HCN3 ion channels: roles in sensory neuronal excitability and pain.

KEY POINTS: HCN ion channels conducting the Ih current control the frequency of firing in peripheral sensory neurons signalling pain. Previous studies have demonstrated a major role for the HCN2 subunit in chronic pain but the potential involvement of HCN3 in pain has not been investigated. HCN3 was found to be widely expressed in all classes of sensory neurons (small, medium, large) where it contributes to Ih . HCN3 deletion increased the firing rate of medium but not small, sensory neurons. Pain sensitivity both acutely and following neuropathic injury was largely unaffected by HCN3 deletion, with the exception of a small decrease of mechanical hyperalgesia in response to a pinprick. We conclude that HCN3 plays little role in either acute or chronic pain sensation. ABSTRACT: HCN ion channels govern the firing rate of action potentials in the pacemaker region of the heart and in pain-sensitive (nociceptive) nerve fibres. Intracellular cAMP promotes activation of the HCN4 and HCN2 isoforms, whereas HCN1 and HCN3 are relatively insensitive to cAMP. HCN2 modulates action potential firing rate in nociceptive neurons and plays a critical role in all modes of inflammatory and neuropathic pain, although the role of HCN3 in nociceptive excitability and pain is less studied. Using antibody staining, we found that HCN3 is expressed in all classes of somatosensory neurons. In small nociceptive neurons, genetic deletion of HCN2 abolished the voltage shift of the Ih current carried by HCN isoforms following cAMP elevation, whereas the voltage shift was retained following deletion of HCN3, consistent with the sensitivity of HCN2 but not HCN3 to cAMP. Deletion of HCN3 had little effect on the evoked firing frequency in small neurons but enhanced the firing of medium-sized neurons, showing that HCN3 makes a significant contribution to the input resistance only in medium-sized neurons. Genetic deletion of HCN3 had no effect on acute thresholds to heat or mechanical stimuli in vivo and did not affect inflammatory pain measured with the formalin test. Nerve-injured HCN3 knockout mice exhibited similar levels of mechanical allodynia and thermal hyperalgesia to wild-type mice but reduced mechanical hyperalgesia in response to a pinprick. These results show that HCN3 makes some contribution to excitability, particularly in medium-sized neurons, although it has no major influence on acute or neuropathic pain processing.

Investigation of the structure activity relationship of flufenamic acid derivatives at the human TRESK channel K2P18.1.

TRESK (Twik RElated Spinal cord K+ channel) is a member of the Twin Pore Domain potassium channel (K2P) family responsible for regulating neuronal excitability in dorsal root ganglion (DRG) and trigeminal (TG) neurons, peripheral neurons involved in pain transmission. As channel opening causes an outward K+ current responsible for cell hyperpolarisation, TRESK represents a potentially interesting target for pain treatment. However, as no crystal structure exists for this protein, the mechanisms involved in the opening action of its ligands are still poorly understood, making the development of new potent and selective openers challenging. In this work we present a structure activity relationship (SAR) of the known TRESK opener flufenamic acid (FFA) and some derivatives, investigating the functional effects of chemical modifications to build a TRESK homology model to support the biological results. A plausible binding mode is proposed, providing the first predictive hypothesis of a human TRESK opener binding site.

Ankyrin-3 is a novel binding partner of the voltage-gated potassium channel Kv1.1 implicated in renal magnesium handling.

The voltage-gated potassium channel, Kv1.1, was recently identified as a causative gene in isolated dominant hypomagnesemia. The channel is situated in the distal convoluted tubule, where it participates in maintaining a favorable electrical gradient for driving magnesium ion into the cell through the transient receptor potential melastatin 6 channel. Pull-down experiments coupled to mass spectrometry using the carboxy-terminal domain of Kv1.1 as bait were used in mouse kidney lysates. Ankyrin-3 (ANK3) was identified as a binding partner of Kv1.1 and was enriched in isolated distal convoluted tubules as compared to whole kidney. Electrophysiology studies performed in HEK293 cells expressing Kv1.1 showed that ANK3 significantly inhibited Kv1.1-mediated currents (267 compared to 125 pA/pF) for control and ANK3, respectively. Finally, to evaluate a potential role of ANK3 in magnesium handling, the intrarenal abundance of ANK3 was measured in mice fed a low-, normal-, or high-magnesium diet for 10 days. Mice maintained on high dietary magnesium significantly doubled their fractional urinary excretion of magnesium, which coincided with a 1.8-fold increase in the renal expression of ANK3 compared to mice on a normal- or low-magnesium diet. Thus, our observations demonstrate a novel role for ANK3 in modulating the biophysical properties of Kv1.1. Such regulation appears to be particularly important in conditions of high dietary magnesium.

CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia.

Familial hypomagnesemia is a rare human disorder caused by renal or intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures, and cardiac arrhythmias. Our knowledge of the physiology of Mg(2+) (re)absorption, particularly the luminal uptake of Mg(2+) along the nephron, has benefitted from positional cloning approaches in families with Mg(2+) reabsorption disorders; however, basolateral Mg(2+) transport and its regulation are still poorly understood. Here, by using a candidate screening approach, we identified CNNM2 as a gene involved in renal Mg(2+) handling in patients of two unrelated families with unexplained dominant hypomagnesemia. In the kidney, CNNM2 was predominantly found along the basolateral membrane of distal tubular segments involved in Mg(2+) reabsorption. The basolateral localization of endogenous and recombinant CNNM2 was confirmed in epithelial kidney cell lines. Electrophysiological analysis showed that CNNM2 mediated Mg(2+)-sensitive Na(+) currents that were significantly diminished in mutant protein and were blocked by increased extracellular Mg(2+) concentrations. Our data support the findings of a recent genome-wide association study showing the CNNM2 locus to be associated with serum Mg(2+) concentrations. The mutations found in CNNM2, its observed sensitivity to extracellular Mg(2+), and its basolateral localization signify a critical role for CNNM2 in epithelial Mg(2+) transport.

A novel KCNA1 mutation causing episodic ataxia type I.

We describe the clinical phenotype of a novel de novo KNCA1 mutation, and functional characterization of the effects of the mutation on Kv1.1 channel function. HEK293 cells were transfected transiently with either wild-type or mutant channels. Representative currents were evoked after application of a series of square voltage steps from -80 mV to +50 mV in 200-ms intervals from Vh = -80 mV. Extracellular K(+) was added to evoke tail currents. Equal amounts of wild-type and Kv1.1(I262M) mutant DNA were transfected transiently in HEK293 cells to evaluate the influence of the mutation. We found that Kv1.1(I262M) leads to a defective voltage-gated potassium channel. Coexpression studies revealed a dominant-negative effect. We describe the phenotype of a novel KCNA1 mutation causing episodic ataxia. Patch-clamp studies confirm the pathogenicity of the mutation in vitro and suggest that it is dominant with respect to wild-type.

Membrane topology and intracellular processing of cyclin M2 (CNNM2).

Recently, mutations in the cyclin M2 (CNNM2) gene were identified to be causative for severe hypomagnesemia. In kidney, CNNM2 is a basolaterally expressed protein with predominant expression in the distal convoluted tubule. Transcellular magnesium (Mg(2+)) reabsorption in the distal convoluted tubule represents the final step before Mg(2+) is excreted into the urine, thus fine-tuning its final excretion via a tightly regulated mechanism. The present study aims to get insight in the structure of CNNM2 and to characterize its post-translational modifications. Here, membrane topology studies using intramolecular epitopes and immunocytochemistry showed that CNNM2 has an extracellular N terminus and an intracellular C terminus. This suggests that one of the predicted transmembrane regions might be re-entrant. By homology modeling, we demonstrated that the loss-of-function mutation as found in patients disturbs the potential ATP binding by the intracellular cystathionine ß-synthase domains. In addition, the cellular processing pathway of CNNM2 was exposed in detail. In the endoplasmic reticulum, the signal peptidase complex cleaves off a large N-terminal signal peptide of about 64 amino acids. Mutagenesis screening showed that CNNM2 is glycosylated at residue Asn-112, stabilizing CNNM2 on the plasma membrane. Interestingly, co-immunoprecipitation studies evidenced that CNNM2a forms heterodimers with the smaller isoform CNNM2b. These new findings on CNNM2 structure and processing may aid to elucidate the physiological role of CNNM2 in Mg(2+) reabsorption in the kidney.

