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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 dimerization is not affected by reducing agents, capsaicin, Ca2+ or transglutaminase inhibitors. (A) Western Blot of the effect of reducing agents on the VR1 dimer. The addition of DTT (100 mM) and TCEP (20 mM) did not modify the ratio of monomer to dimer in VR1-expressing oocytes (p > 0.05, for 3 independent experiments). (B) Effect of Ca2+ on dimer formation in VR1. The addition of Ca2+ to the biochemical assays (in the presence or the absence of capsaicin, lanes 1 and 2) did not modify the ratio of monomer to dimer (p > 0.05, for 3 independent experiment). Addition of EGTA (2 mM) to the assays (lanes 3 and 4) did not modify the monomer to dimer ratio either (p > 0.05, for 3 independent experiments). For the Ca2+-free condition, the expected free Ca2+ concentration was 0.4 nM, calculated using WebMax C version 2.1 . and assuming a contaminant level of 5 μM Ca2+ in our water. For the condition in which Ca2+ was present, we added 1.8 mM Ca2+ and no chelator to the solution (see Materials and Methods). (C) Effects of transglutaminase inhibitors on dimerization of VR1. The addition of cysteamine (20 mM) and MDC (250 μM) to oocytes did not alter the amount of dimer in relation to monomer when compared to the control lane which did not receive any treatment (p > 0.05, for 3 independent experiments).
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Figure 6: VR1 dimerization is not affected by reducing agents, capsaicin, Ca2+ or transglutaminase inhibitors. (A) Western Blot of the effect of reducing agents on the VR1 dimer. The addition of DTT (100 mM) and TCEP (20 mM) did not modify the ratio of monomer to dimer in VR1-expressing oocytes (p > 0.05, for 3 independent experiments). (B) Effect of Ca2+ on dimer formation in VR1. The addition of Ca2+ to the biochemical assays (in the presence or the absence of capsaicin, lanes 1 and 2) did not modify the ratio of monomer to dimer (p > 0.05, for 3 independent experiment). Addition of EGTA (2 mM) to the assays (lanes 3 and 4) did not modify the monomer to dimer ratio either (p > 0.05, for 3 independent experiments). For the Ca2+-free condition, the expected free Ca2+ concentration was 0.4 nM, calculated using WebMax C version 2.1 . and assuming a contaminant level of 5 μM Ca2+ in our water. For the condition in which Ca2+ was present, we added 1.8 mM Ca2+ and no chelator to the solution (see Materials and Methods). (C) Effects of transglutaminase inhibitors on dimerization of VR1. The addition of cysteamine (20 mM) and MDC (250 μM) to oocytes did not alter the amount of dimer in relation to monomer when compared to the control lane which did not receive any treatment (p > 0.05, for 3 independent experiments).

Mentions: A previous study has reported that the presence of capsaicin and chemical cross-linkers influence the formation of multimers in VR1 [23]. Moreover, this study reported that cross-linking could be a Ca2+ mediated process, through the activation of endogenous transglutaminases. In our expression system, the presence of a dimer was seen even in the absence of capsaicin. Thus, we set out to study the factors that could be involved in the formation of this complex. Our first approach was to determine whether this dimer could be due to the presence of a disulfide bond between subunits. Although we have β-mercaptoethanol in the sample buffer for all experiments, it is possible that a disulfide bond refractory to reduction by this reagent was present. We therefore used the stronger reducing agents DTT and TCEP in the biochemical assays, and used them in various steps of the purification (see Materials and methods). Figure 6A shows the results for this experiment. The control lane represents the results obtained from oocytes processed under control conditions (with β-mercaptoethanol as the only reducing agent). The next two lanes shown are those of oocytes exposed to the reducing agents DTT (100 mM) and TCEP (20 mM) both during processing and in the sample buffer. We quantified the ratio of intensity of the monomer band to the dimer band. Neither DTT nor TCEP treatment produced a difference in this ratio compared to the control condition (t-test, p > 0.05 for 3 independent experiments). These data indicate that the presence of the VR1 dimer is likely not due to an intersubunit disulfide bond.


Subunit modification and association in VR1 ion channels.

