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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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Δ2–52 forms functional, capsaicin-activated channels. (A) Current family activated by a saturating capsaicin concentration (4 μM) in an outside-out patch obtained from stepping the voltage from a holding potential of 0 mV to between -100 and +100 mV. (B) Current-voltage relation for currents activated by 4 μM capsaicin for Δ2–52 (blue) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (C) Current family activated by a sub-saturating capsaicin concentration (0.5 μM). (D) Current-voltage relation for currents activated by 0.5 μM capsaicin for Δ2–52 (blue) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (E) Dose-response relations for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K½= 390 nM, Imax = 1630 pA, and n = 1.5. Filled circles represent actual data values. All data in this figure are from the same patch.
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Figure 5: Δ2–52 forms functional, capsaicin-activated channels. (A) Current family activated by a saturating capsaicin concentration (4 μM) in an outside-out patch obtained from stepping the voltage from a holding potential of 0 mV to between -100 and +100 mV. (B) Current-voltage relation for currents activated by 4 μM capsaicin for Δ2–52 (blue) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (C) Current family activated by a sub-saturating capsaicin concentration (0.5 μM). (D) Current-voltage relation for currents activated by 0.5 μM capsaicin for Δ2–52 (blue) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (E) Dose-response relations for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K½= 390 nM, Imax = 1630 pA, and n = 1.5. Filled circles represent actual data values. All data in this figure are from the same patch.

Mentions: Next, as with N604S, we wanted to be certain that Δ2–52 expressed as a functional channel in Xenopus oocytes. The results of electrophysiological experiments are shown in Figure 5. We found Δ2–52 to form functional, capsaicin-activated channels in Xenopus oocytes. Although an increase in rectification was consistently observed with these channels, compared to wild-type VR1 (Figure 5B), the ratio of the current at +100 mV to that at -100 mV is not statistically different from wild-type VR1. Fits of dose-response relations (Figure 5E) with the Hill equation yielded a mean n value of 2.1 ± 0.26, and a mean K½ value of 1.98 ± 0.8 μM; neither n nor K½ were significantly different from VR1 (t-test, p > 0.05, for 5 patches).


Subunit modification and association in VR1 ion channels.

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

Δ2–52 forms functional, capsaicin-activated channels. (A) Current family activated by a saturating capsaicin concentration (4 μM) in an outside-out patch obtained from stepping the voltage from a holding potential of 0 mV to between -100 and +100 mV. (B) Current-voltage relation for currents activated by 4 μM capsaicin for Δ2–52 (blue) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (C) Current family activated by a sub-saturating capsaicin concentration (0.5 μM). (D) Current-voltage relation for currents activated by 0.5 μM capsaicin for Δ2–52 (blue) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (E) Dose-response relations for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K½= 390 nM, Imax = 1630 pA, and n = 1.5. Filled circles represent actual data values. All data in this figure are from the same patch.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Δ2–52 forms functional, capsaicin-activated channels. (A) Current family activated by a saturating capsaicin concentration (4 μM) in an outside-out patch obtained from stepping the voltage from a holding potential of 0 mV to between -100 and +100 mV. (B) Current-voltage relation for currents activated by 4 μM capsaicin for Δ2–52 (blue) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (C) Current family activated by a sub-saturating capsaicin concentration (0.5 μM). (D) Current-voltage relation for currents activated by 0.5 μM capsaicin for Δ2–52 (blue) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (E) Dose-response relations for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K½= 390 nM, Imax = 1630 pA, and n = 1.5. Filled circles represent actual data values. All data in this figure are from the same patch.
Mentions: Next, as with N604S, we wanted to be certain that Δ2–52 expressed as a functional channel in Xenopus oocytes. The results of electrophysiological experiments are shown in Figure 5. We found Δ2–52 to form functional, capsaicin-activated channels in Xenopus oocytes. Although an increase in rectification was consistently observed with these channels, compared to wild-type VR1 (Figure 5B), the ratio of the current at +100 mV to that at -100 mV is not statistically different from wild-type VR1. Fits of dose-response relations (Figure 5E) with the Hill equation yielded a mean n value of 2.1 ± 0.26, and a mean K½ value of 1.98 ± 0.8 μM; neither n nor K½ were significantly different from VR1 (t-test, p > 0.05, for 5 patches).

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