Calcium-permeable acid-sensing ion channel in nociceptive plasticity: A new target for pain control

The development of chronic pain involves increased sensitivity of peripheral nociceptors and elevated neuronal activity in many regions of the central nervous system. Much of these changes are caused by the amplification of nociceptive signals resulting from the modulation and altered expression of specific ion channels and receptors in the central and peripheral nervous system. Understanding the processes by which these ion channels and receptors are regulated and how these mechanisms malfunction may lead to new treatments for chronic pain. Here we review the contribution of the Ca²⁺-permeable acid-sensing ion channel (ASIC_Ca) in the development and persistence of chronic pain, and the potential underlying mechanisms. Accumulating evidence suggests that ASIC_Ca represents an attractive new target for developing effective therapies for chronic pain.     

Targeting of CaV3.2 T-type calcium channels in peripheral sensory neurons for the treatment of painful diabetic neuropathy

Pain-sensing sensory neurons (nociceptors) of the dorsal root ganglion (DRG) can become sensitized (hyperexcitable) in response to pathological conditions such as diabetes, which in turn may lead to the development of painful peripheral diabetic neuropathy (PDN). Because of insufficient knowledge about the mechanisms for this hypersensitization, current treatment for painful PDN has been limited to somewhat nonspecific systemic drugs having significant side effects or potential for abuse. Recent studies have established that the Ca_V3.2 isoform of T-channels makes a previously unrecognized contribution to sensitization of pain responses by enhancing excitability of nociceptors in animal models of type 1 and type 2 PDN. Furthermore, it has been reported that the glycosylation inhibitor neuraminidase can inhibit the native and recombinant Ca_V3.2 T-currents in vitro and completely reverse mechanical and thermal hyperalgesia in diabetic animals with PDN in vivo. Understanding details of posttranslational regulation of nociceptive channel activity via glycosylation may facilitate development of novel therapies for treatment of painful PDN. Pharmacological targeting the specific pathogenic mechanism rather than the channel per se may cause fewer side effects and reduce the potential for drug abuse in patients with diabetes.     

Voltage-gated calcium channels in chronic pain: emerging role of alternative splicing

N- and T-type voltage-gated calcium channels are key established players in chronic pain. Current work suggests that alternative splicing of these channels constitutes an important aspect in the investigation of their roles in the pathogenesis of chronic pain. Recent N-type channel studies describe a nociceptor-enriched alternatively spliced module responsible for voltage-independent G protein modulation and internalization, which is implicated in the control of distinct nociceptive pathways. On the contrary, although a large body of work has demonstrated that peripheral Ca_v3.2-encoded T-type currents are involved in several types of chronic pain, little is known with respect to the expression of numerous newly discovered splice variants in specific pain pathways. The elucidation of the new layers of molecular complexity uncovered in N- and T-type channel splice variants and their respective locations and roles in different pain pathways will allow for the development of better therapeutic strategies for the treatment of chronic pain.     

Modulation of T-type calcium channels by bioactive lipids

T-type calcium channels (T-channels/Ca_V3) have unique biophysical properties allowing a calcium influx at resting membrane potential of most cells. T-channels are ubiquitously expressed in many tissues and contribute to low-threshold spikes and burst firing in central neurons as well as to pacemaker activities in cardiac cells. They also emerged as potential targets to treat cancer and hypertension. Regulation of these channels appears complex, and several studies have indicated that Ca_V3.1, Ca_V3.2, and Ca_V3.3 currents are directly inhibited by multiple endogenous lipids independently of membrane receptors or intracellular pathways. These bioactive lipids include arachidonic acid and ω3 poly-unsaturated fatty acids; the endocannabinoid anandamide and other N-acylethanolamides; the lipoamino-acids and lipo-neurotransmitters; the P450 epoxygenase metabolite 5,6-epoxyeicosatrienoic acid; as well as similar molecules with 18–22 carbons in the alkyl chain. In this review, we summarize evidence for direct effects of these signaling molecules, the molecular mechanisms underlying the current inhibition, and the involved chemical features. The impact of this modulation in physiology and pathophysiology is discussed with a special emphasis on pain aspects and vasodilation. Overall, these data clearly indicate that T-current inhibition is an important mechanism by which bioactive lipids mediate their physiological functions.     

