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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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Related in: MedlinePlus

VR1 forms functional channels in Xenopus oocytes. (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. For scale bar see (C). (B) Current-voltage relation for currents activated by 4 μM capsaicin. 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 (red) with data from currents obtained with 4 μM capsaicin shown also (blue). Data were normalized to the value of the current at -100 mV. (E) Dose-response relation for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K½ = 440 nM, Imax = 567 pA, and n = 2.2. Filled circles represent actual data values. All data in this figure are from the same patch.
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Figure 1: VR1 forms functional channels in Xenopus oocytes. (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. For scale bar see (C). (B) Current-voltage relation for currents activated by 4 μM capsaicin. 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 (red) with data from currents obtained with 4 μM capsaicin shown also (blue). Data were normalized to the value of the current at -100 mV. (E) Dose-response relation for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K½ = 440 nM, Imax = 567 pA, and n = 2.2. Filled circles represent actual data values. All data in this figure are from the same patch.

Mentions: We expressed VR1 channels in Xenopus oocytes in order to characterize their electrophysiological and biochemical properties. Using outside-out patch-clamp recordings, we studied the responses of the channels to capsaicin. Figure 1A depicts a current family obtained using a saturating (4 μM) capsaicin concentration when the voltage was stepped from a holding potential of 0 mV to from -100 to +100 mV. A current-voltage relation measured from these currents (Figure 1B) shows the pronounced outward rectification exhibited by VR1. This rectification has been shown to be independent of external divalent cations [24], and is likely due to a combination of rectification in unitary conductance [9] and voltage-dependent gating [24]. Application of a lower concentration of capsaicin (0.5 μM) activated a smaller fraction of the channels in this patch, giving a smaller overall current (Figure 1C). By plotting the normalized currents in the presence of 4 μM and 0.5 μM capsaicin on the same graph (Figure 1D), we show that the voltage dependence remains unaltered under both experimental conditions. Figure 1E shows a dose-response relation for activation of VR1 channels by capsaicin for the same patch. Fits of dose-response relations with the Hill equation (see Experimental Procedures) yielded values of n = 1.8 ± 0.06 and a K½ = 614 ± 110 nM (mean of 5 patches). These values are similar to those previously reported [20,24]. These data indicate that in our system VR1 RNA is expressed as a functional protein with characteristics similar to those reported by other groups.


Subunit modification and association in VR1 ion channels.

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

VR1 forms functional channels in Xenopus oocytes. (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. For scale bar see (C). (B) Current-voltage relation for currents activated by 4 μM capsaicin. 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 (red) with data from currents obtained with 4 μM capsaicin shown also (blue). Data were normalized to the value of the current at -100 mV. (E) Dose-response relation for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K½ = 440 nM, Imax = 567 pA, and n = 2.2. 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 1: VR1 forms functional channels in Xenopus oocytes. (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. For scale bar see (C). (B) Current-voltage relation for currents activated by 4 μM capsaicin. 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 (red) with data from currents obtained with 4 μM capsaicin shown also (blue). Data were normalized to the value of the current at -100 mV. (E) Dose-response relation for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K½ = 440 nM, Imax = 567 pA, and n = 2.2. Filled circles represent actual data values. All data in this figure are from the same patch.
Mentions: We expressed VR1 channels in Xenopus oocytes in order to characterize their electrophysiological and biochemical properties. Using outside-out patch-clamp recordings, we studied the responses of the channels to capsaicin. Figure 1A depicts a current family obtained using a saturating (4 μM) capsaicin concentration when the voltage was stepped from a holding potential of 0 mV to from -100 to +100 mV. A current-voltage relation measured from these currents (Figure 1B) shows the pronounced outward rectification exhibited by VR1. This rectification has been shown to be independent of external divalent cations [24], and is likely due to a combination of rectification in unitary conductance [9] and voltage-dependent gating [24]. Application of a lower concentration of capsaicin (0.5 μM) activated a smaller fraction of the channels in this patch, giving a smaller overall current (Figure 1C). By plotting the normalized currents in the presence of 4 μM and 0.5 μM capsaicin on the same graph (Figure 1D), we show that the voltage dependence remains unaltered under both experimental conditions. Figure 1E shows a dose-response relation for activation of VR1 channels by capsaicin for the same patch. Fits of dose-response relations with the Hill equation (see Experimental Procedures) yielded values of n = 1.8 ± 0.06 and a K½ = 614 ± 110 nM (mean of 5 patches). These values are similar to those previously reported [20,24]. These data indicate that in our system VR1 RNA is expressed as a functional protein with characteristics similar to those reported by other groups.

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