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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

N604S forms functional channels, with properties like that of wild type. (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 N604S (blue) and wild-type VR1 channels (green). 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 N604S (red) 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 = 905 nM, Imax = 114 pA, and n = 1.8. Filled circles represent actual data values, plotted on a double-log axis. All data in this figure is from the same patch.
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Figure 3: N604S forms functional channels, with properties like that of wild type. (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 N604S (blue) and wild-type VR1 channels (green). 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 N604S (red) 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 = 905 nM, Imax = 114 pA, and n = 1.8. Filled circles represent actual data values, plotted on a double-log axis. All data in this figure is from the same patch.

Mentions: Once we determined that the N604S mutation efficiently removed the glycosylated form of the monomer, we tested to be certain that N604S channels were present in the plasma membrane and had electrophysiological characteristics similar to those observed in wild-type channels. We performed patch-clamp recordings on oocytes injected with N604S RNA. The results of these experiments are shown in Figure 3. Like for wild-type VR1 channels, capsaicin activated N604S channels in a concentration-dependent manner (Figures 3A, 3C, and 3E). Current voltage relations at both a saturating (Figure 3B) and a subsaturating (Figure 3D) concentration of capsaicin show that the voltage dependence of N604S channels is similar to that of wild-type VR1 channels (in Figure 3 compare data from N604S channels [blue and red] to data from wild-type VR1 channels [green]): the ratio of the current at +100 mV to that at -100 mV is not statistically different from wild-type VR1. Furthermore, the dose-response relation in Figure 3E shows that the apparent affinity of N604S channels for capsaicin is not significantly different from wild-type channels (t-test, p > 0.05), with n = 2.3 ± 0.3 and K½ = 780 ± 71 nM (mean of 5 patches). These data indicate that eliminating the N-glycosylation site of the VR1 channel gives rise to a functional protein electrophysiologically similar to wild-type VR1.


Subunit modification and association in VR1 ion channels.

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

N604S forms functional channels, with properties like that of wild type. (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 N604S (blue) and wild-type VR1 channels (green). 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 N604S (red) 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 = 905 nM, Imax = 114 pA, and n = 1.8. Filled circles represent actual data values, plotted on a double-log axis. All data in this figure is from the same patch.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: N604S forms functional channels, with properties like that of wild type. (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 N604S (blue) and wild-type VR1 channels (green). 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 N604S (red) 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 = 905 nM, Imax = 114 pA, and n = 1.8. Filled circles represent actual data values, plotted on a double-log axis. All data in this figure is from the same patch.
Mentions: Once we determined that the N604S mutation efficiently removed the glycosylated form of the monomer, we tested to be certain that N604S channels were present in the plasma membrane and had electrophysiological characteristics similar to those observed in wild-type channels. We performed patch-clamp recordings on oocytes injected with N604S RNA. The results of these experiments are shown in Figure 3. Like for wild-type VR1 channels, capsaicin activated N604S channels in a concentration-dependent manner (Figures 3A, 3C, and 3E). Current voltage relations at both a saturating (Figure 3B) and a subsaturating (Figure 3D) concentration of capsaicin show that the voltage dependence of N604S channels is similar to that of wild-type VR1 channels (in Figure 3 compare data from N604S channels [blue and red] to data from wild-type VR1 channels [green]): the ratio of the current at +100 mV to that at -100 mV is not statistically different from wild-type VR1. Furthermore, the dose-response relation in Figure 3E shows that the apparent affinity of N604S channels for capsaicin is not significantly different from wild-type channels (t-test, p > 0.05), with n = 2.3 ± 0.3 and K½ = 780 ± 71 nM (mean of 5 patches). These data indicate that eliminating the N-glycosylation site of the VR1 channel gives rise to a functional protein electrophysiologically similar to wild-type VR1.

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