Chloride-dependent mechanisms of multimodal sensory discrimination and neuropathic sensitization in Drosophila

Individual sensory neurons can be tuned to many stimuli, each driving unique, stimulus-relevant behaviors, and the ability of multimodal nociceptor neurons to discriminate between potentially harmful and innocuous stimuli is broadly important for organismal survival. Moreover, disruptions in the capacity to differentiate between noxious and innocuous stimuli can result in neuropathic pain. Drosophila larval Class III (CIII) neurons are peripheral noxious cold nociceptors and gentle touch mechanosensors; high levels of activation drive cold-evoked contraction (CT) behavior, while low levels of activation result in a suite of touch-associated behaviors. However, it is unknown what molecular factors underlie CIII multimodality. Here, we show that the TMEM16/anoctamins subdued and white walker (wwk; CG15270) are required for cold-evoked CT, but not for touch-associated behavior, indicating a conserved role for anoctamins in nociception. We also evidence that CIII neurons make use of atypical depolarizing chloride currents to encode cold, and that overexpression of ncc69—a fly homologue of NKCC1—results in phenotypes consistent with neuropathic sensitization, including behavioral hypersensitization and spontaneous nociceptor activity, making Drosophila CIII neurons a candidate system for future studies of the basic mechanisms underlying neuropathic pain.

Substratum stiffness tunes membrane voltage in mammary epithelial cells

Membrane voltage (Vm) plays a critical role in the regulation of several cellular behaviors, including proliferation, apoptosis, and phenotypic plasticity. Many of these same behaviors are affected by the stiffness of the underlying extracellular matrix, but the connections between Vm and the mechanical properties of the microenvironment are unclear. Here, we investigated the relationship between matrix stiffness and Vm by culturing mammary epithelial cells on synthetic substrata, the stiffnesses of which mimicked those of the normal mammary gland and breast tumors. Although proliferation is associated with depolarization, we surprisingly observed that cells are hyperpolarized when cultured on stiff substrata, a microenvironmental condition that enhances proliferation. Accordingly, we found that Vm becomes depolarized as stiffness decreases, in a manner dependent on intracellular calcium. Furthermore, inhibiting calcium-gated chloride currents abolishes the effects of substratum stiffness on Vm. Specifically, we uncovered a role for cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of Vm by substratum stiffness. Together, these results suggest a novel role for CFTR and membrane voltage in the response of mammary epithelial cells to their mechanical microenvironment.

Gender-Dependent Phenotype in Polycystic Kidney Disease Is Determined by Differential Intracellular Ca2+ Signals

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss of function of PKD1 (polycystin 1) or PKD2 (polycystin 2). The Ca2+-activated Cl− channel TMEM16A has a central role in ADPKD. Expression and function of TMEM16A is upregulated in ADPKD which causes enhanced intracellular Ca2+ signaling, cell proliferation, and ion secretion. We analyzed kidneys from Pkd1 knockout mice and found a more pronounced phenotype in males compared to females, despite similar levels of expression for renal tubular TMEM16A. Cell proliferation, which is known to be enhanced with loss of Pkd1−/−, was larger in male when compared to female Pkd1−/− cells. This was paralleled by higher basal intracellular Ca2+ concentrations in primary renal epithelial cells isolated from Pkd1−/− males. The results suggest enhanced intracellular Ca2+ levels contributing to augmented cell proliferation and cyst development in male kidneys. Enhanced resting Ca2+ also caused larger basal chloride currents in male primary cells, as detected in patch clamp recordings. Incubation of mouse primary cells, mCCDcl1 collecting duct cells or M1 collecting duct cells with dihydrotestosterone (DHT) enhanced basal Ca2+ levels and increased basal and ATP-stimulated TMEM16A chloride currents. Taken together, the more severe cystic phenotype in males is likely to be caused by enhanced cell proliferation, possibly due to enhanced basal and ATP-induced intracellular Ca2+ levels, leading to enhanced TMEM16A currents. Augmented Ca2+ signaling is possibly due to enhanced expression of Ca2+ transporting/regulating proteins.

