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Putative binding sites for arachidonic acid on the human cardiac Kv 1.5 channel.

ABSTRACT: BACKGROUND AND PURPOSE:In human heart, the Kv 1.5 channel contributes to repolarization of atrial action potentials. This study examined the electrophysiological and molecular mechanisms underlying arachidonic acid (AA)-induced inhibition of the human Kv 1.5 (hKv 1.5) channel. EXPERIMENTAL APPROACH:Site-directed mutagenesis was conducted to mutate amino acids that reside within the pore domain of the hKv 1.5 channel. Whole-cell patch-clamp method was used to record membrane currents through wild type and mutant hKv 1.5 channels heterologously expressed in CHO cells. Computer docking simulation was conducted to predict the putative binding site(s) of AA in an open-state model of the Kv 1.5 channel. KEY RESULTS:The hKv 1.5 current was minimally affected at the onset of depolarization but was progressively reduced during depolarization by the presence of AA, suggesting that AA acts as an open-channel blocker. AA itself affected the channel at extracellular sites independently of its metabolites and signalling pathways. The blocking effect of AA was attenuated at pH?8.0 but not at pH?6.4. The blocking action of AA developed rather rapidly by co-expression of Kv ?1.3. The AA-induced block was significantly attenuated in H463C, T480A, R487V, I502A, I508A, V512A and V516A, but not in T462C, A501V and L510A mutants of the hKv 1.5 channel. Docking simulation predicted that H463, T480, R487, I508, V512 and V516 are potentially accessible for interaction with AA. CONCLUSIONS AND IMPLICATIONS:AA itself interacts with multiple amino acids located in the pore domain of the hKv 1.5 channel. These findings may provide useful information for future development of selective blockers of hKv 1.5 channels.

SUBMITTER: Bai JY

PROVIDER: S-EPMC5341216 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Putative binding sites for arachidonic acid on the human cardiac Kv 1.5 channel.

Bai Jia-Yu JY Ding Wei-Guang WG Kojima Akiko A Seto Tomoyoshi T Matsuura Hiroshi H

British journal of pharmacology 20151022 22

<h4>Background and purpose</h4>In human heart, the Kv 1.5 channel contributes to repolarization of atrial action potentials. This study examined the electrophysiological and molecular mechanisms underlying arachidonic acid (AA)-induced inhibition of the human Kv 1.5 (hKv 1.5) channel.<h4>Experimental approach</h4>Site-directed mutagenesis was conducted to mutate amino acids that reside within the pore domain of the hKv 1.5 channel. Whole-cell patch-clamp method was used to record membrane curren ...[more]

PMID: 26292661

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Project description:BackgroundUnderstanding transcriptional regulation of gene expression is one of the greatest challenges of modern molecular biology. A central role in this mechanism is played by transcription factors, which typically bind to specific, short DNA sequence motifs usually located in the upstream region of the regulated genes. We discuss here a simple and powerful approach for the ab initio identification of these cis-regulatory motifs. The method we present integrates several elements: human-mouse comparison, statistical analysis of genomic sequences and the concept of coregulation. We apply it to a complete scan of the human genome.ResultsBy using the catalogue of conserved upstream sequences collected in the CORG database we construct sets of genes sharing the same overrepresented motif (short DNA sequence) in their upstream regions both in human and in mouse. We perform this construction for all possible motifs from 5 to 8 nucleotides in length and then filter the resulting sets looking for two types of evidence of coregulation: first, we analyze the Gene Ontology annotation of the genes in the set, searching for statistically significant common annotations; second, we analyze the expression profiles of the genes in the set as measured by microarray experiments, searching for evidence of coexpression. The sets which pass one or both filters are conjectured to contain a significant fraction of coregulated genes, and the upstream motifs characterizing the sets are thus good candidates to be the binding sites of the TF's involved in such regulation. In this way we find various known motifs and also some new candidate binding sites.ConclusionWe have discussed a new integrated algorithm for the "ab initio" identification of transcription factor binding sites in the human genome. The method is based on three ingredients: comparative genomics, overrepresentation, different types of coregulation. The method is applied to a full-scan of the human genome, giving satisfactory results.

Dataset Information

Putative binding sites for arachidonic acid on the human cardiac Kv 1.5 channel.

Publications

Putative binding sites for arachidonic acid on the human cardiac Kv 1.5 channel.

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