Efficient coupling of ligand binding to channel opening by the binding domain of a modulatory (β) subunit of the olfactory cyclic nucleotide-gated channel

E. C. Young, D. M. Sciubba, S. A. Siegelbaum

Research output: Contribution to journalArticlepeer-review

Abstract

CNG channels in vivo are heteromers of homologous α and β subunits that each contain a six-transmembrane segment domain and a COOH-terminal cytoplasmic cyclic nucleotide binding domain (BD). In heterologous expression systems, heteromeric αβ channels activate with greater sensitivity to ligand than do homomeric α channels; however, ligand-gating of channels containing only β subunit BDs has never been studied because β subunits cannot form functional homomeric CNG channels. To characterize directly the contribution of the β subunit BD to ligand-gating, we constructed a chimeric subunit, X-β, whose BD sequence was that of the β subunit CNG5 from rat, but whose sequence outside the BD was derived from α subunits. For comparison, we constructed another chimera, X-α, whose sequence outside the BD was identical to that of X-β, but whose BD sequence was that of the α subunit CNG2 from catfish. When expressed in Xenopus oocytes, X-β and X-α each formed functional homomeric channels activated by both cAMP and cGMP. This is the first demonstration that the β subunit BD can couple ligand binding to activation in the absence of α subunit BD residues. Notably, both agonists activate X-β more effectively than X-α (higher opening efficacy and lower K1/2). The BD is believed to comprise two functionally distinct subdomains: (1) the roll subdomain (β-roll and flanking A- and B-helices) and (2) the C-helix subdomain. Opening efficacy was previously believed to be controlled primarily by the C-helix, but when we made additional chimeras by exchanging the subdomains between X-β and X-α, we found that both subdomains contain significant determinants of efficacy and agonist selectivity. In particular, only channels containing the roll subdomain of the β subunit had high efficacy. Thermodynamic linkage analysis shows that interaction between the two subdomains accounts for a significant portion of their contribution to activation energetics.

Original languageEnglish (US)
Pages (from-to)523-545
Number of pages23
JournalJournal of General Physiology
Volume118
Issue number5
DOIs
StatePublished - 2001

Keywords

  • Allosteric activation
  • Ligand-gated channels
  • Thermodynamic linkage
  • cAMP
  • cGMP

ASJC Scopus subject areas

  • Physiology

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