Olfactory receptor neurons in Atlantic salmon (
Adenylyl cyclase and cAMP appear to dominate odor signal transduction in mammals (for reviews, see [
As potent olfactory stimuli for Atlantic salmon, amino acids and bile acids interact with distinct subclasses of olfactory receptors to begin the process of olfactory reception [
The significance of olfactory PLC activity resides in part with the location and characteristics of receptors for IP3. In most cells, IP3 receptors mediate the release of Ca2+ from internal stores in the endoplasmic reticulum (for review, see [
The goal of the present study was to characterize further the PLC-based olfactory signal transduction system of Atlantic salmon, beginning with the hypothesis that IP3 binding sites would colocalize with odor receptor binding sites in a plasma membrane rich fraction (PMR) that we characterized previously [
Atlantic salmon (
Binding of [3H]IP3 ([inositol-1-3H]; 21.0 Ci/mmol; New England Nuclear, Boston, MA) was measured using conditions described by Kalinoski et al. [
The binding assay for [3H]IP4 ([Inositol-1-3H]; 21.0 Ci/mmol; New England Nuclear, Boston, MA) was performed under conditions identical to those described by Challiss et al. [
Binding assays for both [3H]IP3 and [3H]IP4 were based on conditions optimized by others ([
At a radioligand concentration of 7 nM, no specific binding of IP3 was detectable with the olfactory PMR fraction. At 14 nM radioligand, IP3 binding to the olfactory PMR fraction was at the lower limit of detection in the assay (see data labeled IP3-PMR in Figure
Initial screening of IP3 and IP4 binding sites in membrane fractions from salmon olfactory rosettes. The plasma membrane rich (PMR) fraction and a microsomal (MS) fraction were prepared from olfactory rosettes of Atlantic salmon. Specific binding of [3H]IP3 was determined in incubations with 14 nM [3H]IP3 in the presence and absence of excess unlabeled InsP3. Specific binding of IP4 to the PMR fraction is shown for comparison to IP3. The concentration of [3H]IP4 in the reaction mixture was also 14 nM.
In contrast, specific binding sites for [3H]IP3 were readily detected in a microsomal (MS) preparation from salmon olfactory rosettes (see data labeled IP3-MS in Figure
While IP3 binding to the salmon olfactory PMR fraction was at the lower limit for detection in our assay, binding sites for IP4 were readily detected and were present at high density (see data labeled IP4-PMR in Figure
Experiments performed with increasing concentrations of [3H]IP4 demonstrated that specific binding was saturable (Figure
Saturation binding of [3H]IP4 to a plasma membrane rich fraction from salmon olfactory rosettes. Specific binding of IP4 was determined at each of the IP4 concentrations shown. Results are averaged from a single experiment performed in duplicate using olfactory rosettes from 10 Atlantic salmon and are representative of the results of three independent experiments. The inset shows a Scatchard analysis of the binding of IP4 to the plasma membrane rich fraction. Binding data as in Figure
To further characterize the specificity of IP4 binding to the olfactory PMR fraction, competition experiments were performed using 14 nM [3H]IP4 and various other inositol polyphosphates (InsP
Inositol polyphosphate selectivity in competing for [3H]IP4 binding to a PMR fraction from salmon olfactory rosettes. The relative amount of specific binding of 14 nM [3H]IP4 was determined in the presence of at least three concentrations of different inositol polyphosphates (InsP
InsP5 and InsP6 showed reasonably potent inhibition of [3H]IP4 binding. Other IP4 analogs (Ins(3,4,5,6)P4 and Ins(1,4,5,6)P4) were intermediate in potency as inhibitors, while the IP3 analogs (Ins(1,3,4)P3 and Ins(1,4,5)P3) showed little or no activity. Similarly, Ins(1,4)P2 and Ins(4,5)P2, the dephosphorylation products formed from the inactivation of Ins(1,4,5)P3, had no inhibitory effect on [3H]IP4 binding when incubated at 10
EC50 values for inhibition of IP4 binding by inositol polyphosphates.
Inositol polyphosphate | EC50 |
---|---|
InsP6 | 42.7 nM |
Ins(1,3,4,5,6)P5 | 316 nM |
Ins(3,4,5,6)P4 | 3.2 |
Ins(1,4,5,6)P4 | 31.6 |
Ins(1,4,5)P3 | >100 |
Ins(1,3,4)P3 | >100 |
Ins(1,4)P2 | No inhibition at 10 |
Ins(4,5)P2 | No inhibition at 10 |
Concentrations of the competing inositol polyphosphate (
Previous characterization of the PMR fraction showed high levels of the plasma membrane marker Na, K-ATPase and binding sites for amino acid [
In contrast, IP4 binding sites were abundant in this PMR fraction, which was previously shown to support odor-stimulated PLC activity [
In essentially all animal cells, IP3 is metabolized in a bifurcate pathway that includes phosphorylation by a 3-kinase to produce IP4 [
In summary, we found a unique IP4 binding site that colocalizes with odor receptors in a subcellular fraction derived from the olfactory system of Atlantic salmon. This is the first biochemical evidence of a putative membrane-bound IP4 receptor in a fish olfactory system. The exact plasma membrane location and the colocalization of odor receptors and putative IP4 receptors in the same plasma membrane remain to be shown. In the only other olfactory system in which it has been studied, electrophysiological studies have demonstrated that IP4 gates a calcium channel and helps regulate Ca2+ entry into lobster olfactory neurons [
The authors thank Dr. Terrance Bradley for providing the Atlantic salmon used in this study and Dr. Ching-Shih Chen for providing the inositol phosphate analogs. Initial portions of this paper were supported by the University of Rhode Island. The authors acknowledge the advice and assistance of Dr. Jian Wang and Dr. Chun-Shiang Chung with the brain fractions and Lee-Ju Cheng with IP3 binding assays. The authors also thank Dr. Karl Hartman for helpful discussions and suggestions.