine whether the band(s) we observed corresponded to full-length VR1 subunits. When oocytes injected with VR1 RNA were examined with SDS/PAGE and Western blot, three bands were apparent (Figures 2B,2C,2D). Two of these bands were seen as a doublet at about 80 kDa and 84 kDa (best seen in Figure 2D). A third band of about 200 kDa was also observed. The size of these bands was the same for blots probed with the N-terminal antibody (Figure 2B) or the FLAG antibody (Figure 2C), indicating that they represent full-length VR1 subunits. For the remainder of this study we used the FLAG antibody due to the lower background it produced.


Subunit modification and association in VR1 ion channels.

Rosenbaum T, Awaya M, Gordon SE - BMC Neurosci (2002)

VR1 is glycosylated at N604. (A) Cartoon (not to scale) of proposed subunit topology for a single VR1 subunit. Shown are the epitopes for the N-terminal and FLAG antibodies used in Western blot experiments, and the consensus sequence for glycosylation, located just distal to the fifth transmembrane domain at position 604. The red circle depicts the approximate localization of this consensus sequence. The line parting from the circle points to the sequence of this N-glycosylation site and the yellow box shows the asparagine which was substituted for a serine in order to produce the glycosylation mutant (N604S). (B) Western blot of oocytes expressing VR1 probed with the N-terminal antibody. Uninjected oocytes and oocytes expressing VR1 were prepared as described in Experimental Procedures. The monomer was observed as a doublet at 80 kDa and at 84 kDa in VR1-injected oocytes and a third band of 200 kDa was also observed in these oocytes. No bands were observed in the uninjected oocytes. (C) Western blot of oocytes expressing VR1 probed with the FLAG antibody. Bands are as in (B). (D) Western blot of oocytes expressing VR1 or N604S. Uninjected oocytes and oocytes expressing VR1 or N604S were prepared as described in Experimental Procedures. The monomer doublet in the VR1 is not present in the N604S mutant; only the 80 kDa band is observed. No bands were observed in the uninjected oocytes. This blot was probed with the FLAG antibody.
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Related In: Results  -  Collection

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Figure 2: VR1 is glycosylated at N604. (A) Cartoon (not to scale) of proposed subunit topology for a single VR1 subunit. Shown are the epitopes for the N-terminal and FLAG antibodies used in Western blot experiments, and the consensus sequence for glycosylation, located just distal to the fifth transmembrane domain at position 604. The red circle depicts the approximate localization of this consensus sequence. The line parting from the circle points to the sequence of this N-glycosylation site and the yellow box shows the asparagine which was substituted for a serine in order to produce the glycosylation mutant (N604S). (B) Western blot of oocytes expressing VR1 probed with the N-terminal antibody. Uninjected oocytes and oocytes expressing VR1 were prepared as described in Experimental Procedures. The monomer was observed as a doublet at 80 kDa and at 84 kDa in VR1-injected oocytes and a third band of 200 kDa was also observed in these oocytes. No bands were observed in the uninjected oocytes. (C) Western blot of oocytes expressing VR1 probed with the FLAG antibody. Bands are as in (B). (D) Western blot of oocytes expressing VR1 or N604S. Uninjected oocytes and oocytes expressing VR1 or N604S were prepared as described in Experimental Procedures. The monomer doublet in the VR1 is not present in the N604S mutant; only the 80 kDa band is observed. No bands were observed in the uninjected oocytes. This blot was probed with the FLAG antibody.
Mentions: To examine the size of VR1 with SDS/PAGE, we constructed a VR1 subunit with a FLAG epitope tag on its extreme C-terminal end (Figure 2A). By using both this C-terminal FLAG epitope and an N-terminal epitope for which a commercial antibody is available, we could determine whether the band(s) we observed corresponded to full-length VR1 subunits. When oocytes injected with VR1 RNA were examined with SDS/PAGE and Western blot, three bands were apparent (Figures 2B,2C,2D). Two of these bands were seen as a doublet at about 80 kDa and 84 kDa (best seen in Figure 2D). A third band of about 200 kDa was also observed. The size of these bands was the same for blots probed with the N-terminal antibody (Figure 2B) or the FLAG antibody (Figure 2C), indicating that they represent full-length VR1 subunits. For the remainder of this study we used the FLAG antibody due to the lower background it produced.

Bottom Line: This dimer persisted under strongly reducing conditions, was not affected by capsaicin or calcium, and was refractory to treatment with transglutaminase inhibitors.The persistence of this dimer even under harsh denaturing and reducing conditions indicates a strong interaction among pairs of subunits.This biochemical dimerization is particularly intriguing given that functional channels are almost certainly tetramers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology, Department of Physiology and Biophysics, University of Washington, Box 356485, Seattle, WA 98195-6485, USA. tronsenba@u.washington.edu

ABSTRACT

Background: The capsaicin (vanilloid) receptor, VR1, is an agonist-activated ion channel expressed by sensory neurons that serves as a detector of chemical and thermal noxious stimuli.

Results: In the present study we investigated the properties of VR1 ion channels expressed in Xenopus oocytes. A VR1 subunit with a FLAG epitope tag at the C-terminus was constructed. When examined for size on an SDS gel, VR1-expressing oocytes produced a doublet corresponding to the size of the monomer and a band at about twice the molecular weight of the monomer. A consensus site for N-linked glycosylation was identified in the primary sequence at position 604. In channels in which the putative glycosylation site was mutated from asparagine to serine (N604S), the larger of the two monomer bands could no longer be detected on the gel. Electrophysiological experiments showed these unglycosylated channels to be functional. The high molecular weight band observed on the gel could represent either a dimer or a monomer conjugated to an unknown factor. To distinguish between these possibilities, we coexpressed a truncated VR1 subunit with full-length VR1. A band of intermediate molecular weight (composed of one full-length and one truncated subunit) was observed. This dimer persisted under strongly reducing conditions, was not affected by capsaicin or calcium, and was refractory to treatment with transglutaminase inhibitors.

Conclusions: The persistence of this dimer even under harsh denaturing and reducing conditions indicates a strong interaction among pairs of subunits. This biochemical dimerization is particularly intriguing given that functional channels are almost certainly tetramers.

Show MeSH