Biochemical Evidence for a Putative Inositol 1,3,4,5-Tetrakisphosphate Receptor in the Olfactory System of Atlantic Salmon (Salmo salar)

Olfactory receptor neurons in Atlantic salmon (Salmo salar) appear to use a phosphoinositide-directed phospholipase C (PLC) in odorant signal transduction. The consequences of odor-activated PLC depend on its product, inositol 1,4,5-trisphosphate (IP3). Therefore, a plasma membrane rich (PMR) fraction, previously characterized from salmon olfactory rosettes, was used to study binding sites for IP3 and its phosphorylation product, inositol 1,3,4,5-tetrakisphosphate (IP4). Binding sites for IP3 were present at the lower limit for detection in the PMR fraction but were abundant in a microsomal fraction. Binding sites for IP4 were abundant in the PMR fraction and thus colocalized in the same subcellular fraction with odorant receptors for amino acids and bile acids. Binding of IP4 was saturable and high affinity (K d = 83 nM). The rank order for potency of inhibition of IP4 by other inositol polyphosphates (InsPx) followed the phosphorylation number with InsP6 > InsP5 > other InsP4 isomers > InsP3 isomers > InsP2 isomers, with the latter showing no activity. The consequences of PLC activity in this system may be dictated in part by a putative receptor for IP4.

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 [18,19]. The amino acid and bile acid receptors appear to be coupled through G proteins to the activation of phospholipase C (PLC) and the breakdown of phosphatidylinositol 4,5bisphosphate (PIP 2 ) to generate diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ) [17,18]. Early biochemical data characterizing these as G protein-coupled receptors is now supported by molecular studies characterizing olfactory receptor gene sequences from Atlantic salmon [20][21][22][23]. Underscoring the importance of these receptors in salmon physiology, odorant receptor expression has been shown to change during the parr-smolt transformation, a period characterized by increased olfactory sensitivity and olfactorybased learning [24].
The significance of olfactory PLC activity resides in part with the location and characteristics of receptors for IP 3 . In most cells, IP 3 receptors mediate the release of Ca 2+ from internal stores in the endoplasmic reticulum (for review, see [25]). However, in association with PLC-based olfactory signal transduction, IP 3 receptors have been found in olfactory cilia of catfish [6], carp [14], and lobster [26,27]. From this position, IP 3 may gate Ca 2+ influx through the plasma membrane rather than the release from intracellular stores. Another important part of IP 3 signaling in other systems has been its metabolism, including phosphorylation by a 3-kinase to generate the biologically active inositol 1,3,4,5tetrakisphosphate (IP 4 ) [28][29][30]. While IP 4 continues to be studied in mammalian systems for roles as diverse as regulating nuclear calcium signaling [31], tyrosine kinase [32], and mitochondrial permeability and apoptosis [33,34], Fadool and Ache [26] showed that olfactory receptor neurons of lobster express an IP 4 receptor acting as a functional channel in the plasma membrane. In lobster, plasma membrane IP 3 and IP 4 receptors may interact reciprocally to regulate Ca 2+ entry in olfactory neurons.
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 IP 3 binding sites would colocalize with odor receptor binding sites in a plasma membrane rich fraction (PMR) that we characterized previously [17][18][19][20]35]. Finding that binding of IP 3 was marginal in this fraction, we proceeded to detect and characterize PMR binding sites for IP 4 which may play a critical role in salmon olfactory transduction. Binding sites for IP 3 were subsequently detected in the endoplasmic reticulum-rich microsomal fraction.