CaV3.2 calcium channels control NMDA receptor-mediated transmission: a new mechanism for absence epilepsy

Ca_V3.2 T-type calcium channels, encoded by CACNA1H, are expressed throughout the brain, yet their general function remains unclear. We discovered that Ca_V3.2 channels control NMDA-sensitive glutamatergic receptor (NMDA-R)-mediated transmission and subsequent NMDA-R-dependent plasticity of AMPA-R-mediated transmission at rat central synapses. Interestingly, functional Ca_V3.2 channels primarily incorporate into synapses, replace existing Ca_V3.2 channels, and can induce local calcium influx to control NMDA transmission strength in an activity-dependent manner. Moreover, human childhood absence epilepsy (CAE)-linked hCa_V3.2(C456S) mutant channels have a higher channel open probability, induce more calcium influx, and enhance glutamatergic transmission. Remarkably, cortical expression of hCa_V3.2(C456S) channels in rats induces 2- to 4-Hz spike and wave discharges and absence-like epilepsy characteristic of CAE patients, which can be suppressed by AMPA-R and NMDA-R antagonists but not T-type calcium channel antagonists. These results reveal an unexpected role of Ca_V3.2 channels in regulating NMDA-R-mediated transmission and a novel epileptogenic mechanism for human CAE.     

Short- and long-term roles of phosphatidylinositol 4,5-bisphosphate PIP2 on Cav3.1- and Cav3.2-T-type calcium channel current

T-type calcium (Ca²⁺) channels play important physiological functions in excitable cells including cardiomyocyte. Phosphatidylinositol-4,5-bisphosphate (PIP₂) has recently been reported to modulate various ion channels’ function. However the actions of PIP₂ on the T-type Ca²⁺ channel remain unclear. To elucidate possible effects of PIP₂ on the T-type Ca²⁺ channel, we applied patch clamp method to investigate recombinant Ca_V3.1- and Ca_V3.2-T-type Ca²⁺ channels expressed in mammalian cell lines with PIP₂ in acute- and long-term potentiation. Short- and long-term potentiation of PIP₂ shifted the activation and the steady-state inactivation curve toward the hyperpolarization direction of Ca_V3.1-I_Ca.T without affecting the maximum inward current density. Short- and long-term potentiation of PIP₂ also shifted the activation curve toward the hyperpolarization direction of Ca_V3.2-I_Ca.T without affecting the maximum inward current density. Conversely, long-term but not short-term potentiation of PIP₂ shifted the steady-state inactivation curve toward the hyperpolarization direction of Ca_V3.2-I_Ca.T. Long-term but not short-term potentiation of PIP₂ blunted the voltage-dependency of current decay Ca_V3.1-I_Ca.T. PIP₂ modulates Ca_V3.1- and Ca_V3.2-I_Ca.T not by their current density but by their channel gating properties possibly through its membrane-delimited actions.     

The trafficking of NaV1.8

The α-subunit of tetrodotoxin-resistant voltage-gated sodium channel Na_V1.8 is selectively expressed in sensory neurons. It has been reported that Na_V1.8 is involved in the transmission of nociceptive information from sensory neurons to the central nervous system in nociceptive [1] and neuropathic [24] pain conditions. Thus Na_V1.8 has been a promising target to treat chronic pain. Here we discuss the recent advances in the study of trafficking mechanism of Na_V1.8. These pieces of information are particularly important as such trafficking machinery could be new targets for painkillers.     

Upregulation of the T-type calcium current in small rat sensory neurons after chronic constrictive injury of the sciatic nerve

Recent data indicate that peripheral T-type Ca2+ channels are instrumental in supporting acute pain transmission. However, the function of these channels in chronic pain processing is less clear. To address this issue, we studied the expression of T-type Ca2+ currents in small nociceptive dorsal root ganglion (DRG) cells from L4-5 spinal ganglia of adult rats with neuropathic pain due to chronic constrictive injury (CCI) of the sciatic nerve. In control rats, whole cell recordings revealed that T-type currents, measured in 10 mM Ba2+ as a charge carrier, were present in moderate density (20 +/- 2 pA/pF). In rats with CCI, T-type current density (30 +/- 3 pA/pF) was significantly increased, but voltage- and time-dependent activation and inactivation kinetics were not significantly different from those in controls. CCI-induced neuropathy did not significantly change the pharmacological sensitivity of T-type current in these cells to nickel. Collectively, our results indicate that CCI-induced neuropathy significantly increases T-type current expression in small DRG neurons. Our finding that T-type currents are upregulated in a CCI model of peripheral neuropathy and earlier pharmacological and molecular studies suggest that T-type channels may be potentially useful therapeutic targets for the treatment of neuropathic pain associated with partial mechanical injury to the sciatic nerve.     

