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Conformational Flip of Nonactivated HCN2 Channel Subunits Evoked by Cyclic Nucleotides.

ABSTRACT: Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric proteins that evoke electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are activated by hyperpolarizing voltage but are also receptors for the intracellular ligand adenosine-3',5'-cyclic monophosphate (cAMP) that enhances activation but is unable to activate the channels alone. Using fcAMP, a fluorescent derivative of cAMP, we analyzed the effect of ligand binding on HCN2 channels not preactivated by voltage. We identified a conformational flip of the channel as an intermediate state following the ligand binding and quantified it kinetically. Globally fitting the time courses of ligand binding and unbinding revealed modest cooperativity among the subunits in the conformational flip. The intensity of this cooperativity, however, was only moderate compared to channels preactivated by hyperpolarizing voltage. These data provide kinetic information about conformational changes proceeding in nonactivated HCN2 channels when cAMP binds. Moreover, our approach bears potential for analyzing the function of any other membrane receptor if a potent fluorescent ligand is available.

SUBMITTER: Thon S

PROVIDER: S-EPMC4675818 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Conformational Flip of Nonactivated HCN2 Channel Subunits Evoked by Cyclic Nucleotides.

Thon Susanne S Schulz Eckhard E Kusch Jana J Benndorf Klaus K

Biophysical journal 20151201 11

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric proteins that evoke electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are activated by hyperpolarizing voltage but are also receptors for the intracellular ligand adenosine-3',5'-cyclic monophosphate (cAMP) that enhances activation but is unable to activate the channels alone. Using fcAMP, a fluorescent derivative of cAMP, we analyzed the effect of ligand binding on HCN2 channels no ...[more]

PMID: 26636938

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Project description:BackgroundClinical and animal studies demonstrated that neuroinflammation from anesthesia (sevoflurane) is the main contributor to cause perioperative neurocognitive disorders (PND). Recently, it was reported that microglia respond to hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which was the target of sevoflurane. Whether HCN channels are involved in the induction of neuroinflammation after sevoflurane exposure is still unclear.ResultsSevoflurane exposure had increased cognitive dysfunction and anxiety-like behaviors in rats. Rats inhaled with sevoflurane had activated microglia and increased neuroinflammation (IL-1β, IL-6, and TNF-α) in the hippocampus. RNA sequencing identified 132 DEGs (86 up-regulated and 46 down-regulated DEGs [differentially expressed genes]) in the hippocampus of PND rats. RNA-sequencing also uncovered that sevoflurane exposure down-regulates HCN2 expression. Pathway and process enrichment analysis suggests DEGs are mainly enriched in regulation of system process, positive regulation of glutamate secretion, secretion, regulation of synaptic transmission, regulation of nervous system process, behavior, negative regulation of sodium ion transport, and learning or memory. We validated that sevoflurane exposure can down-regulate the levels of PEX5R/Trip8b (an interaction partner and auxiliary subunit of HCN channels) and HCN1-4 channels in the hippocampus of PND rats. We used immunofluorescence staining to identify that HCN2 co-labels with neurons (Neun), astrocytes (GFAP), and microglia (iba1). We observed that the co-labeling of HCN2 with neurons or microglia decreased in the hippocampus and cortex after sevoflurane exposure. Blocking HCN2 by ZD7288 treatment further activated microglia and aggravated sevoflurane exposure-induced anxiety-like behavior, cognitive impairment, and neuroinflammation.ConclusionsWe concluded that sevoflurane exposure can induce an increased level of neuroinflammation, microglial activation, cognitive dysfunction, and anxiety-like behaviors in rats. HCN2 channel, as the target of sevoflurane action, mediates this process. HCN2 might be a target for the treatment and prevention of sevoflurane-induced PND.

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Conformational Flip of Nonactivated HCN2 Channel Subunits Evoked by Cyclic Nucleotides.

Publications

Conformational Flip of Nonactivated HCN2 Channel Subunits Evoked by Cyclic Nucleotides.

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