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

VR1 dimerization is not affected by reducing agents, capsaicin, Ca2+ or transglutaminase inhibitors. (A) Western Blot of the effect of reducing agents on the VR1 dimer. The addition of DTT (100 mM) and TCEP (20 mM) did not modify the ratio of monomer to dimer in VR1-expressing oocytes (p > 0.05, for 3 independent experiments). (B) Effect of Ca2+ on dimer formation in VR1. The addition of Ca2+ to the biochemical assays (in the presence or the absence of capsaicin, lanes 1 and 2) did not modify the ratio of monomer to dimer (p > 0.05, for 3 independent experiment). Addition of EGTA (2 mM) to the assays (lanes 3 and 4) did not modify the monomer to dimer ratio either (p > 0.05, for 3 independent experiments). For the Ca2+-free condition, the expected free Ca2+ concentration was 0.4 nM, calculated using WebMax C version 2.1 . and assuming a contaminant level of 5 μM Ca2+ in our water. For the condition in which Ca2+ was present, we added 1.8 mM Ca2+ and no chelator to the solution (see Materials and Methods). (C) Effects of transglutaminase inhibitors on dimerization of VR1. The addition of cysteamine (20 mM) and MDC (250 μM) to oocytes did not alter the amount of dimer in relation to monomer when compared to the control lane which did not receive any treatment (p > 0.05, for 3 independent experiments).
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Figure 6: VR1 dimerization is not affected by reducing agents, capsaicin, Ca2+ or transglutaminase inhibitors. (A) Western Blot of the effect of reducing agents on the VR1 dimer. The addition of DTT (100 mM) and TCEP (20 mM) did not modify the ratio of monomer to dimer in VR1-expressing oocytes (p > 0.05, for 3 independent experiments). (B) Effect of Ca2+ on dimer formation in VR1. The addition of Ca2+ to the biochemical assays (in the presence or the absence of capsaicin, lanes 1 and 2) did not modify the ratio of monomer to dimer (p > 0.05, for 3 independent experiment). Addition of EGTA (2 mM) to the assays (lanes 3 and 4) did not modify the monomer to dimer ratio either (p > 0.05, for 3 independent experiments). For the Ca2+-free condition, the expected free Ca2+ concentration was 0.4 nM, calculated using WebMax C version 2.1 . and assuming a contaminant level of 5 μM Ca2+ in our water. For the condition in which Ca2+ was present, we added 1.8 mM Ca2+ and no chelator to the solution (see Materials and Methods). (C) Effects of transglutaminase inhibitors on dimerization of VR1. The addition of cysteamine (20 mM) and MDC (250 μM) to oocytes did not alter the amount of dimer in relation to monomer when compared to the control lane which did not receive any treatment (p > 0.05, for 3 independent experiments).
Mentions: A previous study has reported that the presence of capsaicin and chemical cross-linkers influence the formation of multimers in VR1 [23]. Moreover, this study reported that cross-linking could be a Ca2+ mediated process, through the activation of endogenous transglutaminases. In our expression system, the presence of a dimer was seen even in the absence of capsaicin. Thus, we set out to study the factors that could be involved in the formation of this complex. Our first approach was to determine whether this dimer could be due to the presence of a disulfide bond between subunits. Although we have β-mercaptoethanol in the sample buffer for all experiments, it is possible that a disulfide bond refractory to reduction by this reagent was present. We therefore used the stronger reducing agents DTT and TCEP in the biochemical assays, and used them in various steps of the purification (see Materials and methods). Figure 6A shows the results for this experiment. The control lane represents the results obtained from oocytes processed under control conditions (with β-mercaptoethanol as the only reducing agent). The next two lanes shown are those of oocytes exposed to the reducing agents DTT (100 mM) and TCEP (20 mM) both during processing and in the sample buffer. We quantified the ratio of intensity of the monomer band to the dimer band. Neither DTT nor TCEP treatment produced a difference in this ratio compared to the control condition (t-test, p > 0.05 for 3 independent experiments). These data indicate that the presence of the VR1 dimer is likely not due to an intersubunit disulfide bond.

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
Related in: MedlinePlus