The family of mammalian genes related to the Drosophila Shaker gene, consisting of four subfamilies, is thought to encode subunits of tetrameric voltage-gated K+ channels. There is compelling evidence that subunits of the same subfamily, but not of different subfamilies, form heteromultimeric channels in vitro, and thus, each gene subfamily is postulated to encode components of an independent channel system. In order to identify cells with native channels containing subunits of one of these subfamilies (Shaw-related or ShIII), the cellular distribution of ShIII transcripts was examined by Northern blot analysis and in situ hybridization. Three of four ShIII genes (KV3.1, KV3.2, and KV3.3) are expressed mainly in the CNS. KV3.4 transcripts are also present in the CNS but are more abundant in skeletal muscle. In situ hybridization studies in the CNS reveal discrete and specific neuronal populations that prominently express ShIII mRNAs, both in projecting and in local circuit neurons. In the cerebral cortex, hippocampus, and caudate- putamen, subsets of neurons can be distinguished by the expression of specific ShIII mRNAs. Each ShIII gene exhibits a unique pattern of expression; however, many neuronal populations expressing KV3.1 transcripts also express KV3.3 mRNAs. Furthermore, KV3.4 transcripts are present, albeit at lower levels, in several of the neuronal populations that also express KV3.1 and/or KV3.3 mRNAs, revealing a high potential for heteromultimer formation between the products of three of the four genes. Expression of ShIII cRNAs in Xenopus oocytes was used to explore the functional consequences of heteromultimer formation between ShIII subunits. Small amounts of KV3.4 cRNA, which expresses small, fast-inactivating currents when injected alone, produced fast-inactivating currents that are severalfold larger when coinjected with an excess of KV3.1 or KV3.3 cRNA. This amplification is due to both an increase in single-channel conductance in the heteromultimeric channels and the observation that less than four, perhaps even a single KV3.4 subunit is sufficient to impart fast- inactivating properties to the channel. The oocyte experiments indicate that the apparently limited, low-level expression of KV3.4 in the CNS is potentially significant. The anatomical studies suggest that heteromultimer formation between ShIII proteins might be a common feature in the CNS. Moreover, the possibility that the subunit composition of heteromultimers varies in different neurons should be considered, since the ratios of overlapping signals change from one neuronal population to another. In order to proceed with functional analysis of native ShIII channels, it is important to known which subunit compositions might occur in vivo. The studies presented here provide important clues for the identification of native homo- and heteromultimeric ShIII channels in neurons.

Olfactory marker protein (OMP) is an abundant, phylogenetically conserved, cytoplasmic protein of unknown function expressed almost exclusively in mature olfactory sensory neurons. To address its function, we generated OMP-deficient mice by gene targeting in embryonic stem cells. We report that these OMP-null mice are compromised in their ability to respond to odor stimuli, providing insight to OMP function. The maximal electroolfactogram response of the olfactory neuroepithelium to several odorants was 20-40% smaller in the mutants compared with controls. In addition, the onset and recovery kinetics following isoamyl acetate stimulation are prolonged in the null mice. Furthermore, the ability of the mutants to respond to the second odor pulse of a pair is impaired, over a range of concentrations, compared with controls. These results imply that neural activity directed toward the olfactory bulb is also reduced. The bulbar phenotype observed in the OMP-null mouse is consistent with this hypothesis. Bulbar activity of tyrosine hydroxylase, the rate limiting enzyme of catecholamine biosynthesis, and content of the neuropeptide cholecystokinin are reduced by 65% and 50%, respectively. This similarity to postsynaptic changes in gene expression induced by peripheral olfactory deafferentation or naris blockade confirms that functional neural activity is reduced in both the olfactory neuroepithelium and the olfactory nerve projection to the bulb in the OMP-null mouse. These observations provide strong support for the conclusion that OMP is a novel modulatory component of the odor detection/signal transduction cascade.

The olfactory cyclic nucleotide-gated channel subunit 1 (OCNC1) is required for signal transduction in olfactory receptor cells. To further investigate the role of this channel in the olfactory system, the biochemical and morphological consequences of targeted disruption of OCNC1 were investigated in adult mice. Null as compared to wild-type mice had smaller olfactory bulbs, suggesting compromised development of the central target of the receptor cells. Ectopic olfactory marker protein (OMP)-stained fibers localized to the external plexiform layer reflected the relative immaturity of the olfactory bulb in the null mice. The olfactory epithelium of the knock-out mouse was thinner and showed lower expression of olfactory marker protein and growth-associated protein 43, indicating decreases in both generation and maturation of receptor cells. Tyrosine hydroxylase (TH) expression in the olfactory bulb, examined as a reflection of afferent activity, was reduced in the majority of periglomerular neurons but retained in atypical or "necklace" glomeruli localized to posterior aspects of the olfactory bulb. Double label studies demonstrated that the remaining TH-immunostained neurons received their innervation from a subset of receptor cells previously shown to express a phosphodiesterase that differs from that found in most receptor cells. These data indicate that expression of OCNC1 is required for normal development of the olfactory epithelium and olfactory bulb. The robust expression of TH in some periglomerular cells in the OCNC1-null mice suggests that receptor cells innervating these glomeruli may use an alternate signal transduction pathway.