Subunit modification and association in VR1 ion channels.
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.
Rosenbaum
Tamara
T
Department of Ophthalmology, Department of Physiology and Biophysics, University of Washington, Box 356485, Seattle, WA 98195-6485, USA. tronsenba@u.washington.edu
Awaya
Mika
M
Gordon
Sharona E
SE
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002211:Capsaicin / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011955:Receptors, Drug / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D014982:Xenopus laevis
D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011955:Receptors, Drug; D002211:Capsaicin; D002118:Calcium