Objective: To explore the effects of 3-phosphate dependent protein kinase 1-protein kinase B (PDK1-Akt) signaling pathway on the transcription, expression and function of cardiac hyperpolarized activated cyclic nucleotide gated 4 (HCN4) ion channels. Methods: Atrial myocytes were obtained from healthy male wild-type C57 mice and heart-specific PDK1 knockout mice (PDK1-KO) by enzymolysis. Then the atrial myocytes were divided into blank control group and PDK1-KO group. In further studies, the isolated atrial myocytes were cultured and further divided into drug control group (treated with dimethyl sulfoxide (DMSO)) and PDK1 knockdown group (treated with 1 μg/ml PDK1 short hairpin RNA (shRNA) interference plasmid), SC79 group (treated with 8 μmol/ml SC79), GSK2334470 group (treated with 10 nmol/L GSK2334470) and PDK1 knockdown+SC79 group (8 μmol/ml SC79 and 1 μg/ml PDK1 shRNA interference plasmid). Real time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of PDK1 and HCN4, Western blot was used to detect the protein expression levels of PDK1, Akt and HCN4, the whole cell patch clamp was used to detecte the current density of HCN, and immunofluorescence was used to detecte the expression of HCN4 protein on atrial cells. Results: (1) the expression levels of HCN4 mRNA (1.46±0.03 vs. 0.99±0.01, P<0.001) and protein (1.14±0.02 vs. 1.00±0.06, P=0.017) in PDK1-KO group were higher than those in blank control group. The HCN current density in PDK1-KO group was higher than that in blank control group((-17.47±2.00) pA/pF vs. (-12.15±2.25) pA/pF, P=0.038). (2) The functions of PDK1 shRNA and specific Akt agonist SC79 were verified by comparing the PDK1 knockdown group and SC79 group with the drug control group. The results showed that the expression levels of PDK1 mRNA and protein in PDK1 knockdown group were lower than those in drug control group, and the expression level of phosphorylated Akt (Thr 308) protein in SC79 group was higher than that in drug control group. (3) The expression levels of HCN4 mRNA (3.61±0.46 vs. 1.00±0.08, P<0.001) and protein (2.33±0.11 vs. 1.00±0.05, P<0.001) in GSK2334470 group were higher than those in drug control group. (4) To reduce the effect of drug-miss target, the cultured atrial myocytes were transfected with shRNA plasmid of PDK1 and intervened with SC79. The results showed that the expression of HCN4 mRNA in PDK1 knockdown group was higher than that in the drug control group (1.76±0.11 vs. 1.00±0.06, P<0.001), and PDK1 knockdown+SC79 group (1.76±0.11 vs. 1.33±0.07, P=0.003). In PDK1 knockdown+SC79 group, the mRNA expression level was also higher than that in the drug control group (1.33±0.07 vs. 1.00±0.06, P<0.001). The expression level of HCN4 protein in PDK1 knockdown group was higher than that in drug control group (1.15±0.04 vs. 1.00±0.05, P=0.003). As for the The expression level of HCN4 protein, there was no significantly statistical difference between the PDK1 knockdown+SC79 group and the drug control group (P>0.05), but PDK1 knockdown+SC79 group was lower than PDK1 knockdown group (0.95±0.01 vs. 1.15±0.04, P<0.001). In patch clamp experiments, the results showed that the HCN current density was (-13.27±1.28) pA/pF in the drug control group, (-18.76±2.03) pA/pF in the PDK1 knockdown group, (-13.50±2.58) pA/pF in the PDK1 knockdown+SC79 group; the HCN current density of PDK1 knockdown group was higher than that of drug control group (P<0.001), but there was no significant difference between PDK1 knockdown+SC79 group and drug control group (P>0.05). (5) The results of immunofluorescence showed that the brightness of green fluorescence of PDK1 knockdown group was higher than that of drug control group, indicating that the expression of HCN4 localized on cell membrane was increased. However, the green fluorescence of PDK1 knockdown+SC79 group was lighter than that of PDK1 knockdown group, suggesting that the expression of HCN4 in PDK1-knockdown cell membrane decreased after further activating Akt. Conclusion: PDK1-Akt signaling pathway is involved in the regulation of HCN4 ion channel transcription, expression and function.
Animals ; Cyclic Nucleotide-Gated Cation Channels ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism* ; Male ; Mice ; Myocytes, Cardiac/metabolism* ; Potassium Channels/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction

