Biomedical subjects

Haoyu Zeng

Publications and source records attributed to Haoyu Zeng.

6 recordsLinked to original sources

PubMed2006

Mallotoxin is a novel human ether-a-go-go-related gene (hERG) potassium channel activator.

Human ether-a-go-go-related gene (hERG) encodes a rapidly activating delayed rectifier potassium channel that plays important roles in cardiac action potential repolarization. Although many drugs and compounds block hERG channels, activators of the channel have only recently been described. Three structurally diverse synthetic compounds have been reported to activate hERG channels by altering deactivation or inactivation or by unidentified mechanisms. Here, we describe a novel, naturally occurring hERG channel activator, mallotoxin (MTX). The effects of MTX on hERG channels were investigated using the patch-clamp technique. MTX increased both step and tail hERG currents with EC(50) values of 0.34 and 0.52 microM, respectively. MTX leftward shifted the voltage dependence of hERG channel activation to less depolarized voltages ( approximately 24 mV at 2.5 microM). In addition, MTX increased hERG deactivation time constants. MTX did not change the half-maximal inactivation voltage of the hERG channel, but it reduced the slope of the voltage-dependent inactivation curve. All of these factors contribute to the enhanced activity of hERG channels. During a voltage-clamp protocol using prerecorded cardiac action potentials, 2.5 microM MTX increased the total potassium ions passed through hERG channels by approximately 5-fold. In conclusion, MTX activates hERG channels through distinct mechanisms and with significantly higher potency than previously reported hERG channel activators.

Acetophenones↗

PubMed2006

Mechanisms of two modulatory actions of the channel-binding protein Slob on the Drosophila Slowpoke calcium-dependent potassium channel.

Slob57 is an ion channel auxiliary protein that binds to and modulates the Drosophila Slowpoke calcium-dependent potassium channel (dSlo). We reported recently that residues 1-39 of Slob57 comprise the key domain that both causes dSlo inactivation and shifts its voltage dependence of activation to more depolarized voltages. In the present study we show that removal of residues 2-6 from Slob57 abolishes the inactivation, but the ability of Slob57 to rightward shift the voltage dependence of activation of dSlo remains. A synthetic peptide corresponding in sequence to residues 1-6 of Slob57 blocks dSlo in a voltage- and dose-dependent manner. Two Phe residues and at least one Lys residue in this peptide are required for the blocking action. These data indicate that the amino terminus of Slob57 directly blocks dSlo, thereby leading to channel inactivation. Further truncation to residue Arg(16) eliminates the modulation of voltage dependence of activation. Thus these two modulatory actions of Slob57 are independent. Mutation within the calcium bowl of dSlo greatly reduces its calcium sensitivity (Bian, S., I. Favre, and E. Moczydlowski. 2001. Proc. Natl. Acad. Sci. USA. 98:4776-4781). We found that Slob57 still causes inactivation of this mutant channel, but does not shift its voltage dependence of activation. This result confirms further the independence of the inactivation and the voltage shift produced by Slob57. It also suggests that the voltage shift requires high affinity Ca(2+) binding to an intact calcium bowl. Furthermore, Slob57 inhibits the shift in the voltage dependence of activation of dSlo evoked by Ca(2+), and this inhibition by Slob57 is greater at higher free Ca(2+) concentrations. These results implicate distinct calcium-dependent and -independent mechanisms in the modulation of dSlo by Slob.

Amino Acids↗

PubMed2005

Expression and function of variants of slob, slowpoke channel binding protein, in Drosophila.

Slob binds to and modulates the Drosophila Slowpoke (dSlo) calcium-activated potassium channel and also recruits the ubiquitous signaling protein 14-3-3 to the channel regulatory complex. RT-PCR reveals the presence of multiple slob transcripts in Drosophila heads. The transcripts are predicted to encode proteins that we call Slob51 (kDa), Slob57, Slob65, and Slob71. Slob51 and Slob65 are splice variants that lack a motif important for the binding of 14-3-3. Previous microarray analyses demonstrated the circadian cycling of slob mRNA, and we show by quantitative PCR that more than one transcript cycles in fly heads. Using in situ hybridization, we observe differences in the expression patterns of the different transcripts. Immunohistochemistry on Drosophila heads reveals Slob71/65 protein to be enriched in the lateral neurons, in contrast to Slob57/51 protein, which is expressed most prominently in the pars intercerebralis neurons and dorsal giant interneurons. Using a heterologous expression system, we show that different Slobs bind to different extents to dSlo and 14-3-3. These data reveal an unexpected diversity of the dSlo/Slob/14-3-3 dynamic regulatory complex.

