Xiaona Du

The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl– channels

Bradykinin (BK) is an inflammatory mediator and one of the most potent endogenous pain-inducing substances. When released at sites of tissue damage or inflammation, or applied exogenously, BK produces acute spontaneous pain and causes hyperalgesia (increased sensitivity to potentially painful stimuli). The mechanisms underlying spontaneous pain induced by BK are poorly understood. Here we report that in small nociceptive neurons from rat dorsal root ganglia, BK, acting through its B2 receptors, PLC, and release of calcium from intracellular stores, robustly inhibits M-type K+ channels and opens Ca2+-activated Cl- channels (CaCCs) encoded by Tmem16a (also known as Ano1). Summation of these two effects accounted for the depolarization and increase in AP firing induced by BK in DRG neurons. Local injection of inhibitors of CaCC and specific M-channel openers both strongly attenuated the nociceptive effect of local injections of BK in rats. These results provide a framework for understanding spontaneous inflammatory pain and may suggest new drug targets for treatment of such pain.

Characteristic Interactions with Phosphatidylinositol 4,5-Bisphosphate Determine Regulation of Kir Channels by Diverse Modulators

The activity of specific inwardly rectifying potassium (Kir) channels is regulated by any of a number of different modulators, such as protein kinase C, Gq -coupled receptor stimulation, pH, intracellular Mg2+ or the βγ-subunits of G proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) is an essential factor for maintenance of the activity of all Kir channels. Here, we demonstrate that the strength of channel-PIP2 interactions determines the sensitivity of Kir channels to regulation by the various modulators. Furthermore, our results suggest that differences among Kir channels in their specific regulation by a given modulator may reflect differences in their apparent affinity of interactions with PIP2.

EGF enhances the migration of cancer cells by up-regulation of TRPM7.

Ion channels involved in the migration of tumor cells that is required for their invasion and metastasis. In this paper, we describe the interaction of TRPM7 channel and epidermal growth factor (EGF), an important player in cancer development in the migration of lung cancer cells. The TRPM7 currents in A549 cells were first characterized by means of electrophysiology, pharmacology and RNA interference. Removing Ca(2+) from the extracellular solution not only potentiated a large inward current, but also abolished the outward rectification. 200μM 2-APB inhibited the outward and the inward TRPM7 currents and at the same time restored the property of outward rectification. EGF greatly enhanced the migration of A549 cells, and also markedly up-regulated the membrane protein expression of TRPM7 and the amplitude of TRPM7 currents. Depressing the function of TRPM7 with RNA interference or pharmacological agents not only reversed the EGF-enhanced migration of A549 cells but also inhibited the basal migration of A549 cells in the absence of EGF. Thus it seems that TRPM7 plays a pivotal role in the migration of A549 cells induced by EGF and thus could be a potential therapeutic target in lung cancers.

Potassium Channels in Peripheral Pain Pathways: Expression, Function and Therapeutic Potential

Electrical excitation of peripheral somatosensory nerves is a first step in generation of most pain signals in mammalian nervous system. Such excitation is controlled by an intricate set of ion channels that are coordinated to produce a degree of excitation that is proportional to the strength of the external stimulation. However, in many disease states this coordination is disrupted resulting in deregulated peripheral excitability which, in turn, may underpin pathological pain states (i.e. migraine, neuralgia, neuropathic and inflammatory pains). One of the major groups of ion channels that are essential for controlling neuronal excitability is potassium channel family and, hereby, the focus of this review is on the K+ channels in peripheral pain pathways. The aim of the review is threefold. First, we will discuss current evidence for the expression and functional role of various K+ channels in peripheral nociceptive fibres. Second, we will consider a hypothesis suggesting that reduced functional activity of K+ channels within peripheral nociceptive pathways is a general feature of many types of pain. Third, we will evaluate the perspectives of pharmacological enhancement of K+ channels in nociceptive pathways as a strategy for new analgesic drug design.