Neuropathic pain is a complex chronic pain state caused by the dysfunction of somatosensory nervous system, and it affects the millions of people worldwide. At present, there are very few medical treatments available for neuropathic pain management and the intolerable side effects of medications may further worsen the symptoms. Despite the presence of profound knowledge that delineates the pathophysiology and mechanisms leading to neuropathic pain, the unmet clinical needs demand more research in this field that would ultimately assist to ameliorate the pain conditions. Efforts are being made globally to explore and understand the basic molecular mechanisms responsible for somatosensory dysfunction in preclinical pain models. The present review highlights some of the novel molecular targets like D-amino acid oxidase, endoplasmic reticulum stress receptors, sigma receptors, hyperpolarization-activated cyclic nucleotide-gated cation channels, histone deacetylase, Wnt/β-catenin and Wnt/Ryk, ephrins and Eph receptor tyrosine kinase, Cdh-1 and mitochondrial ATPase that are implicated in the induction of neuropathic pain. Studies conducted on the different animal models and observed results have been summarized with an aim to facilitate the efforts made in the drug discovery. The diligent analysis and exploitation of these targets may help in the identification of some promising therapies that can better manage neuropathic pain and improve the health of patients.
Adenosine Triphosphatases ; Chronic Pain ; Cyclic Nucleotide-Gated Cation Channels ; Drug Discovery ; Endoplasmic Reticulum Stress ; Ephrins ; Histone Deacetylases ; Humans ; Models, Animal ; Nervous System ; Neuralgia* ; Oxidoreductases ; Receptors, Eph Family ; Receptors, sigma

OBJECTIVETo investigate the rebound depolarization of substantia gelatinosa (SG) neurons in rat spinal dorsal horn and explore its modulatory mechanisms to provide better insights into rebound depolarization-related diseases.

METHODSParasagittal slices of the spinal cord were prepared from 3- to 5-week-old Sprague-Dawley rats. The electrophysiologic characteristics and responses to hyperpolarization stimulation were recorded using whole-cell patch-clamp technique. The effects of hyperpolarization-activated cyclic nucleotide gated cation (HCN) channel blockers and T-type calcium channel blockers on rebound depolarization of the neurons were studied.

RESULTSA total of 63 SG neurons were recorded. Among them, 23 neurons showed no rebound depolarization, 19 neurons showed rebound depolarization without spikes, and 21 neurons showed rebound depolarization with spikes. The action potential thresholds of the neurons without rebound depolarization were significantly higher than those of the neurons with rebound depolarization and spikes (-28.7∓1.6 mV vs -36.0∓2.0 mV, P<0.05). The two HCN channel blockers CsCl and ZD7288 significantly delayed the latency of rebound depolarization with spike from 45.9∓11.6 ms to 121.6∓51.3 ms (P<0.05) and from 36.2∓10.3 ms to 73.6∓13.6 ms (P<0.05), respectively. ZD7288 also significantly prolonged the latency of rebound depolarization without spike from 71.9∓35.1 ms to 267.0∓68.8 ms (P<0.05). The T-type calcium channel blockers NiCl2 and mibefradil strongly decreased the amplitude of rebound depolarization with spike from 19.9∓6.3 mV to 9.5∓4.5 mV (P<0.05) and from 26.1∓9.4 mV to 15.5∓5.0 mV (P<0.05), respectively. Mibefradil also significantly decreased the amplitude of rebound depolarization without spike from 14.3∓3.0 mV to 7.9∓2.0 mV (P<0.05).

CONCLUSIONNearly two-thirds of the SG neurons have rebound depolarizations modulated by HCN channel and T-type calcium channel.

Action Potentials ; Animals ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, T-Type ; Cell Polarity ; Cesium ; pharmacology ; Chlorides ; pharmacology ; Cyclic Nucleotide-Gated Cation Channels ; antagonists & inhibitors ; Neurons ; cytology ; Patch-Clamp Techniques ; Pyrimidines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Dorsal Horn ; cytology ; Substantia Gelatinosa ; cytology

OBJECTIVE: To investigate the expression of hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) and connexin43 (Cx43) in the sinoatrial node of electric shock death. METHODS: As experimental group, 34 cases of electric shock death who had definite current mark evidence were selected from pathology department of Xuzhou Medical College from 2010 to 2013. As the control group, 20 cases of fatal severe craniocerebral injury in traffic accidents were chosen. The expressions of HCN4 and Cx43 in the sinoatrial node were observed by immunohistochemical technology. RESULTS: HCN4 positive cells expressed in the cell membrane and cytoplasm of the sinoatrial node. Cx43 positive cells expressed in the cell membrane and cytoplasm of T cells and myocardial cells. The expression of HCN4 was significantly higher than that of the control group (P < 0.05) and the expression of Cx43 was significantly lower than that of the control group (P < 0.05). CONCLUSION The changes of HCN4 and Cx43 expressions in the sinoatrial node illustrate electric shock death might be related to the abnormalities of cardiac electrophysiology and conduction.
Connexin 43/metabolism* ; Cyclic Nucleotide-Gated Cation Channels ; Heart Rate ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism* ; Immunohistochemistry/methods* ; Myocardium/metabolism* ; Myocytes, Cardiac ; Sinoatrial Node/physiopathology*