Isolation of the Plasma Membrane Rich (PMR) and
Microsomal Fractions. Atlantic salmon (Salmo salar) were raised under conditions of simulated natural photoperiod and temperature in the aquaculture facility of University of Rhode Island. Using a modification of a method devised originally for rainbow trout by Cagan and Zeiger [36], a plasma membrane rich (PMR) fraction was obtained from the olfactory rosettes as described previously [19]. Rosettes were pooled from ten salmon for each analysis. The microsomal fraction was isolated from the olfactory rosettes using the method of Kalinoski et al. [6]. For comparative purposes, PMR fractions and microsomal fractions were also prepared from salmon brain and rat brain. Concentrations of proteins were determined by the method of Bradford (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard. 3 Binding. Binding of [ 3 H]IP 3 ([inositol-1-3 H]; 21.0 Ci/ mmol; New England Nuclear, Boston, MA) was measured using conditions described by Kalinoski et al. [6] except that microsomal fractions (100 g protein per assay) or PMR fractions (100-300 g protein per assay) were from salmon olfactory rosettes or from salmon or rat brain. Digitonin (50 g/mL) was added to permeabilize any membrane vesicles and insure that all binding sites are accessible [6]. The incubation buffer consisted of 110 mM KCl, 1 mM EGTA/0.2 mM CaCl 2 (free Ca 2+ concentration = 20 nM), and 10 mM HEPES, pH 7.4. Incubations were carried out for 30 min at 4 ∘ C. Separation of bound and free [ 3 H]IP 3 was achieved by rapidly filtering through Whatman GF/C filters and washing 3 times with assay buffer. Filters were extracted in scintillation cocktail for 4 hr, and the amount of associated radioactivity was determined by scintillation spectrometry. The amount of binding was determined in the absence (total binding) and presence (nonspecific binding) of excess (120 M) unlabeled InsP 3 . Two concentrations of [ 3 H]IP 3 (7 and 14 nM) were tested. The calculated difference between total and nonspecific binding was operationally defined as a specific binding.  3 Binding. At a radioligand concentration of 7 nM, no specific binding of IP 3 was detectable with the olfactory PMR fraction. At 14 nM radioligand, IP 3 binding to the olfactory PMR fraction was at the lower limit of detection in the assay (see data labeled IP 3 -PMR in Figure 1). Nonspecific binding accounted for almost 90% of the small amount of total binding of [ 3 H]IP 3 to the PMR fraction. Similar results were obtained with a salmon brain PMR fraction, analyzed as a negative control. The specific binding of [ 3 H]IP 3 corresponded to a maximum of 16 fmol bound per mg olfactory PMR protein and 10 fmol per mg salmon brain fraction.

IP
In contrast, specific binding sites for [ 3 H]IP 3 were readily detected in a microsomal (MS) preparation from salmon olfactory rosettes (see data labeled IP 3 -MS in Figure 1). In this preparation, specific binding accounted for at least 75% of the total binding of [ 3 H]IP 3 and corresponded to 1.2 pmol IP 3 bound per mg MS protein, a level nearly 100 times higher than the PMR fraction. This compares favorably to the level of IP 3 binding measured in a rat brain microsomal fraction that was analyzed as a positive control. 4 Binding. While IP 3 binding to the salmon olfactory PMR fraction was at the lower limit for detection in our assay, binding sites for IP 4 were readily detected and were present at high density (see data labeled IP 4 -PMR in Figure 1). At comparable ligand concentration (14 nM), the olfactory PMR fraction supported binding of 364 fmol IP 4 per mg protein (contrasted with 16 fmol IP 3 per mg protein). Nonspecific binding represented less than 20% of total binding. In a single trial with the microsomal preparation from salmon olfactory rosettes, specific binding of [ 3 H]IP 4 was at the lower limit of detection (not shown). Thus, IP 4 sites were readily detected in the PMR but not the microsomal fraction, a result opposite of that for IP 3 binding. Experiments performed with increasing concentrations of [ 3 H]IP 4 demonstrated that specific binding was saturable ( Figure 2). Scatchard analysis of the binding data (Figure 2, inset) yielded 83 nM for the and 3811 fmol/mg protein for the max for IP 4 binding to the olfactory PMR fraction.