Properties and role of voltage-dependent calcium channels during mouse skeletal muscle differentiation

Skeletal muscle differentiation depends on calcium ions, but it is yet unclear whether calcium entry through voltage-dependent calcium channels (VDCCs) contributes to the myoblast fusion process. In this study, we investigate whether calcium influx through functional T-type VDCCs precedes and affects mouse satellite cell fusion. We report here on the properties and the role of the VDCCs expressed in differentiating mouse muscular cells using both the C2C12 cell line and primary cultures of satellite cells. We present electrophysiological and biochemical evidence demonstrating that T-type and L-type VDCCs are not present in C2C12 and primary cultures of mouse satellite cells prior to the fusion stage. Although mRNA for the T-type Ca_V3.2 subunit was detected in differentiated C2C12 cells, no T-type calcium currents could be recorded, while both T-type and L-type calcium currents were detected after the fusion process in primary cultures. In addition, chronic application of 30 μM nickel, known to inhibit T-type Ca_V3.2 channels, did not alter the fusion of C2C12 cells and mouse satellite cells in primary culture. Overall, the data indicate that, unlike in humans, Ca_V3.2 T-type calcium channels play no role in mouse satellite cell fusion.     

The neuropathic pain triad: neurons, immune cells and glia

Nociceptive pain results from the detection of intense or noxious stimuli by specialized high-threshold sensory neurons (nociceptors), a transfer of action potentials to the spinal cord, and onward transmission of the warning signal to the brain. In contrast, clinical pain such as pain after nerve injury (neuropathic pain) is characterized by pain in the absence of a stimulus and reduced nociceptive thresholds so that normally innocuous stimuli produce pain. The development of neuropathic pain involves not only neuronal pathways, but also Schwann cells, satellite cells in the dorsal root ganglia, components of the peripheral immune system, spinal microglia and astrocytes. As we increasingly appreciate that neuropathic pain has many features of a neuroimmune disorder, immunosuppression and blockade of the reciprocal signaling pathways between neuronal and non-neuronal cells offer new opportunities for disease modification and more successful management of pain.     

Surface expression and function of Cav3.2 T-type calcium channels are controlled by asparagine-linked glycosylation

Low-voltage-activated T-type calcium channels play important roles in neuronal physiology where they control cellular excitability and synaptic transmission. Alteration in T-type channel expression has been linked to various pathophysiological conditions such as pain arising from diabetic neuropathy. In the present study, we looked at the role of asparagine (N)-linked glycosylation on human Ca_v3.2 T-type channel expression and function. Manipulation of N-glycans on cells expressing a recombinant Ca_v3.2 channel revealed that N-linked glycosylation is critical for proper functional expression of the channel. Using site-directed mutagenesis to disrupt the canonical N-linked glycosylation sites of Ca_v3.2 channel, we show that glycosylation at asparagine N192 is critical for channel expression at the surface, whereas glycosylation at asparagine N1466 controls channel activity. Moreover, we demonstrate that N-linked glycosylation of Ca_v3.2 not only controls surface expression and activity of the channel but also underlies glucose-dependent potentiation of T-type Ca²⁺ current. Our data suggest that N-linked glycosylation of T-type channels may play an important role in aberrant upregulation of T-type channel activity in response to glucose elevations.     

T-type Ca2+ channels in spermatogenic cells and sperm

Cell function is importantly regulated by the intracellular concentration of Ca²⁺ ([Ca²⁺]i). Sperm development and function are deeply influenced by [Ca²⁺]i which is modulated amongst other ion transporters by plasma membrane Ca²⁺ permeable channels. The presence and role of voltage-dependent Ca²⁺ channels (Ca_V) of the T-type (Ca_V3) in sperm physiology have become a matter of debate in recent years. Though they are functionally present in later stages of development in spermatogenic cells and testicular sperm and their mRNAs and proteins detected from spermatogenic cells to mature mammalian spermatozoa, their currents have not been recorded in mature spermatozoa. This review critically summarizes the evidence for the involvement of Ca_V3 channels in sperm development and function.     

