This study set out to identify structures that are relevant for permeation and gating in the cation channel TRPC3. Point mutations were introduced by side-directed mutagenesis and whole-cell voltage clamp recordings were made from HEK293 cells, transfected either transiently or stably with wild-type TRPC3 or mutated channels. TRPC3 consists of six hydrophobic membrane spanning regions of which transmembrane segment 5 and 6 are likely to form the central pore region. According a hypothetical model of the TRPC3 pore structure, the short helical loop between TM5 and TM6, which lines the immediate permeation pathway, might constitute the selectivity filter. Several negatively charged amino acid residues (glutamate and aspartate) within this loop are potential key-elements of this selectivity filter, which governs permeation of TRPC3.The results of the present study show that neutralization of glutamate to glutamine at position 630 of TRPC3 generates a channel with completely altered current-voltage relationship and significantly decreased barium permeability. By contrast, introduction of positively charged lysine at position 630 eliminated channel function. In essence, these findings demonstrate the great importance of E630 as part of the permeation pathway of TRPC3. A glutamate-to-glutamine mutation at position 616 resulted in a nonfunctional channel, which does not longer respond to carbachol. Thus glutamate at position 616, hypothetically located at the outer pore vestibule, appears to be important for PLC-mediated TRPC3 activation.Moreover, exchange of negatively charged glutamate to either neutral glutamine or positive lysine at position 644 did not have any significant effect on permeation properties, confirming the hypothetical localization of E644 within the hydrophobic region of the TRPC3 complex, which excludes a participation in ion permeation within TRPC3.