Discussion
Previous characterization of the PMR fraction showed high levels of the plasma membrane marker Na, K-ATPase and binding sites for amino acid [19,20] and bile acid [18] odors. This fraction had minimal contamination with endoplasmic reticulum as suggested by the absence of thapsigarginsensitive Ca 2+ -ATPase [35]. The low level of observed IP 3 binding can also be considered as evidence of the lack of ER especially when compared to the microsomal fraction which is traditionally used as a source of endoplasmic reticulum and IP 3 receptors [25]. A comparison of IP 3 binding to the two subcellular fractions is consistent with the presence of IP 3 receptors in endoplasmic reticulum rather than plasma membranes. This does not rule out the possibility that IP 3 receptors would be detected at a higher level in isolated cilia [6] rather than the PMR fraction, but the low level of IP 3 binding to the olfactory PMR fraction contrasts sharply with the high density of binding sites corresponding to odorant amino acid receptors [19,20]. Clearly, IP 3 receptors do not colocalize with odorant receptors in this fraction. Thus, our initial hypothesis that IP 3 binding sites would be abundant in the PMR fraction from the olfactory rosettes of Atlantic salmon was not supported by this study.
In contrast, IP 4 binding sites were abundant in this PMR fraction, which was previously shown to support odorstimulated PLC activity [17,18,20]. Thus, it is an IP 4 binding that colocalizes with odor receptors in the PMR fraction from salmon. Although the binding sites for IP 4 appear in the PMR fraction with odor binding sites, we cannot confirm from this result alone that they appear together on the same membrane. In the only other olfactory system in which it has been characterized, IP 4 gated a calcium channel in the lobster olfactory system [26]. If in salmon, the colocalization of odor and IP 4 binding sites in the PMR fraction extends to a common membrane location, then an IP 4 receptor could be an important downstream element in salmon olfactory transduction. The pH optimum and high affinity value for IP 4 binding are similar to what has been reported in mammalian brain, but the profile for the competition by other InsP is somewhat different [37]. The max for IP 4 binding reflects a density of sites comparable to the density of IP 3 binding sites in the olfactory plasma membrane of catfish ( max = 17.6 pmol/mg protein from Kalinoski et al. [6]). The value for IP 4 binding is much lower (i.e., the affinity is much higher) than the for IP 3 binding sites in catfish ( = 1.1 M from Kalinoski et al. [6]), which is consistent with the lower level of IP 4 produced relative to IP 3 [38].
In essentially all animal cells, IP 3 is metabolized in a bifurcate pathway that includes phosphorylation by a 3-kinase to produce IP 4 [28,39]. Higher-order inositol polyphosphates are also produced in cells along with an array of dephosphorylation products. We included many of these inositol polyphosphates in competition analyses to further characterize the olfactory IP 4 binding site. Among the inositol polyphosphates tested, InsP 5 and InsP 6 showed reasonably potent inhibition of [ 3 H]IP 4 binding. These are formed by the sequential actions of specific kinases, are inhibitors of IP 4 3-phosphatase and IP 4 5-phosphatase [40], and are active in other cellular systems [41]. In contrast, Ins(1,3,4)P 3 , Ins(1,4)P 2 , and Ins(4,5)P 2 showed little or no ability to interact with the IP 4 site. This is not surprising because these are regarded as the products of inactivating phosphatases. Marginal inhibition of [ 3 H]IP 4 binding by IP 3 (Ins(1,4,5)P 3 ) confirmed the independence of the IP 4 and IP 3 binding sites in this system and supported the conclusions from direct measurements of [ 3 H]IP 3 binding at optimal pH that these sites are not present in the PMR fraction. In summary, we found a unique IP 4 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 IP 4 receptor in a fish olfactory system. The exact plasma membrane location and the colocalization of odor receptors and putative IP 4 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 IP 4 gates a calcium channel and helps regulate Ca 2+ entry into lobster olfactory neurons [26], a similar role to that ascribed to IP 3 in lobster [27], catfish [6], and carp [14]. This provides the only context with which to interpret the significance of finding IP 4 binding sites in membranes of the salmon olfactory system and to begin to suggest that IP 4 rather than (or in addition to) IP 3 may be a key downstream element for olfactory signal transduction in Atlantic salmon.