New TRPC6 gain-of-function mutation in a non-consanguineous Dutch family with late-onset focal segmental glomerulosclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a leading cause of steroid-resistant nephrotic syndrome. Hereditary FSGS is frequently caused by mutations in important structural podocyte proteins, including the slit diaphragm-associated transient receptor potential channel C6 (TRPC6). METHODS: In five patients with biopsy-proven autosomal-dominant FSGS from five different Dutch families, all 13 exons of TRPC6 were sequenced. Upon identification of a novel TRPC6 sequence variant, the resultant amino acid change was introduced in the wild-type TRPC6 protein and functionally tested using patch-clamp analyses and cell-surface biotinylation experiments. RESULTS: None of the previously described TRPC6 mutations were found in our cohort. In one family, we identified a novel c.524G>A sequence variant resulting in a p.Arg175Gln (R175Q) substitution in the TRPC6 protein. This sequence variant was absent in 449 control subjects and from public SNP databases. The mutation was located in the third ankyrin repeat domain (ANK3) in the cytoplasmic N-tail of TRPC6, important for protein-protein interaction and regulation of ion channel activity. Patch-clamp analyses of the mutant channel indeed showed an increased TRPC6 channel-mediated current. However, cell-surface expression of the mutant channel was not increased. CONCLUSIONS: We identified a novel TRPC6 p.Arg175Gln gain-of-function mutation that shows increased TRPC6-mediated current, which is not due to altered cell-surface expression. This is the first mutation identified in ANK3 of the TRPC6 N-tail and is most likely responsible for the late-onset autosomal dominant FSGS in this family.

Urinary plasmin inhibits TRPV5 in nephrotic-range proteinuria.

Urinary proteins that leak through the abnormal glomerulus in nephrotic syndrome may affect tubular transport by interacting with membrane transporters on the luminal side of tubular epithelial cells. Patients with nephrotic syndrome can develop nephrocalcinosis, which animal models suggest may develop from impaired transcellular Ca(2+) reabsorption via TRPV5 in the distal convoluted tubule (DCT). In nephrotic-range proteinuria, filtered plasminogen reaches the luminal side of DCT, where it is cleaved into active plasmin by urokinase. In this study, we found that plasmin purified from the urine of patients with nephrotic-range proteinuria inhibits Ca(2+) uptake in TRPV5-expressing human embryonic kidney 293 cells through the activation of protease-activated receptor-1 (PAR-1). Preincubation with a plasmin inhibitor, a PAR-1 antagonist, or a protein kinase C (PKC) inhibitor abolished the effect of plasmin on TRPV5. In addition, ablation of the PKC phosphorylation site S144 rendered TRPV5 resistant to the action of plasmin. Patch-clamp experiments showed that a decreased TRPV5 pore size and a reduced open probability accompany the plasmin-mediated reduction in Ca(2+) uptake. Furthermore, high-resolution nuclear magnetic resonance spectroscopy demonstrated specific interactions between calmodulin and residues 133-154 of the N-terminus of TRPV5 for both wild-type and phosphorylated (S144pS) peptides. In summary, PAR-1 activation by plasmin induces PKC-mediated phosphorylation of TRPV5, thereby altering calmodulin-TRPV5 binding, resulting in decreased channel activity. These results indicate that urinary plasmin could contribute to the downstream effects of proteinuria on the tubulointerstitium by negatively modulating TRPV5.

Activated spinal cord ependymal stem cells rescue neurological function.

Spinal cord injury (SCI) is a major cause of paralysis. Currently, there are no effective therapies to reverse this disabling condition. The presence of ependymal stem/progenitor cells (epSPCs) in the adult spinal cord suggests that endogenous stem cell-associated mechanisms might be exploited to repair spinal cord lesions. epSPC cells that proliferate after SCI are recruited by the injured zone, and can be modulated by innate and adaptive immune responses. Here we demonstrate that when epSPCs are cultured from rats with a SCI (ependymal stem/progenitor cells injury [epSPCi]), these cells proliferate 10 times faster in vitro than epSPC derived from control animals and display enhanced self renewal. Genetic profile analysis revealed an important influence of inflammation on signaling pathways in epSPCi after injury, including the upregulation of Jak/Stat and mitogen activated protein kinase pathways. Although neurospheres derived from either epSPCs or epSPCi differentiated efficiently to oligodendrocites and functional spinal motoneurons, a better yield of differentiated cells was consistently obtained from epSPCi cultures. Acute transplantation of undifferentiated epSPCi or the resulting oligodendrocyte precursor cells into a rat model of severe spinal cord contusion produced a significant recovery of motor activity 1 week after injury. These transplanted cells migrated long distances from the rostral and caudal regions of the transplant to the neurofilament-labeled axons in and around the lesion zone. Our findings demonstrate that modulation of endogenous epSPCs represents a viable cell-based strategy for restoring neuronal dysfunction in patients with spinal cord damage.

