Molecular cloning and functional expression of a potassium channel cDNA isolated from a rat cardiac library

A full-length K+ channel cDNA (RHK1) was isolated from a rat cardiac library using the polymerase chain reaction (PCR) method and degenerate oligonucleotide primers derived from K+ channel sequences conserved between Drosophila Shaker H4 and mouse brain MBK1. Although RHK1 was isolated from heart, its expression was found in both heart and brain. The RHK1-encoded protein, when expressed in Xenopus oocytes, gated a 4-aminopyridine (4-AP)-sensitive transient outward current. This current is similar to the transient outward current measured in rat ventricular myocytes with respect to voltage-dependence of activation and inactivation, time course of activation and inactivation, and pharmacology.     

DAG lipase activity is necessary for TRP channel regulation in Drosophila photoreceptors

In Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses.     

Role of protein synthesis in the carbohydrate-induced changes in the activities of acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase in cultured rat hepatocytes.

Changes in the activities of acetyl-CoA carboxylase and HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase were studied in primary cultures of adult-rat hepatocytes after exposure of the cells to insulin and/or carbohydrates. To determine the contribution of protein synthesis to changes in enzyme activity, the relative rate of synthesis of each enzyme was measured and the amount of translatable mRNA coding for the enzymes was determined by translation in vitro and immunoprecipitation. Addition of insulin to the culture medium increased the activities of acetyl-CoA carboxylase and HMG-CoA reductase by approx. 4- and 3-fold respectively. Although similar increases in the relative rate of synthesis of each protein and template activity were noted, initial increases in the activity of each enzyme occurred before any changes in protein synthesis were observed, suggesting the involvement of post-translational modification of enzyme activity in addition to changes in protein synthesis. The addition of fructose to the culture medium, in the absence of insulin, increased the activity of the carboxylase and the reductase approx. 3-fold, similar to the effects of insulin. However, the effect of fructose was to increase the rate of synthesis and the amount of translatable mRNA coding for acetyl-CoA carboxylase, whereas the increase in the activity of HMG-CoA reductase was not accompanied by any changes in the rate of synthesis or template activity. The effects of fructose could not be mimicked by glucose unless insulin was also present in the culture medium. Similar to observations in vitro, the injection of insulin or the feeding of a high-fructose diet to rats made diabetic by the injection of streptozotocin produced an increase in the activities of acetyl-CoA carboxylase and HMG-CoA reductase, and only the increase in the activity of the carboxylase was accompanied by an increase in the amount of translatable mRNA coding for the enzyme. The results are discussed in terms of the effects of fructose on the synthesis of enzymes involved in lipogenesis.     

Evolution of androgen-regulated mRNA expression in mouse kidney

To gain information on the evolution of mammalian gene expression patterns, we studied the androgen-inducible expression of three kidney mRNAs in several mouse species (genus Mus). The RP2, ornithine decarboxylase, and beta-glucuronidase mRNAs have each evolved independently, in that the pattern of variation among species is unique for each. This suggests a role for gene-specific, cis-acting genetic elements. Relationships between the regulatory phenotypes and the species phylogeny suggest that the variations in hormone-inducible mRNA expression were generated by a series of independent mutations that occurred in specific lineages, resulting in modifications of the progenitor phenotype. Alternatively, the variations may have preexisted within the progenitor population as polymorphisms that were fixed during establishment of individual lineages. Thus, significant alterations in the androgen-regulated mRNA phenotype have occurred either prior to or during speciation within the Mus genus. These alterations are presumed to be in regulatory sequences that control the expression of the corresponding genes and their response to testosterone; as such, they should be useful in further studying the genetic determinants of gene expression and its evolution.      

Multiple products of the Drosophila Shaker gene may contribute to potassium channel diversity

K+ channels are known through electrophysiology and pharmacology to be an exceptionally diverse group of channels. Molecular studies of the Shaker (Sh) locus in Drosophila have provided the first glimpse of K+ channel structure. The sequences of several Sh cDNA clones have been reported; none are identical. We have isolated and examined 18 additional Sh cDNAs in an attempt to understand the origin, extent, and significance of the variability. The diversity is extensive: we have already identified cDNAs representing at least nine distinct types, and Sh could potentially encode 24 or more products. This diversity, however, fits a simple pattern in which variable 3' and 5' ends are spliced onto a central constant region to yield different cDNA types. These different Sh cDNAs encode proteins with distinct structural features.