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, distributing in a variety of tissues, especially in excitable cells such as heart cells and many kinds of neurons, have an important role in the modulation of heart rate and neuronal excitability. Different from typical voltage-gated sodium channels and potassium channels, HCN channels were evoked inward currents when the cell was hyperpolarized. More and more recent studies have disclosed that HCN channels play important roles in the nervous system, which were linked with its special electrophysiological features as well as its regulatory effect on the cellular membrane excitability. HCN channels could be modulated by many factors including both extracellular molecules and intracellular signaling cascades, which made its functions complicated in the different condition. Based on its role, HCN channels are presumed to be a promising target for chronic pain and brain disorders. In this paper, we will focus on the advancement of roles of HCN channels in the neural system as well as its complex modulator factors.
Cyclic Nucleotide-Gated Cation Channels ; physiology ; Humans ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; physiology ; Membrane Potentials ; Neurons ; physiology ; Potassium Channels ; physiology

The cyclic nucleotide-gated (CNG) channel is a nonselective cation channel and one of the main entrances of Ca2+ influxion into cells. CNG channels are opened by direct binding of cyclic nucleotides. Six different genes encode the CNG protein, 4 A subunits and 2 B subunits. The activity of CNG channels can be regulated by Ca2+/Ca(2+)-binding proteins (CaM) and phosphorylation/dephosphorylation. Recently, extensive attention has been drawn to the researches on CNG channels in the reproductive system, and many studies show that CNG channels play a pivotal role in sperm motility, capacitation and acrosome reaction. This article focuses on the relationship of CNG channels with sperm function.
Animals ; Calcium ; metabolism ; Cyclic Nucleotide-Gated Cation Channels ; physiology ; Humans ; Male ; Spermatozoa ; metabolism ; physiology

To investigate the effects of novel intravenous general anesthetic propofol on membrane electrophysiological characteristics and action potential (AP) of the supraoptic nucleus (SON) neurons and possible ionic mechanisms, intracellular recordings were conducted in SON neurons from the coronal hypothalamic slice preparation of adult male Sprague Dawley (SD) rats. The results showed that bath application of 0.1 mmol/L propofol induced a significant decline in resting potential (P < 0.01), and higher concentrations of propofol (0.3 and 1.0 mmol/L) decreased time constant and slope resistance of cell membrane (P < 0.01). Under the hyperpolarizing current pulses exceeding 0.5 nA, an anomalous rectification was induced by hyperpolarization-activated cation channel (I(h) channel) in 11 out of 18 tested SON neurons. Bath of propofol reversibly decreased the anomalous rectification. Moreover, 0.1 mmol/L propofol elevated threshold level (P < 0.01) and decreased Max L. slope (P < 0.05) of the spike potential in SON neurons. Interestingly, 0.3 and 1.0 mmol/L propofol nullified APs in 6% (1/18) and 71% (12/17) tested SON neurons, respectively. In the SON neurons where APs were not nullified, propofol (0.3 mmol/L) decreased the amplitude of spike potential (P < 0.05). The higher concentrations of propofol (0.3 and 1.0 mmol/L) decreased firing frequencies evoked by depolarizing current pulses (0.1-0.7 nA), and shifted the current intensity-firing frequency relation curves downward and to the right. These results suggest that propofol decreases the excitability of SON neurons by inhibiting I(h) and sodium channels.
Action Potentials ; drug effects ; Anesthetics, Intravenous ; pharmacology ; Animals ; Cyclic Nucleotide-Gated Cation Channels ; antagonists & inhibitors ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; Hypothalamus ; drug effects ; physiology ; In Vitro Techniques ; Male ; Potassium Channels ; Propofol ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sodium Channel Blockers ; pharmacology ; Supraoptic Nucleus ; drug effects ; physiology

OBJECTIVETo investigate the transcriptional regulation of pacemaker channel I(f) mediated by vasoactive peptide endothelin-1 (ET-1) in neonatal rat ventricular myocytes and its mechanism.

