Zhanfeng Jia

NGF Inhibits M/KCNQ Currents and Selectively Alters Neuronal Excitability in Subsets of Sympathetic Neurons Depending on their M/KCNQ Current Background

M/KCNQ currents play a critical role in the determination of neuronal excitability. Many neurotransmitters and peptides modulate M/KCNQ current and neuronal excitability through their G protein–coupled receptors. Nerve growth factor (NGF) activates its receptor, a member of receptor tyrosine kinase (RTK) superfamily, and crucially modulates neuronal cell survival, proliferation, and differentiation. In this study, we studied the effect of NGF on the neuronal (rat superior cervical ganglion, SCG) M/KCNQ currents and excitability. As reported before, subpopulation SCG neurons with distinct firing properties could be classified into tonic, phasic-1, and phasic-2 neurons. NGF inhibited M/KCNQ currents by similar proportion in all three classes of SCG neurons but increased the excitability only significantly in tonic SCG neurons. The effect of NGF on excitability correlated with a smaller M-current density in tonic neurons. The present study indicates that NGF is an M/KCNQ channel modulator and the characteristic modulation of the neuronal excitability by NGF may have important physiological implications.

Activation of Epidermal Growth Factor Receptor Inhibits KCNQ2/3 Current through Two Distinct Pathways: Membrane PtdIns(4,5)P2 Hydrolysis and Channel Phosphorylation

KCNQ2/3 currents are the molecular basis of the neuronal M currents that play a critical role in neuron excitability. Many neurotransmitters modulate M/KCNQ currents through their G-protein-coupled receptors. Membrane PtdIns(4,5)P2 hydrolysis and channel phosphorylation are two mechanisms that have been proposed for modulation of KCNQ2/3 currents. In this study, we studied regulation of KCNQ2/3 currents by the epidermal growth factor (EGF) receptor, a member of another family of membrane receptors, receptor tyrosine kinases. We demonstrate here that EGF induces biphasic inhibition of KCNQ2/3 currents in human embryonic kidney 293 cells and in rat superior cervical ganglia neurons, an initial fast inhibition and a later slow inhibition. Additional studies indicate that the early and late inhibitions resulted from PtdIns(4,5)P2 hydrolysis and tyrosine phosphorylation, respectively. We further demonstrate that these two processes are mutually dependent. This study indicates that EGF is a potent modulator of M/KCNQ currents and provides a new dimension to the understanding of the modulation of these channels.

Multiple target of hAmylin on rat primary hippocampal neurons

&NA; Alzheimers disease (AD) and type II diabetes mellitus (DM2) are the most common aging‐related diseases and are characterized by &bgr;‐amyloid and amylin accumulation, respectively. Multiple studies have indicated a strong correlation between these two diseases. Amylin oligomerization in the brain appears to be a novel risk factor for developing AD. Although amylin aggregation has been demonstrated to induce cytotoxicity in neurons through altering Ca2+ homeostasis, the underlying mechanisms have not been fully explored. In this study, we investigated the effects of amylin on rat hippocampal neurons using calcium imaging and whole‐cell patch clamp recordings. We demonstrated that the amylin receptor antagonist AC187 abolished the Ca2+ response induced by low concentrations of human amylin (hAmylin). However, the Ca2+ response induced by higher concentrations of hAmylin was independent of the amylin receptor. This effect was dependent on extracellular Ca2+. Additionally, blockade of L‐type Ca2+ channels partially reduced hAmylin‐induced Ca2+ response. In whole‐cell recordings, hAmylin depolarized the membrane potential. Moreover, application of the transient receptor potential (TRP) channel antagonist ruthenium red (RR) attenuated the hAmylin‐induced increase in Ca2+. Single‐cell RT‐PCR demonstrated that transient receptor potential vanilloid 4 (TRPV4) mRNA was expressed in most of the hAmylin‐responsive neurons. In addition, selective knockdown of TRPV4 channels inhibited the hAmylin‐evoked Ca2+ response. These results indicated that different concentrations of hAmylin act through different pathways. The amylin receptor mediates the excitatory effects of low concentrations of hAmylin. In contrast, for high concentrations of hAmylin, hAmylin aggregates precipitated on the neuronal membrane, activated TRPV4 channels and subsequently triggered membrane voltage‐gated calcium channel opening followed by membrane depolarization. Therefore, our data suggest that TRPV4 is a key molecular mediator for the cytotoxic effects of hAmylin on hippocampal neurons. HighlightsHigh and low concentration of hAmylin acted on the hippocampal neurons through different mechanisms.Action of low concentration hAmylin depending on Amylin receptor whereas high concentration hAmylin via hAmylin oligomers.TRPV4 is a key molecular target mediating cytotoxic effects of hAmylin oligomers on hippocampal neurons.

