Episodic ataxia (EA) is a human genetic disease characterized by episodes of hemiplegia, seizures and paroxysmal cerebellar incoordination. So far six genetically and clinically forms of EA can be distinguished. Recently, a proline to arginine substitution (P290R) in human EAAT1 has been proposed to be the molecular elicitor of EA6. Here we examined the effects of P290R on transport and channel function in human EAAT1. We used patch-clamp, confocal microscopy and biochemical techniques for analysis of the transporter/channel function and the subcellular distribution of WT and P290R hEAAT1 in heterologous expression systems. Patch clamp analysis indicates that the P/R mutation modifies the current amplitudes of hEAAT1 anion channels up to fourfold, but leaves their anion selectivity unaffected. The relative glutamate-independent anion current amplitude is significantly increased in P290R hEAAT1 and the apparent k_D for L-glutamate is reduced to a tenth. The findings indicate a gain-of-function of the EAAT1 anion channel in the mutant. Therefore we used noise analysis to study the underlying unitary properties of WT and P290R hEAAT1 anion channels. The unitary conductance of WT hEAAT1 anion channels is not changed by P290R, but voltage dependence of open probability is dramatically altered in mutant hEAAT1 anion channels. To further test EAAT1 function we used confocal microscopy analysis and biochemical approaches on P290R hEAAT1. We observed an impairment of surface membrane expression by a third and an almost complete reduction of L-glutamate uptake to background levels in the mutant.