Sex differences in the involvement of skeletal and cardiac muscles in myopathic Ano5-/- mice.

Limb-girdle muscular dystrophy R12 (LGMD-R12) is caused by recessive mutations in the Anoctamin-5 gene (ANO5, TMEM16E). Although ANO5 myopathy is not X-chromosome linked, we performed a meta-analysis of the research literature and found that three-quarters of patients with LGMD-R12 are males. Females are less likely to present with moderate to severe skeletal muscle and/or cardiac pathology. Because these sex differences could be explained in several ways, we compared males and females in a mouse model of LGMD-R12. This model recapitulates the sex differences in human LGMD-R12. Only male Ano5-/- mice had elevated serum creatine kinase after exercise and exhibited defective membrane repair after laser injury. In contrast, by these measures, female Ano5-/- mice were indistinguishable from wild type. Despite these differences, both male and female Ano5-/- mice exhibited exercise intolerance. Although exercise intolerance of male mice can be explained by skeletal muscle dysfunction, echocardiography revealed that Ano5-/- female mice had features of cardiomyopathy that may be responsible for their exercise intolerance. These findings heighten concerns that mutations of ANO5 in humans may be linked to cardiac disease.

ANO5 ensures trafficking of annexins in wounded myofibers.

Mutations in ANO5 (TMEM16E) cause limb-girdle muscular dystrophy R12. Defective plasma membrane repair is a likely mechanism. Using myofibers from Ano5 knockout mice, we show that trafficking of several annexin proteins, which together form a cap at the site of injury, is altered upon loss of ANO5. Annexin A2 accumulates at the wound to nearly twice the level observed in WT fibers, while annexin A6 accumulation is substantially inhibited in the absence of ANO5. Appearance of annexins A1 and A5 at the cap is likewise diminished in the Ano5 knockout. These changes are correlated with an alteration in annexin repair cap fine structure and shedding of annexin-positive vesicles. We conclude that loss of annexin coordination during repair is disrupted in Ano5 knockout mice and underlies the defective repair phenotype. Although ANO5 is a phospholipid scramblase, abnormal repair is rescued by overexpression of a scramblase-defective ANO5 mutant, suggesting a novel, scramblase-independent role of ANO5 in repair.

TMEM16B regulates anxiety-related behavior and GABAergic neuronal signaling in the central lateral amygdala.

TMEM16B (ANO2) is the Ca2+-activated chloride channel expressed in multiple brain regions, including the amygdala. Here we report that Ano2 knockout mice exhibit impaired anxiety-related behaviors and context-independent fear memory, thus implicating TMEM16B in anxiety modulation. We found that TMEM16B is expressed in somatostatin-positive (SOM+) GABAergic neurons of the central lateral amygdala (CeL), and its activity modulates action potential duration and inhibitory postsynaptic current (IPSC). We further provide evidence for TMEM16B actions not only in the soma but also in the presynaptic nerve terminals of GABAergic neurons. Our study reveals an intriguing role for TMEM16B in context-independent but not context-dependent fear memory, and supports the notion that dysfunction of the amygdala contributes to anxiety-related behaviors.

Impaired Motor Coordination and Learning in Mice Lacking Anoctamin 2 Calcium-Gated Chloride Channels.

Neurons communicate through excitatory and inhibitory synapses. Both lines of communication are adjustable and allow the fine tuning of signal exchange required for learning processes in neural networks. Several distinct modes of plasticity modulate glutamatergic and GABAergic synapses in Purkinje cells of the cerebellar cortex to promote motor control and learning. In the present paper, we present evidence for a role of short-term ionic plasticity in the cerebellar circuit activity. This type of plasticity results from altered chloride driving forces at the synapses that molecular layer interneurons form on Purkinje cell dendrites. Previous studies have provided evidence for transiently diminished chloride gradients at these GABAergic synapses following climbing fiber activity. Electrical stimulation of climbing fibers in acute slices caused a decline of inhibitory postsynaptic currents recorded from Purkinje cells. Dendritic calcium-gated chloride channels of the type anoctamin 2 (ANO2) were proposed to mediate this short-term modulation of inhibition, but the significance of this process for motor control has not been established yet. Here, we report results of behavioral studies obtained from Ano2 -/- mice, a mouse line that was previously shown to lack this particular mode of ionic plasticity. The animals display motor coordination deficits that constitute a condition of mild ataxia. Moreover, motor learning is severely impaired in Ano2 -/- mice, suggesting cerebellar dysfunction. This reduced motor performance of Ano2 -/- mice highlights the significance of inhibitory control for cerebellar function and introduces calcium-dependent short-term ionic plasticity as an efficient control mechanism for neural inhibition.

Cellular defects by deletion of ANO10 are due to deregulated local calcium signaling.

TMEM16K (ANO10) belongs to a family of ion channels and phospholipid scramblases. Mutations in ANO10 cause neurological and immunological defects, and abrogated ion transport. Here we show that Ano10 knockout in epithelial cells leads to defective ion transport, attenuated volume regulation and deranged Ca2+ signaling. Intestinal epithelial cells from Ano10 null mice are reduced in size and demonstrate an almost abolished spontaneous and TNFα-induced apoptosis. Similar defects were found in mouse peritoneal Ano10 null macrophages and in human THP1 macrophages with reduced ANO10 expression. A cell cycle dependent colocalization of Ano10 with acetylated tubulin, centrioles, and a submembranous tubulin containing compartment was observed in Fisher rat thyroid cells. Axs, the Drosophila ortholog of ANO10 is known for its role in mitotic spindle formation and association with the endoplasmic reticulum and Ca2+ signaling. We therefore propose that mutations in ANO10 cause cellular defects and genetic disorders through deranged local Ca2+ signaling.