Anoctamin 2-chloride channels reduce simple spike activity and mediate inhibition at elevated calcium concentration in cerebellar Purkinje cells

Modulation of neuronal excitability is a prominent way of shaping the activity of neuronal networks. Recent studies highlight the role of calcium-activated chloride currents in this context, as they can both increase or decrease excitability. The calcium-activated chloride channel Anoctamin 2 (ANO2 alias TMEM16B) has been described in several regions of the mouse brain, including the olivo-cerebellar system. In inferior olivary neurons, ANO2 was proposed to increase excitability by facilitating the generation of high-threshold calcium spikes. An expression of ANO2 in cerebellar Purkinje cells was suggested, but its role in these neurons remains unclear. In the present study, we confirmed the expression of Ano2 mRNA in Purkinje cells and performed electrophysiological recordings to examine the influence of ANO2-chloride channels on the excitability of Purkinje cells by comparing wildtype mice to mice lacking ANO2. Recordings were performed in acute cerebellar slices of adult mice, which provided the possibility to study the role of ANO2 within the cerebellar cortex. Purkinje cells were uncoupled from climbing fiber input to assess specifically the effect of ANO2 channels on Purkinje cell activity. We identified an attenuating effect of ANO2-mediated chloride currents on the instantaneous simple spike activity both during strong current injections and during current injections close to the simple spike threshold. Moreover, we report a reduction of inhibitory currents from GABAergic interneurons upon depolarization, lasting for several seconds. Together with the role of ANO2-chloride channels in inferior olivary neurons, our data extend the evidence for a role of chloride-dependent modulation in the olivo-cerebellar system that might be important for proper cerebellum-dependent motor coordination and learning.

TMEM16A deficiency: a potentially fatal neonatal disease resulting from impaired chloride currents

IntroductionTMEM16A is a calcium-activated chloride channel expressed in various secretory epithelia. Two siblings presented in early infancy with reduced intestinal peristalsis and recurrent episodes of haemorrhagic diarrhoea. In one of them, the episodes were characterised by hepatic pneumatosis with gas bubbles in the portal vein similar to necrotising enterocolitis of the newborn.MethodsExome sequencing identified a homozygous truncating pathogenic variant in ANO1. Expression analysis was performed using reverse transcription PCR, western blot and immunohistochemistry. Electrophysiological and cell biological studies were employed to characterise the effects on ion transport both in patient respiratory epithelial cells and in transfected HEK293 cells.ResultsThe identified variant led to TMEM16A dysfunction, which resulted in abolished calcium-activated Cl− currents. Secondarily, CFTR function is affected due to the close interplay between both channels without inducing cystic fibrosis (CF).ConclusionTMEM16A deficiency is a potentially fatal disorder caused by abolished calcium-activated Cl− currents in secretory epithelia. Secondary impairment of CFTR function did not cause a CF phenotyp, which may have implications for CF treatment.

Pathogenesis of hypertension in a mouse model for human CLCN2 related hyperaldosteronism

Human primary aldosteronism (PA) can be caused by mutations in several ion channel genes but mouse models replicating this condition are lacking. We now show that almost all known PA-associated CLCN2 mutations markedly increase ClC-2 chloride currents and generate knock-in mice expressing a constitutively open ClC-2 Cl− channel as mouse model for PA. The Clcn2op allele strongly increases the chloride conductance of zona glomerulosa cells, provoking a strong depolarization and increasing cytoplasmic Ca2+ concentration. Clcn2op mice display typical features of human PA, including high serum aldosterone in the presence of low renin activity, marked hypertension and hypokalemia. These symptoms are more pronounced in homozygous Clcn2op/op than in heterozygous Clcn2+/op mice. This difference is attributed to the unexpected finding that only ~50 % of Clcn2+/op zona glomerulosa cells are depolarized. By reproducing essential features of human PA, Clcn2op mice are a valuable model to study the pathological mechanisms underlying this disease.