Biophys J, May 2000, p. 2307-2320, Vol. 78, No. 5

Structural Basis for Ligand Selectivity of Heteromeric Olfactory Cyclic Nucleotide-Gated Channels

Mark S. Shapiro^* and William N. Zagotta

 ^*Department of Physiology and Biophysics and  Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195-7290 USA

In vertebrate olfactory receptors, cAMP produced by odorants opens cyclic nucleotide-gated (CNG) channels, which allow Ca²⁺ entry and depolarization of the cell. These CNG channels are composed of  subunits and at least two types of  subunits that are required for increased cAMP selectivity. We studied the molecular basis for the altered cAMP selectivity produced by one of the  subunits (CNG5, CNC4, OCNC2) using cloned rat olfactory CNG channels expressed in Xenopus oocytes. Compared with  subunit homomultimers ( channels), channels composed of  and  subunits (+ channels) were half-activated (K_1/2) by eightfold less cAMP and fivefold less cIMP, but similar concentrations of cGMP. The K_1/2 values for heteromultimers of the  subunit and a chimeric  subunit with the  subunit cyclic nucleotide-binding region (CNBR) (+-CNBR channels) were restored to near the values for  channels. Furthermore, a single residue in the CNBR could account for the altered ligand selectivity. Mutation of the methionine residue at position 475 in the  subunit to a glutamic acid as in the  subunit (-M475E) reverted the K_1/2,cAMP/K_1/2,cGMP and K_1/2,cIMP/K_1/2,cGMP ratios of +-M475E channels to be very similar to those of  channels. In addition, comparison of +-CNBR channels with +-M475E channels suggests that the CNBR of the  subunit contains amino acid differences at positions other than 475 that produce an increase in the apparent affinity for each ligand. Like the wild-type  subunit, the chimeric / subunits conferred a shallow slope to the dose-response curves, increased voltage dependence, and caused desensitization. In addition, as for + channels, block of +CNBR channels by internal Mg²⁺ was not steeply voltage-dependent (z ~1e) as compared to block of  channels (z 2.7e). Thus, the ligand-independent effects localize outside of the CNBR. We propose a molecular model to explain how the  subunit alters ligand selectivity of the heteromeric channels.

Biophys J, May 2000, p. 2307-2320, Vol. 78, No. 5
© 2000 by the Biophysical Society   0006-3495/00/05/2307/14  $2.00

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