Arachidonic Acid Activates Kir2.3 Channels by Enhancing Channel-Phosphatidyl-inositol 4,5-bisphosphate Interactions

Kir2.0 channels play a significant role in setting the resting membrane potential, modulating action potential wave form, and buffering extracellular potassium. One member of this family, Kir2.3, is highly expressed in the heart and brain and is modulated by a variety of factors, including arachidonic acid (AA). Using two-electrode voltage clamp and inside-out patch clamp recordings from Xenopus laevis oocytes expressing Kir2.3 channels, we found that AA selectively activated Kir2.3 channels with an EC50 of 0.59 μM and that this activation required phosphatidyl inositol 4,5-bisphosphate (PIP2). We found that AA activated Kir2.3 by enhancing channel-PIP2 interactions as demonstrated by a shift in PIP2 activation curve. EC50 for channel activation by PIP2 were 36 and 12 μM in the absence and presence of AA, respectively. A single point mutation on the channel C terminus that enhanced basal channel-PIP2 interactions reduced the sensitivity of the channel to AA. Effects of AA are mediated through cytoplasmic sites on the channel by increasing the open probability, mainly due to more frequent bursts of opening in the presence of PIP2. Therefore, enhanced interaction with PIP2 is the molecular mechanism for Kir2.3 channel activation by AA.

Agonist-Dependent Potentiation of Vanilloid Receptor Transient Receptor Potential Vanilloid Type 1 Function by Stilbene Derivatives

Transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel activated by capsaicin, low pH, and noxious heat and plays a key role in nociception. Understanding mechanisms for functional modulation of TRPV1 has important implications. One characteristic of TRPV1 is that channel activity induced by either capsaicin or other activators can be sensitized or modulated by factors involving different cell signaling mechanisms. In this study, we describe a novel mechanism for the modulation of TRPV1 function: TRPV1 function is modulated by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and its analogs. We found that, in rat dorsal root ganglion neurons, although DIDS did not induce the activation of TRPV1 per se but drastically increased the TRPV1 currents induced by either capsaicin or low pH. DIDS also blocked the tachyphylaxis of the low pH-induced TRPV1 currents. 4-Acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), a DIDS analog, failed to enhance the capsaicin-evoked TRPV1 current but increased the low pH-evoked TRPV1 currents, with an effect comparable with that of DIDS. SITS also blocked the low pH-induced tachyphylaxis. DIDS also potentiated the currents of TRPV1 channels expressed in human embryonic kidney 293 cells, with an effect of left-shifting the concentration-response curve of the capsaicin-induced TRPV1 currents. This study demonstrates that DIDS and SITS, traditionally used chloride channel blockers, can modify TRPV1 channel function in an agonist-dependent manner. The results provide new input for understanding TRPV1 modulation and developing new modulators of TRPV1 function.

Tannic acid modulates excitability of sensory neurons and nociceptive behavior and the Ionic mechanism.

M/Kv7 K(+) channels, Ca(2+)-activated Cl(-) channels (CaCCs) and voltage gated Na(+) channels expressed in dorsal root ganglia (DRG) play an important role in nociception. Tannic acid has been proposed to be involved in multiple beneficial health effects; tannic acid has also been described to be analgesic. However the underlying mechanism is unknown. In this study, we investigated the effects of tannic acid on M/Kv7 K(+), Na(+) currents and CaCCs, and the effects on bradykinin-induced nociceptive behavior. A perforated patch technique was used. The bradykinin-induced rat pain model was used to assess the analgesic effect of tannic acid. We demonstrated that tannic acid enhanced M/Kv7 K(+) currents but inhibited bradykinin-induced activation of CaCC/TMEM16A currents in rat small DRG neurons. Tannic acid potentiated Kv7.2/7.3 and Kv7.2 currents expressed in HEK293B cells, with an EC50 of 7.38 and 5.40 µM, respectively. Tannic acid inhibited TTX-sensitive and TTX-insensitive currents of small DRG neurons with IC50 of 5.25 and 8.43 µM, respectively. Tannic acid also potently suppressed the excitability of small DRG neurons. Furthermore, tannic acid greatly reduced bradykinin-induced pain behavior of rats. This study thus demonstrates that tannic acid is an activator of M/Kv7 K(+) and an inhibitor of voltage-gated Na(+) channels and CaCC/TMEM16A, which may underlie its inhibitory effects on excitability of DRG neurons and its analgesic effect. Tannic acid could be a useful agent in treatment of inflammatory pain conditions such as osteoarthritis, rheumatic arthritis and burn pain.