Cloning and characterization of voltage‐gated calcium channel alpha1 subunits in Xenopus laevis during development

Voltage‐gated calcium channels play a critical role in regulating the Ca²⁺ activity that mediates many aspects of neural development, including neural induction, neurotransmitter phenotype specification, and neurite outgrowth. Using Xenopus laevis embryos, we describe the spatial and temporal expression patterns during development of the 10 pore‐forming alpha1 subunits that define the channels' kinetic properties. In situ hybridization indicates that Ca_V1.2, Ca_V2.1, Ca_V2.2, and Ca_V3.2 are expressed during neurula stages throughout the neural tube. These, along with Ca_V1.3 and Ca_V2.3, beginning at early tail bud stages, and Ca_V3.1 at late tail bud stages, are detected in complex patterns within the brain and spinal cord through swimming tadpole stages. Additional expression of various alpha1 subunits was observed in the cranial ganglia, retina, olfactory epithelium, pineal gland, and heart. The unique expression patterns for the different alpha1 subunits suggests they are under precise spatial and temporal regulation and are serving specific functions during embryonic development. Developmental Dynamics 238:2891–2902, 2009. © 2009 Wiley‐Liss, Inc.     

P2X4R(+) microglia drive neuropathic pain

Neuropathic pain, the most debilitating of all clinical pain syndromes, may be a consequence of trauma, infection or pathology from diseases that affect peripheral nerves. Here we provide a framework for understanding the spinal mechanisms of neuropathic pain as distinct from those of acute pain or inflammatory pain. Recent work suggests that a specific microglia response phenotype characterized by de novo expression of the purinergic receptor P2X4 is critical for the pathogenesis of pain hypersensitivity caused by injury to peripheral nerves. Stimulating P2X4 receptors initiates a core pain signaling pathway mediated by release of brain-derived neurotrophic factor, which produces a disinhibitory increase in intracellular chloride in nociceptive (pain-transmitting) neurons in the spinal dorsal horn. The changes caused by signaling from P2X4R(+) microglia to nociceptive transmission neurons may account for the main symptoms of neuropathic pain in humans, and they point to specific interventions to alleviate this debilitating condition.     

Voltage-activated ion channels and Ca2+-induced Ca2+ release shape Ca2+ signaling in Merkel cells

Ca²⁺ signaling and neurotransmission modulate touch-evoked responses in Merkel cell–neurite complexes. To identify mechanisms governing these processes, we analyzed voltage-activated ion channels and Ca²⁺ signaling in purified Merkel cells. Merkel cells in the intact skin were specifically labeled by antibodies against voltage-activated Ca²⁺ channels (Ca_V2.1) and voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels. Voltage-clamp recordings revealed small Ca²⁺ currents, which produced Ca²⁺ transients that were amplified sevenfold by Ca²⁺-induced Ca²⁺ release. Merkel cells’ voltage-activated K⁺ currents were carried predominantly by BK_Ca channels with inactivating and non-inactivating components. Thus, Merkel cells, like hair cells, have functionally diverse BK_Ca channels. Finally, blocking K⁺ channels increased response magnitude and dramatically shortened Ca²⁺ transients evoked by mechanical stimulation. Together, these results demonstrate that Ca²⁺ signaling in Merkel cells is governed by the interplay of plasma membrane Ca²⁺ channels, store release and K⁺ channels, and they identify specific signaling mechanisms that may control touch sensitivity.     

Molecular mechanisms and signaling by comenic acid in nociceptive neurons influence the pathophysiology of neuropathic pain

Comenic acid (CA), a specific agonist of opioid-like receptors, effectively and safely relieves neuropathic pain by decreasing the Na_V1.8 channel voltage sensitivity in the primary sensory neuron membrane. CA triggers downstream signaling cascades, in which the Na,K-ATPase/Src complex plays a key role. After leaving the complex, the signal diverges ‘tangentially’ and ‘radially’. It is directed ‘tangentially’ along the neuron membrane to Na_V1.8 channels, decreasing the effective charge of their activation gating system. In the radial direction moving towards the cell genome, the signal activates the downstream signaling pathway involving PKC and ERK1/2. A remarkable feature of CA is its ability to modulate Na_V1.8 channels, which relieves neuropathic pain while simultaneously stimulating neurite growth via the receptor-coupled activation of the ERK1/2-dependent signaling pathway.     