Control of somatic membrane potential in nociceptive neurons and its implications for peripheral nociceptive transmission

&NA; We identified major ion channels influencing the resting membrane potential of nociceptive sensory neurons and demonstrated that changes of somatic/perisomatic membrane potential of these neurons can strongly influence peripheral nociceptive transmission. &NA; Peripheral sensory ganglia contain somata of afferent fibres conveying somatosensory inputs to the central nervous system. Growing evidence suggests that the somatic/perisomatic region of sensory neurons can influence peripheral sensory transmission. Control of resting membrane potential (Erest) is an important mechanism regulating excitability, but surprisingly little is known about how Erest is regulated in sensory neuron somata or how changes in somatic/perisomatic Erest affect peripheral sensory transmission. We first evaluated the influence of several major ion channels on Erest in cultured small‐diameter, mostly capsaicin‐sensitive (presumed nociceptive) dorsal root ganglion (DRG) neurons. The strongest and most prevalent effect on Erest was achieved by modulating M channels, K2P and 4‐aminopiridine‐sensitive KV channels, while hyperpolarization‐activated cyclic nucleotide‐gated, voltage‐gated Na+, and T‐type Ca2+ channels to a lesser extent also contributed to Erest. Second, we investigated how varying somatic/perisomatic membrane potential, by manipulating ion channels of sensory neurons within the DRG, affected peripheral nociceptive transmission in vivo. Acute focal application of M or KATP channel enhancers or a hyperpolarization‐activated cyclic nucleotide‐gated channel blocker to L5 DRG in vivo significantly alleviated pain induced by hind paw injection of bradykinin. Finally, we show with computational modelling how somatic/perisomatic hyperpolarization, in concert with the low‐pass filtering properties of the t‐junction within the DRG, can interfere with action potential propagation. Our study deciphers a complement of ion channels that sets the somatic Erest of nociceptive neurons and provides strong evidence for a robust filtering role of the somatic and perisomatic compartments of peripheral nociceptive neuron.

Design, synthesis and biological activity of pyrazolo(1,5-a)pyrimidin-7(4H)-ones as novel Kv7/KCNQ potassium channel activators

Voltage-gated Kv7/KCNQ/M-potassium channels play a pivotal role in controlling neuronal excitability. Genetic reduction of KCNQ channel activity as a result of mutations causes various human diseases such as epilepsy and arrhythmia. Therefore, discovery of small molecules that activate KCNQ channels is an important strategy for clinical intervention of membrane excitability related disorders. In this study, a series of pyrazolo[1,5-a]pyrimidin-7(4H)-ones (PPOs) have been found to be novel activators (openers) of KCNQ2/3 potassium channels through high-throughput screening by using atomic absorption rubidium efflux assay. Based on structure-activity relationship (SAR), the substituted PPOs have been optimized. The 5-(2,6-dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one (17) was identified as a novel, potent, and selective KCNQ2/3 potassium channel opener by patch-clamp recording assay.

Activation of Ca2+-activated Cl− channel ANO1 by localized Ca2+ signals

Ca2+‐activated chloride channels (CaCCs) regulate numerous physiological processes including epithelial transport, smooth muscle contraction and sensory processing. Anoctamin‐1 (ANO1, TMEM16A) is a principal CaCC subunit in many cell types, yet our understanding of the mechanisms of ANO1 activation and regulation are only beginning to emerge. Ca2+ sensitivity of ANO1 is rather low and at negative membrane potentials the channel requires several micromoles of intracellular Ca2+ for activation. However, global Ca2+ levels in cells rarely reach such levels and, therefore, there must be mechanisms that focus intracellular Ca2+ transients towards the ANO1 channels. Recent findings indeed indicate that ANO1 channels often co‐localize with sources of intracellular Ca2+ signals. Interestingly, it appears that in many cell types ANO1 is particularly tightly coupled to the Ca2+ release sites of the intracellular Ca2+ stores. Such preferential coupling may represent a general mechanism of ANO1 activation in native tissues.

