Faculty Bibliography

1. Xenopus oocytes injected with rat brain mRNA express a transient K+ current similar to the A current that activates transiently near the threshold for Na+ action potential generation (ISA) seen in somatic recordings from neurons. We used hybrid arrest with antisense oligonucleotides to investigate which of the cloned K+ channel proteins might be components of the channels responsible for the ISA expressed from brain mRNA. An oligonucleotide complementary to a sequence common to all known mammalian Shal-related mRNAs [KV4.1, KV4.2, and KV4.3 (the nomenclature of Sh K+ channel genes of Chandy and colleagues was used in this paper)] blocked the expression of the ISA. An oligonucleotide complementary only to the KV4.2 mRNA, the most abundant Shal-related transcript in rat brain RNA preparations, was also quite efficient in arresting the expression of the ISA from brain. These experiments indicate that Shal-related proteins are important components of the channels carrying the ISA expressed in oocytes injected with brain mRNA. However, there are several significant differences between this ISA and the currents expressed in the same oocytes by in vitro transcribed KV4.1 or KV4.2 cRNA. Most of these differences are eliminated if KV4.1 or KV4.2 cRNA is coinjected with brain poly-(A) RNA treated with antisense oligonucleotides which arrest the expression of the ISA, or with a 2-4Kb rat brain poly-(A) RNA fraction which does not express detectable K+ currents under the same recording conditions. These data support the hypothesis that ISA channels such as those expressed from brain mRNA contain Shal proteins that can be modified by proteins encoded in RNAs that by themselves do not express K+ currents

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

Human endothelin (ET) A receptor (hETAR) is a G-protein-mediated receptor that binds ET1 with high affinity and ET2 and ET3 with lower affinities. ET1 is the most potent endogenous vasoconstrictor known at this time. When expressed in Xenopus laevis oocytes, hETAR is rapidly desensitized after stimulation with ET1. This desensitization lasts 90-110 min. Human neurokinin A (hNKAR) and human serotonin type 2 receptors were also expressed in the Xenopus system for comparison to hETAR. hNKAR desensitizes for 25-35 min, while the serotonin receptor does not appear to desensitize. To examine the role of the cytoplasmic tail of hETAR in desensitization, deletion mutations were constructed which remove 11, 36, and 51 amino acids from the cytoplasmic tail. The mutations removing 11 and 36 residues were functional, but the mutation removing 51 amino acids was not functional. The two functional mutations have a resensitization time similar to that of hETAR. In summary, the prolonged desensitization time of hETAR is unique for G-protein-mediated receptors and may attenuate the adverse physiological effects of the endothelin family. In addition, the cytoplasmic tail of hETAR does not appear to play a role in desensitization or resensitization of this receptor