Diabetes-induced abnormalities in ER calcium mobilization in primary and secondary nociceptive neurons

Development of diabetic sensory polyneuropathy is associated with alterations in intracellular calcium homeostasis in primary and secondary nociceptive neurons. We have shown previously that in a model of streptozotocin (STZ)-induced diabetes, the calcium signal is prolonged and calcium release from ryanodine-sensitive calcium stores down-regulated in neurons of the nociceptive system. The aim of the present study was a more detailed characterization of calcium homeostasis in primary (dorsal root ganglia, DRG) and secondary (dorsal horn, DH) nociceptive neurons in STZ-induced diabetes. Fluorescence video-imaging was used to measure free cytosolic [Ca²⁺] ([Ca²⁺]_i) in lumbar nociceptive neurons of control and streptozotocin-diabetic rats. Resting [Ca²⁺]_i rose progressively in these neurons with the duration of diabetes and calcium mobilization from the endoplasmic reticulum (ER) decreased during diabetes. The amplitude of calcium release from both ryanodine- and IP₃-sensitive calcium stores induced by caffeine, ionomycin, ATP or glutamate was significantly (P<0.01) lower in DRG and DH neurons from 6-week STZ-diabetic rats. Diabetes-induced changes in the calcium homeostasis were similar in DRG and DH neurons indicating that they might be general for many types of neurons from the central and peripheral nervous systems.     

Localization of L-type calcium channel CaV1.3 in cat lumbar spinal cord – with emphasis on motoneurons

Voltage-dependent persistent inward currents (PICs) which underlie the plateau potentials are an important intrinsic property of spinal motoneurons. Electrophysiological experiments have indicated that a subtype of the low threshold L-type calcium channel, Ca_V1.3, mediates this current. In mouse and turtle lumbar spinal cord it has been shown that these channel proteins are mainly found on motoneuron dendrites. In the present study we have used immunohistochemistry to locate these channels in lumbar spinal neurons, especially motoneurons, of the cat. The results indicate that Ca_V1.3 immunoreactivity was unevenly distributed among the laminae of the spinal grey matter. The small neurons in superficial dorsal horn (laminae I–III) were sparsely and weakly labelled, while large neurons in ventral horn were frequently and densely labelled. Groups of motoneurons in lamina IX that were immunoreactive to choline acetyltransferase also co-expressed Ca_V1.3. The immmunoreactivity was mainly associated with neuronal somata and proximal dendrites. Double staining with antibodies against Ca_V1.3 and MAP2 (a dendritic marker) showed that some fine fibres, which may include distal dendrites, were also labelled. These results in the cat spinal cord show some differences from studies in mouse and turtle motoneurons where the immunoreactivity against this channel was mainly localized to the dendrites.     

Distribution of calcium channel CaV1.3 immunoreactivity in the rat spinal cord and brain stem

The function of local networks in the CNS depends upon both the connectivity between neurons and their intrinsic properties. An intrinsic property of spinal motoneurons is the presence of persistent inward currents (PICs), which are mediated by non-inactivating calcium (mainly Ca_V1.3) and/or sodium channels and serve to amplify neuronal input signals. It is of fundamental importance for the prediction of network function to determine the distribution of neurons possessing the ion channels that produce PICs. Although the distribution pattern of Ca_V1.3 immunoreactivity (Ca_V1.3-IR) has been studied in some specific central nervous regions in some species, so far no systematic investigations have been performed in both the rat spinal cord and brain stem. In the present study this issue was investigated by immunohistochemistry. The results indicated that the Ca_V1.3-IR neurons were widely distributed across different parts of the spinal cord and the brain stem although with variable labeling intensities. In the spinal gray matter large neurons in the ventral horn (presumably motoneurons) tended to display higher levels of immunoreactivity than smaller neurons in the dorsal horn. In the white matter, a subset of glial cells labeled by an oligodendrocyte marker was also Ca_V1.3-positive. In the brain stem, neurons in the motor nuclei appeared to have higher levels of immunoreactivity than those in the sensory nuclei. Moreover, a number of nuclei containing monoaminergic cells, for example the locus coeruleus, were also strongly immunoreactive. Ca_V1.3-IR was consistently detected in the neuronal perikarya regardless of the neuronal type. However, in the large neurons in the spinal ventral horn and the cranial motor nuclei the Ca_V1.3-IR was clearly detectable in first and second order dendrites. These results indicate that in the rat spinal cord and brain stem Ca_V1.3 is probably a common calcium channel used by many kinds of neurons to facilitate the neuronal information processing via certain intracellular mechanisms, for instance, PICs.