Limits...
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.

Show MeSH
The 200 kDa band represents a dimer of VR1 subunits. Western blot of oocytes expressing wild-type VR1, Δ2–52, or wild-type VR1 + Δ2–52. Oocytes were prepared as described in Experimental Procedures. The band observed for VR1-express ing oocytes was at 200 kDa, the band observed for the Δ2–52-expressing oocytes was at 180 kDa, while oocytes injected with a 1:1 ratio of VR1 and Δ2–52 RNA yield three bands of 200 kDa, 180 kDa, and 190 kDa. This blot was probed with the FLAG antibody.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC102763&req=5

Figure 4: The 200 kDa band represents a dimer of VR1 subunits. Western blot of oocytes expressing wild-type VR1, Δ2–52, or wild-type VR1 + Δ2–52. Oocytes were prepared as described in Experimental Procedures. The band observed for VR1-express ing oocytes was at 200 kDa, the band observed for the Δ2–52-expressing oocytes was at 180 kDa, while oocytes injected with a 1:1 ratio of VR1 and Δ2–52 RNA yield three bands of 200 kDa, 180 kDa, and 190 kDa. This blot was probed with the FLAG antibody.

Mentions: At 200 kDa, the third band observed in Figure 2 was approximately twice the molecular weight of the monomer. We therefore suspected that it might represent a dimer of VR1 subunits. However, it could also arise from a monomer in combination with another cellular factor. To distinguish between these possibilities, we engineered a VR1 mutant in which amino acids 2–52 in the N-terminal were deleted (termed Δ2–52). This construct gave rise to a high molecular weight band of 180 kDa, 20 kDa smaller than that observed for wild-type VR1 (Figure 4). When coexpressed with VR1, heteromeric channels composed of both types of subunits are expected to form. If the higher molecular weight band does represent a dimer of subunits, the heteromeric VR1/Δ2–52 ought to be intermediate in size between the homomeric dimers. When equal amounts of VR1 and Δ2–52 RNA's were coinjected into oocytes, three bands were observed: 200, 190 and 180 kDa (Figure 4). The 200 kDa band represents the homomeric VR1 dimer whereas the 180 kDa band represent the homomeric Δ2–52 dimer. The middle band, at 190 kDa, is indeed intermediate in size. Our interpretation is that this 190 kDa band is composed of one wild-type VR1 subunit and one Δ2–52 subunit, indicating that the high molecular weight band represents subunit dimers. Furthermore, quantification of the intensity of the bands on the gel revealed a 1:2:1 ratio of the three bands. This ratio of the three ways in which the two types of subunit can assemble is what would be expected if assembly between the different types of subunits were random.


Subunit modification and association in VR1 ion channels.

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

The 200 kDa band represents a dimer of VR1 subunits. Western blot of oocytes expressing wild-type VR1, Δ2–52, or wild-type VR1 + Δ2–52. Oocytes were prepared as described in Experimental Procedures. The band observed for VR1-express ing oocytes was at 200 kDa, the band observed for the Δ2–52-expressing oocytes was at 180 kDa, while oocytes injected with a 1:1 ratio of VR1 and Δ2–52 RNA yield three bands of 200 kDa, 180 kDa, and 190 kDa. This blot was probed with the FLAG antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC102763&req=5

Figure 4: The 200 kDa band represents a dimer of VR1 subunits. Western blot of oocytes expressing wild-type VR1, Δ2–52, or wild-type VR1 + Δ2–52. Oocytes were prepared as described in Experimental Procedures. The band observed for VR1-express ing oocytes was at 200 kDa, the band observed for the Δ2–52-expressing oocytes was at 180 kDa, while oocytes injected with a 1:1 ratio of VR1 and Δ2–52 RNA yield three bands of 200 kDa, 180 kDa, and 190 kDa. This blot was probed with the FLAG antibody.
Mentions: At 200 kDa, the third band observed in Figure 2 was approximately twice the molecular weight of the monomer. We therefore suspected that it might represent a dimer of VR1 subunits. However, it could also arise from a monomer in combination with another cellular factor. To distinguish between these possibilities, we engineered a VR1 mutant in which amino acids 2–52 in the N-terminal were deleted (termed Δ2–52). This construct gave rise to a high molecular weight band of 180 kDa, 20 kDa smaller than that observed for wild-type VR1 (Figure 4). When coexpressed with VR1, heteromeric channels composed of both types of subunits are expected to form. If the higher molecular weight band does represent a dimer of subunits, the heteromeric VR1/Δ2–52 ought to be intermediate in size between the homomeric dimers. When equal amounts of VR1 and Δ2–52 RNA's were coinjected into oocytes, three bands were observed: 200, 190 and 180 kDa (Figure 4). The 200 kDa band represents the homomeric VR1 dimer whereas the 180 kDa band represent the homomeric Δ2–52 dimer. The middle band, at 190 kDa, is indeed intermediate in size. Our interpretation is that this 190 kDa band is composed of one wild-type VR1 subunit and one Δ2–52 subunit, indicating that the high molecular weight band represents subunit dimers. Furthermore, quantification of the intensity of the bands on the gel revealed a 1:2:1 ratio of the three bands. This ratio of the three ways in which the two types of subunit can assemble is what would be expected if assembly between the different types of subunits were random.

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