Publisher Correction: Molecular and functional variation in iPSC-derived sensory neurons.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regulation of Kv4.3 and hERG potassium channels by KChIP2 isoforms and DPP6 and response to the dual K+ channel activator NS3623.

Transient outward potassium current (Ito) contributes to early repolarization of many mammalian cardiac action potentials, including human, whilst the rapid delayed rectifier K+ current (IKr) contributes to later repolarization. Fast Ito channels can be produced from the Shal family KCNDE gene product Kv4.3s, although accessory subunits including KChIP2.x and DPP6 are also needed to produce a near physiological Ito. In this study, the effect of KChIP2.1 & KChIP2.2 (also known as KChIP2b and KChIP2c respectively), alone or in conjunction with the accessory subunit DPP6, on both Kv4.3 and hERG were evaluated. A dual Ito and IKr activator, NS3623, has been recently proposed to be beneficial in heart failure and the action of NS3623 on the two channels was also investigated. Whole-cell patch-clamp experiments were performed at 33 ±â€¯1 °C on HEK293 cells expressing Kv4.3 or hERG in the absence or presence of these accessory subunits. Kv4.3 current magnitude was augmented by co-expression with either KChIP2.2 or KChIP2.1 and KChIP2/DPP6 with KChIP2.1 producing a greater effect than KChIP2.2. Adding DPP6 removed the difference in Kv4.3 augmentation between KChIP2.1 and KChIP2.2. The inactivation rate and recovery from inactivation were also altered by KChIP2 isoform co-expression. In contrast, hERG (Kv11.1) current was not altered by co-expression with KChIP2.1, KChIP2.2 or DPP6. NS3623 increased Kv4.3 amplitude to a similar extent with and without accessory subunit co-expression, however KChIP2 isoforms modulated the compound's effect on inactivation time course. The agonist effect of NS3623 on hERG channels was not affected by KChIP2.1, KChIP2.2 or DPP6 co-expression.

Molecular and functional variation in iPSC-derived sensory neurons.

Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools for modeling biological processes, particularly in cell types that are difficult to obtain from living donors. Here we present a map of regulatory variants in iPSC-derived neurons, based on 123 differentiations of iPSCs to a sensory neuronal fate. Gene expression was more variable across cultures than in primary dorsal root ganglion, particularly for genes related to nervous system development. Using single-cell RNA-sequencing, we found that the number of neuronal versus contaminating cells was influenced by iPSC culture conditions before differentiation. Despite high differentiation-induced variability, our allele-specific method detected thousands of quantitative trait loci (QTLs) that influenced gene expression, chromatin accessibility, and RNA splicing. On the basis of these detected QTLs, we estimate that recall-by-genotype studies that use iPSC-derived cells will require cells from at least 20-80 individuals to detect the effects of regulatory variants with moderately large effect sizes.

Hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) ion channels drive pain in mouse models of diabetic neuropathy.

Diabetic patients frequently suffer from continuous pain that is poorly treated by currently available analgesics. We used mouse models of type 1 and type 2 diabetes to investigate a possible role for the hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) ion channels as drivers of diabetic pain. Blocking or genetically deleting HCN2 channels in small nociceptive neurons suppressed diabetes-associated mechanical allodynia and prevented neuronal activation of second-order neurons in the spinal cord in mice. In addition, we found that intracellular cyclic adenosine monophosphate (cAMP), a positive HCN2 modulator, is increased in somatosensory neurons in an animal model of painful diabetes. We propose that the increased intracellular cAMP drives diabetes-associated pain by facilitating HCN2 activation and consequently promoting repetitive firing in primary nociceptive nerve fibers. Our results suggest that HCN2 may be an analgesic target in the treatment of painful diabetic neuropathy.

A human-specific TNF-responsive promoter for Goodpasture antigen-binding protein.

The Goodpasture antigen-binding protein, GPBP, is a serine/threonine kinase whose relative expression increases in autoimmune processes. Tumor necrosis factor (TNF) is a pro-inflammatory cytokine implicated in autoimmune pathogenesis. Here we show that COL4A3BP, the gene encoding GPBP, maps head-to-head with POLK, the gene encoding for DNA polymerase kappa (pol kappa), and shares with it a 140-bp promoter containing a Sp1 site, a TATA-like element, and a nuclear factor kappa B (NFkappaB)-like site. These three elements cooperate in the assembly of a bidirectional transcription complex containing abundant Sp1 and little NFkappaB that is more efficient in the POLK direction. Tumour necrosis factor cell induction is associated with Sp1 release, NFkappaB recruitment and assembly of a complex comparatively more efficient in the COL4A3BP direction. This is accomplished by competitive binding of Sp1 and NFkappaB to a DNA element encompassing a NFkappaB-like site that is pivotal for the 140-bp promoter to function. Consistently, a murine homologous DNA region, which contains the Sp1 site and the TATA-like element but is devoid of the NFkappaB-like site, does not show transcriptional activity in transient gene expression assays. Our findings identify a human-specific TNF-responsive transcriptional unit that locates GPBP in the signalling cascade of TNF and substantiates previous observations, which independently related TNF and GPBP with human autoimmunity.