Depolarization Increases Phosphatidylinositol (PI) 4,5-Bisphosphate Level and KCNQ Currents through PI 4-Kinase Mechanisms

A growing body of evidence shows that membrane phosphatidylinositol 4,5-bisphosphates (PtdIns(4,5)P2, PIP2) play an important role in cell signaling. The presence of PIP2 is fundamentally important for maintaining the functions of a large number of ion channels and transporters, and for other cell processes such as vesicle trafficking, mobility, and endo- and exocytosis. PIP2 levels in the membrane are dynamically modulated, which is an important signaling mechanism for modulation of PIP2-dependent cellular processes. In this study, we describe a novel mechanism of membrane PIP2 modulation. Membrane depolarization induces an elevation in membrane PIP2, and subsequently increases functions of PIP2-sensitive KCNQ potassium channels expressed in Xenopus oocytes. Further evidence suggests that the depolarization-induced elevation of membrane PIP2 occurs through increased activity of PI4 kinase. With increased recognition of the importance of PIP2 in cell function, the effect of membrane depolarization in PIP2 metabolism is destined to have important physiological implications.

Local GABAergic signaling within sensory ganglia controls peripheral nociceptive transmission

The integration of somatosensory information is generally assumed to be a function of the central nervous system (CNS). Here we describe fully functional GABAergic communication within rodent peripheral sensory ganglia and show that it can modulate transmission of pain-related signals from the peripheral sensory nerves to the CNS. We found that sensory neurons express major proteins necessary for GABA synthesis and release and that sensory neurons released GABA in response to depolarization. In vivo focal infusion of GABA or GABA reuptake inhibitor to sensory ganglia dramatically reduced acute peripherally induced nociception and alleviated neuropathic and inflammatory pain. In addition, focal application of GABA receptor antagonists to sensory ganglia triggered or exacerbated peripherally induced nociception. We also demonstrated that chemogenetic or optogenetic depolarization of GABAergic dorsal root ganglion neurons in vivo reduced acute and chronic peripherally induced nociception. Mechanistically, GABA depolarized the majority of sensory neuron somata, yet produced a net inhibitory effect on the nociceptive transmission due to the filtering effect at nociceptive fiber T-junctions. Our findings indicate that peripheral somatosensory ganglia represent a hitherto underappreciated site of somatosensory signal integration and offer a potential target for therapeutic intervention.

GABAB receptors inhibit low-voltage activated and high-voltage activated Ca2+ channels in sensory neurons via distinct mechanisms

Growing evidence suggests that mammalian peripheral somatosensory neurons express functional receptors for gamma-aminobutyric acid, GABAA and GABAB. Moreover, local release of GABA by pain-sensing (nociceptive) nerve fibres has also been suggested. Yet, the functional significance of GABA receptor triggering in nociceptive neurons is not fully understood. Here we used patch-clamp recordings from small-diameter cultured DRG neurons to investigate effects of GABAB receptor agonist baclofen on voltage-gated Ca(2+) currents. We found that baclofen inhibited both low-voltage activated (LVA, T-type) and high-voltage activated (HVA) Ca(2+) currents in a proportion of DRG neurons by 22% and 32% respectively; both effects were sensitive to Gi/o inhibitor pertussis toxin. Inhibitory effect of baclofen on both current types was about twice less efficacious as compared to that of the μ-opioid receptor agonist DAMGO. Surprisingly, only HVA but not LVA current modulation by baclofen was partially prevented by G protein inhibitor GDP-β-S. In contrast, only LVA but not HVA current modulation was reversed by the application of a reducing agent dithiothreitol (DTT). Inhibition of T-type Ca(2+) current by baclofen and the recovery of such inhibition by DTT were successfully reconstituted in the expression system. Our data suggest that inhibition of LVA current in DRG neurons by baclofen is partially mediated by an unconventional signaling pathway that involves a redox mechanism. These findings reinforce the idea of targeting peripheral GABA receptors for pain relief.