METHODSNeonatal rat ventricular myocytes were enzymatically isolated. I(f) current was recorded using the whole-cell patch-clamp technique. The expression of hyperpolarization-activated cyclic nucleotide-gated channel (HCN) isoforms HCN2 and HCN4 were measured by quantitative RT-PCR.

RESULTSET-1 increased the expression of HCN2 and HCN4 mRNA in a dose- and time-dependent manner. These effects were blocked by specific ETA receptor antagonist BQ-123 but not the ETB receptor antagonist BQ-788. The effects of ET-1 on HCN2 and HCN4 mRNA expression were not affected by the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB-203580).

CONCLUSIONThese findings indicate that ET-1 stimulates the expression of pacemaker channel I(f) in cardiomyocytes via ETA receptor through a p38 MAPK-independent signaling pathway, which might be linked to the intrinsic arrhythmogenic potential of ET-1.

Animals ; Animals, Newborn ; Cyclic Nucleotide-Gated Cation Channels ; drug effects ; Endothelin-1 ; metabolism ; Imidazoles ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; Oligopeptides ; pharmacology ; Patch-Clamp Techniques ; Piperidines ; pharmacology ; Pyridines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

Ras proteins are signal-transducing GTPases that cycle between inactive GDP-bound and active GTP-bound forms. Ras is a prolific signaling molecule interacting with a spectrum of effector molecules and acting through more than one signaling pathway. The Ras-effector proteins contain a Ras-associating (RA) domain through which these associate with Ras in a GTP-dependent manner. The RA domain is highly conserved among the members of the growth factor receptor-bound (Grb) 7 family of proteins which includes Grb7, Grb10 and Grb14. Our laboratory has reported an unusual observation that RA domain of Grb14 binds to the C-terminal nucleotide binding site of cyclic nucleotide gated channel (CTRCNGA1) and inhibits the channel activity. Molecular modeling of the CTR-CNGA1 displays 50%-70% tertiary structural similarity towards Ras proteins. We named this region as Ras-like domain (RLD). The interaction between RA-Grb14 and RLD-CNGA1 is mediated through a simple protein-protein interaction temporally and spatially regulated by light and cGMP. It is interesting to note that Grb14 binds to GTPase-mutant Rab5, a Ras-related small GTPase whereas Grb10 binds only to GTP-bound form of active Rab5 but not to GTPase-defective mutant Rab5. These results suggest that Grb14 might have been evolved later in the evolution that binds to both Ras and nucleotide binding proteins such as CNGA1. Our studies also suggest that eukaryotic CNG channels could be evolved through a gene fusion between prokaryotic ion channels and cyclic nucleotide binding proteins, both of which might have undergone several sequence variations for functional adaptation during evolution.
Amino Acid Sequence ; Animals ; Cattle ; Cell Membrane ; metabolism ; radiation effects ; Conserved Sequence ; Cyclic Nucleotide-Gated Cation Channels ; genetics ; metabolism ; Evolution, Molecular ; Female ; GRB7 Adaptor Protein ; chemistry ; genetics ; metabolism ; HEK293 Cells ; Humans ; Light ; Male ; Models, Molecular ; Molecular Sequence Data ; Protein Binding ; radiation effects ; Protein Structure, Tertiary ; Protein Transport ; Rats ; Rod Cell Outer Segment ; radiation effects ; rab5 GTP-Binding Proteins ; metabolism ; ras Proteins ; metabolism

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide-gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In the current study we show that this RA domain-mediated inhibition of rod CNG channel is electrostatic in nature. Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg(559) through a negatively charged β-turn at its RA domain. Moreover, the three Glu residues (180-182) in Grb14 are absolutely critical for electrostatic interaction with the cGMP binding pocket and resultant inhibition. Our study also demonstrates that substitution of Lys140 for Ala or in combination with polyglutamte mutants of Grb14 results in a significantly reduced binding with CNGA1. These results suggest that in addition to Glu(180-182) and Lys(140), other residues in Grb14 may be involved in the electrostatic interaction with CNGA1. The RA domain is highly conserved among the members of Grb7 family of proteins, which includes Grb7, Grb10 and Grb14. Further, only Grb14 is able to modulate the channel activity, but not Grb7 and Grb10. All together, it suggests the existence of a divergence in RA domains among the members of the Grb7 family.
Animals ; Calcium ; metabolism ; Cattle ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide-Gated Cation Channels ; chemistry ; genetics ; metabolism ; GRB10 Adaptor Protein ; genetics ; metabolism ; Humans ; Kidney ; cytology ; metabolism ; Models, Molecular ; Protein Conformation ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Static Electricity