Pharmacological treatment with acetazolamide or transgenic complementation with wild-typeKcna2cDNA partially rescued the motor incoordination inPgumice

Pharmacological treatment with acetazolamide or transgenic complementation with wild-typeKcna2cDNA partially rescued the motor incoordination inPgumice. in the motor incoordination inPgumice. In line with immunochemical analyses showing a significant reduction in the expression of Kv1 channels in the basket cell terminals ofPgumice, expression of homomeric and heteromeric channels containing the Kv1.2(I402T) -subunit in cultured CHO cells revealed subtle changes in biophysical properties but a dramatic decrease in the amount of functional Kv1 channels. Pharmacological treatment with acetazolamide or transgenic complementation with wild-typeKcna2cDNA partially rescued the motor incoordination inPgumice. These results suggest that independent of known mutations inKcna1encoding Kv1.1,Kcna2mutations may be important molecular correlates underlying human cerebellar ataxic disease. Keywords:Mouse Genetics, Mutant, Neurological Diseases, Neuron, Potassium Channels, CHO Cells, Inhibitory Post-synaptic Currents, Voltage-gated Potassium Channel Kv1.2, Missense Mutation == Introduction == Voltage-gated potassium channels play a key role in neuronal excitability and plasticity and are critical in establishing resting membrane potential and firing thresholds, repolarizing action potentials, and limiting excitability (1). Channels are unevenly distributed throughout the brain as a whole and also within individual neurons (210). Therefore, the particular utility of any given channel depends not only on its specific channel properties and stoichiometry but also on its particular localization and density within a cell or cellular compartment. In the cerebellum, the genesKcna1andKcna2encode the voltage-gated potassium channel subunits Kv1.1 and Kv1.2, respectively, which contribute to the low voltage-activated potassium currentIKv1and are coexpressed in the presynaptic GABAergic pinceaus of spontaneously firing basket cell interneurons that provide a strong inhibitory input to Purkinje cells (59). The shunting effect of this inhibitory conductance has been shown through modeling to have a steep correlation with the prolongation of Purkinje cell interspike intervalsin vitro(1113). The cerebellum is involved in the regulation of the initiation and timing of movements and is important for maintaining balance and posture (14). At the core of the cerebellar computational circuitry, the spontaneously spiking Purkinje cells integrate cerebral cortical and sensory, excitatory and inhibitory inputs encoding relevant information in their action potential discharge and communicate the information to the deep cerebellar nuclei for the final output of the cerebellum (15). The total synaptic PF429242 dihydrochloride conductance invading a Purkinje cell effectively functions to clamp the subthreshold membrane voltage, thereby controlling the state of the active conductances that determine each Purkinje cell intrinsic pacemaking activity, which in turn shapes the tonic GABAergic inhibition targeted to the deep cerebellar nuclei (11). Missense mutations ofKcna1are associated with type 1 PF429242 dihydrochloride episodic ataxia (EA1)3(16), whereas mice carrying the EA1-associated V408A mutation show stress-induced loss of motor coordination and a greater frequency and amplitude of spontaneous GABAergic IPSCs in cerebellar Purkinje cells (17). However, the effects of mutations inKcna2are unknown despite Rabbit Polyclonal to BAX the fact that Kv1.1 and Kv1.2 are commonly present within the same tetramers (18,19). To this end, we characterized thein vivoandin vitroeffects of a missense mutation (I402T;Pguallele) in the S6 segment of the Kv1.2 -subunit in mice. Here, we report that mice carrying thePgumutant allele ofKcna2exhibit dominantly inherited chronic motor incoordination, due at least in part to an enhanced GABAergic inhibitory strengthen from basket cells onto Purkinje cells in the cerebellum. == EXPERIMENTAL PROCEDURES == == Mice and ENU Mutagenesis == Male C57BL/6J (B6) mice (The Jackson Laboratory) received three intraperitoneal injections of ENU (85 mg/kg) as previously described (20). Ten weeks after the last ENU injection, the mutagenized males were bred to untreated C3H/HeJ (C3H) female mice (The Jackson Laboratory). G1 progeny (C3HB6F1) of this cross were screened at postnatal day 28 for abnormalities in behavior or appearance using a modified SHIRPA protocol (Centre for Modeling Human Disease, Toronto Centre for Phenogenomics). A male mouse exhibiting an abnormal gait with splayed hind limbs was discovered and namedPingu(Pgu). This founder male was bred to C3H females, and the resulting G2 progeny were put through the same behavior and appearance screen as their sire. Mice exhibiting the characteristic abnormal gait of thePgufounder were classified as affected PF429242 dihydrochloride and further backcrossed to C3H for.