Selective activation of vascular Kv7.4/Kv7.5 K+ channels by fasudil contributes to its vasorelaxant effect

Kv7 (Kv7.1–7.5) channels play an important role in the regulation of neuronal excitability and the cardiac action potential. Growing evidence suggests Kv7.4/Kv7.5 channels play a crucial role in regulating vascular smooth muscle contractility. Most of the reported Kv7 openers have shown poor selectivity across these five subtypes. In this study, fasudil – a drug used for cerebral vasospasm – has been found to be a selective opener of Kv7.4/Kv7.5 channels.

Characterization and structure-activity relationship of natural flavonoids as hERG K+ channel modulators

Objectives: Flavonoids are present in varying concentrations in plant foods and have been reported to have numerous pharmacological activities, such as anti‐cancer, antioxidant, anti‐inflammatory, hepatoprotective, and vasodilator effects. We found that quercetin, fisetin, and some related flavonoid derivatives could inhibit human ether‐à‐go‐go‐related gene (hERG) K+ channels. Key findings: In this study, we tested the effects of a series of flavonoids on the hERG K+ channel expressed in HEK293 cells. For the first time, we demonstrate that quercetin and fisetin (Fise) are potent hERG current blockers. The 50% inhibiting concentration (IC50) and maximum efficacy (Emax) of quercetin were 11.8 ± 0.9 &mgr;M and 82 ± 2%, while those of fisetin were 38.4 ± 6 &mgr;M and 100 ± 6%, respectively. Luteolin (Lute) was a less potent inhibitor of hERG current (48 ± 1% at 100 &mgr;M). Galangin, kaempferol, and isorhamnetin (100 &mgr;M) showed weaker activity on the hERG currents. Conclusion: These results suggest that quercetin, fisetin, and luteolin are potent hERG K+ channel inhibitors and reveal the structure‐activity relationship of natural flavonoids. HighlightsFlavonoid derivatives quercetin, fisetin, and luteolin are potent hERG K+ channel inhibitors.The present work reveal the structure‐activity relationship of natural flavonoids.The results also provide new mechanistic evidence for understanding the effects of these natural products.

Inhibition of transmembrane member 16A calcium-activated chloride channels by natural flavonoids contributes to flavonoid anticancer effects.

Natural flavonoids are ubiquitous in dietary plants and vegetables and have been proposed to have antiviral, antioxidant, cardiovascular protective and anticancer effects. Transmembrane member 16A (TMEM16A)‐encoded Ca2+‐activated Cl− channels play a variety of physiological roles in many organs and tissues. Overexpression of TMEM16A is also believed to be associated with cancer progression. Therefore, inhibition of TMEM16A current may be a potential target for cancer therapy. In this study, we screened a broad spectrum of flavonoids for their inhibitory activities on TMEM16A currents.

Inhibition of TMEM16A calcium‐activated chloride channels by natural flavonoids contributes to flavonoid anticancer effects

Natural flavonoids are ubiquitous in dietary plants and vegetables and have been proposed to have antiviral, antioxidant, cardiovascular protective and anticancer effects. Transmembrane member 16A (TMEM16A)‐encoded Ca2+‐activated Cl− channels play a variety of physiological roles in many organs and tissues. Overexpression of TMEM16A is also believed to be associated with cancer progression. Therefore, inhibition of TMEM16A current may be a potential target for cancer therapy. In this study, we screened a broad spectrum of flavonoids for their inhibitory activities on TMEM16A currents.