14-3-3 Proteins↗

PubMed2005

The amino terminus of Slob, Slowpoke channel binding protein, critically influences its modulation of the channel.

The Drosophila Slowpoke calcium-dependent potassium channel (dSlo) binding protein Slob was discovered by a yeast two-hybrid screen using the carboxy-terminal tail region of dSlo as bait. Slob binds to and modulates the dSlo channel. We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD). The larger variants, which are initiated at the first translational start site and are called Slob71 and Slob65, shift the voltage dependence of dSlo activation, measured by the whole cell conductance-voltage relationship, to the left (less depolarized voltages). Slob53 and Slob47, initiated at the third translational start site, also shift the dSlo voltage dependence to the left. In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance-voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel. These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.

Amino Acid Sequence↗

PubMed2004

The slowpoke channel binding protein Slob from Drosophila melanogaster exhibits regulatable protein kinase activity.

The slowpoke channel binding protein Slob from Drosophila melanogaster contains a putative protein kinase domain within its amino acid sequence. We find that Slob exhibits weak and barely detectable protein kinase activity in vitro, as evidenced by autophosphorylation and by phosphorylation of exogenously added histone as substrate. The phosphorylation of histone is enhanced markedly when Slob is pretreated with the catalytic subunit of cyclic AMP-dependent protein kinase (PKAc). Mass spectrometric and mutational analysis demonstrates that the major site of phosphorylation by PKAc within Slob is serine 54. The enhancement of Slob kinase activity by PKAc pretreatment is eliminated when serine 54 in Slob is mutated to alanine (S54A). Furthermore, Slob kinase activity is enhanced in an S54E mutant that mimics phosphorylation at serine 54, and there is no further enhancement of S54E Slob kinase activity by pretreatment with PKAc. The results are consistent with the hypothesis that Slob exhibits regulatable protein kinase activity, whose activity is enhanced by phosphorylation at serine 54.

Amino Acid Sequence↗

PubMed2002

NMR-based binding screen and structural analysis of the complex formed between alpha-cobratoxin and an 18-mer cognate peptide derived from the alpha 1 subunit of the nicotinic acetylcholine receptor from Torpedo californica.

The alpha18-mer peptide, spanning residues 181-198 of the Torpedo nicotinic acetylcholine receptor alpha1 subunit, contains key binding determinants for agonists and competitive antagonists. To investigate whether the alpha18-mer can bind other alpha-neurotoxins besides alpha-bungarotoxin, we designed a two-dimensional (1)H-(15)N heteronuclear single quantum correlation experiment to screen four related neurotoxins for their binding ability to the peptide. Of the four toxins tested (erabutoxin a, erabutoxin b, LSIII, and alpha-cobratoxin), only alpha-cobratoxin binds the alpha18-mer to form a 1:1 complex. The NMR solution structure of the alpha-cobratoxin.alpha18-mer complex was determined with a backbone root mean square deviation of 1.46 A. In the structure, alpha-cobratoxin contacts the alpha18-mer at the tips of loop I and II and through C-terminal cationic residues. The contact zone derived from the intermolecular nuclear Overhauser effects is in agreement with recent biochemical data. Furthermore, the structural models support the involvement of cation-pi interactions in stabilizing the complex. In addition, the binding screen results suggest that C-terminal cationic residues of alpha-bungarotoxin and alpha-cobratoxin contribute significantly to binding of the alpha18-mer. Finally, we present a structural model for nicotinic acetylcholine receptor-alpha-cobratoxin interaction by superimposing the alpha-cobratoxin.alpha18-mer complex onto the crystal structure of the acetylcholine-binding protein (Protein Data Bank code ).

Amino Acid Sequence↗