New TRPM6 missense mutations linked to hypomagnesemia with secondary hypocalcemia.

Despite recent progress in our understanding of renal magnesium (Mg(2+)) handling, the molecular mechanisms accounting for transepithelial Mg(2+) transport are still poorly understood. Mutations in the TRPM6 gene, encoding the epithelial Mg(2+) channel TRPM6 (transient receptor potential melastatin 6), have been proven to be the molecular cause of hypomagnesemia with secondary hypocalcemia (HSH; OMIM 602014). HSH manifests in the newborn period being characterized by very low serum Mg(2+) levels (<0.4 mmol/l) accompanied by low serum calcium (Ca(2+)) concentrations. A proportion of previously described TRPM6 mutations lead to a truncated TRPM6 protein resulting in a complete loss-of-function of the ion channel. In addition, five-point mutations have been previously described. The aim of this study was to complement the current clinical picture by adding the molecular data from five new missense mutations found in five patients with HSH. To this end, patch-clamp analysis and cell surface measurements were performed to assess the effect of the various mutations on TRPM6 channel function. All mutant channels, expressed in HEK293 cells, showed loss-of-function, whereas no severe trafficking impairment to the plasma membrane surface was observed. We conclude that the new TRPM6 missense mutations lead to dysregulated intestinal/renal Mg(2+) (re)absorption as a consequence of loss of TRPM6 channel function.

Efficient differentiation of human embryonic stem cells into functional cerebellar-like cells.

The cerebellum has critical roles in motor and sensory learning and motor coordination. Many cerebellum-related disorders indicate cell therapy as a possible treatment of neural loss. Here we show that application of inductive signals involved in early patterning of the cerebellar region followed by application of different factors directs human embryonic stem cell differentiation into cerebellar-like cells such as granule neurons, Purkinje cells, interneuron, and glial cells. Neurons derived using our protocol showed a T-shaped polarity phenotype and express similar markers to the developed human cerebellum. Electrophysiological measurements confirmed functional electrical properties compatible with these cells. In vivo implantation of differentiated human embryonic stem cells transfected with MATH1-GFP construct into neonatal mice resulted in cell migration across the molecular and the Purkinje cell layers and settlement in the internal molecular layers. Our findings demonstrate that the universal mechanisms involved in the development of cerebellum can be efficiently recapitulated in vitro, which enables the design of new strategies for cell replacement therapy, to study early human development and pathogenesis of neurodegenerative diseases.

Differentiation of human embryonic stem cells to regional specific neural precursors in chemically defined medium conditions.

BACKGROUND: Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury. METHODOLOGY AND PRINCIPAL FINDINGS: The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both, rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis. CONCLUSIONS/SIGNIFICANCE: These findings suggest that our differentiation protocol has the capacity to generate region-specific and electrophysiologically active neurons under in vitro conditions without embryoid body formation, co-culture with stromal cells and without presence of cells of mesodermal or endodermal lineages.

Differentiation of postnatal neural stem cells into glia and functional neurons on laminin-coated polymeric substrates.

A series of polymeric biomaterials, including poly(methyl acrylate), chitosan, poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate, hydroxyethyl acrylate, and methyl acrylate were tested in vitro as culture substrates and compared for their effect on the differentiation of neural stem cells (NSCs) obtained from the subventricular zone of postnatal rats. Immunocytochemical assay for specific markers and scanning electron microscopy techniques were employed to determine the adhesion of the cultured NSCs to the different biomaterials and the respective neuronal differentiation. The functional properties and the membrane excitability of differentiated NSCs were investigated using a patch-clamp. The results show that the substrate's surface chemistry influences cell attachment and neuronal differentiation, probably through its influence on adsorbed laminin, and that copolymers based on PEA and PHEA in a narrow composition window are suitable substrates to promote cell attachment and differentiation of adult NSCs into functional neurons and glia.