Dissociation of phospholamban regulation of cardiac sarcoplasmic reticulum Ca2+ATPase by quercetin.

Quercetin had a biphasic effect on Ca2+ uptake and calcium-stimulated ATP hydrolysis in isolated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ATPase was observed at low quercetin concentrations (<25 microM) followed by inhibition at higher concentrations. The effects were dependent upon the SR protein concentration, the MgATP concentration, and intact phospholamban regulation of cardiac Ca2+ATPase. Only the inhibitory effects at higher quercetin concentrations were observed in skeletal muscle SR which lacks phospholamban and in cardiac SR treated to remove phospholamban regulation. Stimulation was additive with monoclonal antibody 1D11 (directed against phospholamban) at submaximal antibody concentrations; however, the maximal antibody and quercetin stimulation were identical. Quercetin increased the calcium sensitivity of the Ca2+ATPase like that observed with phosphorylation of phospholamban or treatment with monoclonal antibody 1D11. In addition, low concentrations of quercetin increased the steady-state formation of phosphoenzyme from ATP or Pi, but higher quercetin decreased phosphoenzyme levels. Quercetin, even under stimulatory conditions, was a competitive inhibitor of ATP, but appears to relieve the Ca2+ATPase from phospholamban inhibition, thereby, producing an activation. The subsequent inhibitory action of higher quercetin concentrations results from competition of quercetin with the nucleotide binding site of the Ca2+ATPase. The data suggest that quercetin interacts with the nucleotide binding site to mask phospholamban's inhibition of the SR Ca2+ATPase and suggests that phospholamban may interact at or near the nucleotide binding site.

Alternative splicing contributes to K+ channel diversity in the mammalian central nervous system.

In an attempt to define the molecular basis of the functional diversity of K+ channels, we have isolated overlapping rat brain cDNAs that encoded a neuronal delayed rectifier K+ channel, K,4, that is structurally related to the Drosophila Shaw protein. Unlike previously characterized mammalian K+ channel genes, which each contain a single protein-coding exon, K,4 arises from alternative exon usage at a locus that also encodes another mammalian Shaw homolog, NGK2. Thus, the enormous diversity of K+ channels in mammals can be generated not just through gene duplication and divergence but also through alternative splicing of RNA.

Cloning and expression of cDNA and genomic clones encoding three delayed rectifier potassium channels in rat brain.

Rat brain cDNA and genomic clones encoding three K+ channels, Kv1, Kv2, and Kv3, have been isolated by screening with Shaker probes and encode proteins of 602, 530, and 525 amino acids. Each of the deduced protein sequences contains six hydrophobic domains (including an S4-type region characteristic of many voltage-gated channels) and are 68%-72% identical to each other overall. Transcripts of approximately 3.5, approximately 6.5, and approximately 9.5 kb encode Kv1, Kv2, and Kv3, respectively. The Kv2 mRNA is expressed only in brain, whereas the Kv1 and Kv3 transcripts are found in several other tissues as well. There is a marked increase in the amount of Kv1 mRNA in cardiac tissue during development and a similar, but less pronounced, increase of both this mRNA and the Kv2 transcript in brain. RNAs synthesized in vitro from the three clones induce voltage- and time-dependent, delayed rectifier-like K+ currents when injected into Xenopus oocytes, demonstrating that they encode functional K+ channels.

Cloning and expression of the delayed-rectifier IsK channel from neonatal rat heart and diethylstilbestrol-primed rat uterus.

cDNAs encoding a delayed-rectifier-type K+ channel were cloned from both neonatal rat heart and ovariectomized, diethylstilbestrol-primed rat uterus by using the polymerase chain reaction. Both clones have nucleotide sequences identical to that encoding the rat kidney IsK channel [Takumi, T., Ohkubo, H. & Nakanishi, S. (1988) Science 242, 1042-1045] and encode a putative protein of 130 amino acids. Injection of RNA transcripts of the cDNAs into Xenopus oocytes resulted in the expression of a slowly activating, voltage-dependent K+ current. An antisense oligonucleotide, derived from the sequence of the clone, specifically inhibited the expression of the slow, outward current observed in cells injected with mRNAs isolated from the parent tissues (i.e., kidney, heart, and uterus), indicating that the cloned gene underlies the major K+ current expressed from RNA isolated from these tissues.

Factors affecting interaction of staphylococcal alpha toxin with membranes.

Staphylococcal alpha toxin interacts not only with membranes of erythrocytes but also with membranes of other kinds of mammalian cells (platelets, hepatocytes, and lysosomes from polymorphonuclear leukocytes) with the formation of characteristic ring-like structures that can be seen by electron microscopy. Such structures are not observed when alpha toxin is added to membranes derived from various bacteria. The rings seen on mammalian cell membranes tend to be either randomly disposed or in square array. The frequency with which square arrays are seen is influenced by the presence of staphylococcal delta toxin, by the negative staining agent, and by the kind of cell from which the membrane is derived. Synthetic membranes in the form of liposomes, prepared individually from phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, and cardiolipin, produced randomly disposed rings upon addition of alpha toxin. Liposomes made from phosphatidyl ethanolamine did not yield rings. Alpha toxin-treated liposomes prepared from chloroform-methanol extracts of brain white matter consistently showed rings that were rectangularly ordered. Ordered rings on membranes derived from toxin-treated platelets and those on toxin-treated brain extract liposomes were seen in freeze-etched as well as in negatively stained preparations.