The hereditary ataxias are a complex group of neurological disorders characterized by the degeneration of the cerebellum and its associated connections. The molecular mechanisms that trigger the loss of Purkinje cells in this group of diseases remain incompletely understood. Here, we report a previously undescribed dominant mouse model of cerebellar ataxia, moonwalker (Mwk), that displays motor and coordination defects and loss of cerebellar Purkinje cells. Mwk mice harbor a gain-of-function mutation (T635A) in the Trpc3 gene encoding the nonselective transient receptor potential cation channel, type C3 (TRPC3), resulting in altered TRPC3 channel gating. TRPC3 is highly expressed in Purkinje cells during the phase of dendritogenesis. Interestingly, growth and differentiation of Purkinje cell dendritic arbors are profoundly impaired in Mwk mice. Our findings define a previously unknown role for TRPC3 in both dendritic development and survival of Purkinje cells, and provide a unique mechanism underlying cerebellar ataxia.cerebellum ͉ dendritogenesis ͉ trp channel ͉ mouse mutant T he inherited cerebellar ataxias are a complex group of neurodegenerative disorders characterized by loss of balance and coordination (1-3). Cerebellar ataxia is caused by the degeneration of Purkinje cells, which form the sole output of the cerebellum. To date, more than 50 different inherited forms of cerebellar ataxia are known (4). Importantly, increasing evidence points to the existence of common pathological pathways in different forms of ataxia, including transcriptional regulation, protein aggregation, and calcium homeostasis, which trigger the degeneration of Purkinje cells in these disorders (1, 5). However, the molecular mechanisms mediating these pathways remain poorly understood.To identify gene products that might be key to cerebellar degeneration, we used a phenotype-driven approach to screen for ataxic behavior in a large cohort of N-ethyl-N-nitrosourea (ENU)-mutagenized mice (6). Here, we report that a point mutation (T635A) in the C3-type transient receptor potential (TRPC3) channel in the mouse results in Purkinje cell degeneration and cerebellar ataxia. We also find that the development of dendrites is severely impaired in mutant Purkinje cells. Notably, the identified dominant gain-of-function mutation in TRPC3 provides insight into the function of TRPC3 that powerfully complements the findings obtained from the recently published TRPC3 knockout mouse (7). Our findings suggest that TRPC3 is a regulator of development and survival of Purkinje cells, and link aberrant TRPC3 function to cerebellar disease.

NMDA receptors (NMDARs) are generally believed to mediate exclusively postsynaptic effects at brain synapses. Here we searched for presynaptic effects of NMDA at inhibitory synapses in rat cerebellar slices. In Purkinje cells, application of NMDA enhanced the frequency of miniature IPSCs (mIPSCs) but not that of miniature EPSCs (mEPSCs). This increase in frequency was dependent on the external Mg2+ concentration. In basket and stellate cells, NMDA induced an even larger mIPSC frequency increase than in Purkinje cells, whereas mEPSCs were again not affected. Moreover, NMDA induced an inward current in both types of interneuron, which translated into a small depolarization (approximately 10 mV for 30 microM NMDA) under current-clamp conditions. In paired recordings of connected basket cell-Purkinje cell synapses, depolarizations of 10-30 mV applied to the basket cell soma enhanced the frequency of postsynaptic mIPSCs, suggesting that somatic depolarization was partially transmitted to the terminals in the presence of tetrodotoxin. However, this effect was small and unlikely to account fully for the effects of NMDA on mIPSCs. Consistent with a small number of dendritic NMDARs, evoked EPSCs in interneurons had a remarkably small NMDA component. Evoked IPSCs at interneuron-interneuron synapses were inhibited by NMDA, and the rate of failures was increased, indicating again a presynaptic site of action. We conclude that activation of NMDARs in interneurons exerts complex presynaptic effects, and that the corresponding receptors are most likely located in the axonal domain of the cell.

Store-operated Ca 2+ channels, which are activated by the emptying of intracellular Ca 2+ stores, provide one major route for Ca 2+ in¯ux. Under physiological conditions of weak intracellular Ca 2+ buffering, the ubiquitous Ca 2+ releasing messenger InsP 3 usually fails to activate any store-operated Ca 2+ entry unless mitochondria are maintained in an energized state. Mitochondria rapidly take up Ca 2+ that has been released by InsP 3 , enabling stores to empty suf®ciently for store-operated channels to activate. Here, we report a novel role for mitochondria in regulating store-operated channels under physiological conditions. Mitochondrial depolarization suppresses storeoperated Ca 2+ in¯ux independently of how stores are depleted. This role for mitochondria is unrelated to their actions on promoting InsP 3 -sensitive store depletion, can be distinguished from Ca 2+ -dependent inactivation of the store-operated channels and does not involve changes in intracellular ATP, oxidants, cytosolic acidi®cation, nitric oxide or the permeability transition pore, but is suppressed when mitochondrial Ca 2+ uptake is impaired. Our results suggest that mitochondria may have a more fundamental role in regulating store-operated in¯ux and raise the possibility of bidirectional Ca 2+ -dependent crosstalk between mitochondria and store-operated Ca 2+ channels.

In many non-excitable cells, a Ca 2+ influx pathway is activated following the process of store emptying, and has been called store-operated or capacitative Ca 2+ entry (Putney, 1986 1. One popular model for the activation of store-operated Ca 2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP 3 ) receptors on the stores to bind to, and thus activate, store-operated Ca 2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I CRAC , the most widely distributed and best characterised store-operated Ca 2+ current, in a model system, the RBL-1 rat basophilic cell line.2. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I CRAC . Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment.3. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution.4. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I CRAC was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores.5. Pre-treatment with the membrane-permeable InsP 3 receptor antagonist 2-APB blocked the activation of I CRAC . However, intracellular dialysis with 2-APB failed to prevent I CRAC from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I CRAC had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La 3+ .6. Studies with the more conventional InsP 3 receptor antagonist heparin revealed that occupation of the intracellular InsP 3 -sensitive receptors was not necessary for the activation or maintenance of I CRAC . Similarly, the InsP 3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I CRAC . Exposure to Li + , which reduces InsP 3 levels by interfering with inositol monophosphatase, also failed to alter I CRAC . Caffeine and Li + did not affect the size of the intracellular Ca 2+ signal that arose when external Ca 2+ was re-admitted to cells which had been pre-exposed to thapsigargin/ionomycin in Ca 2+ -free external solution.7. Our findings demonstrate that the cytoskeleton does not seem to regulate calcium influx and that functional InsP 3 receptors are not required for activation of I CRAC . If the secretion-like coupling model indeed accounts for the activation of I CRAC in RBL-1 cells, ...

Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel, cause neonatal diabetes. Many patients also suffer from hypotonia (weak and flaccid muscles) and balance problems. The diabetes arises from suppressed insulin secretion by overactive KATP channels in pancreatic beta-cells, but the source of the motor phenotype is unknown. By using mice carrying a human Kir6.2 mutation (Val59-->Met59) targeted to either muscle or nerve, we show that analogous motor impairments originate in the central nervous system rather than in muscle or peripheral nerves. We also identify locomotor hyperactivity as a feature of KATP channel overactivity. These findings suggest that drugs targeted against neuronal, rather than muscle, KATP channels are needed to treat the motor deficits and that such drugs require high blood-brain barrier permeability.