TRPC3 is a member of the TRP-channel superfamily, which forms non-selective cation channels with significant permeability for Ca2+ and Na+. TRPC3 is supposed to play important roles in physiological / pathophysiological processes and seems to be an interesting potential pharmacological target. TRPC3 channels can apparently be activated through different ways: in response to receptor stimulation of Gq-protein coupled receptors, such as the muscarinic acetylcholine receptors M1, M3, and M5, or through store depletion of intracellular Ca2+-stores. The aim of the present study was to characterize the impact of different TRPC3-activators on the channel's permeation properties. Therefore, we compared the permeation properties of TRPC3 when activated by two different stimuli: the cholinergic agonist Carbachol and the novel TRPC3-agonist GSK1702934A. Besides investigations with wild-type TRPC3 channels, we also intended to characterize permeation and gating as well a linkage between these functions in TRPC3G652A, a mutant of TRPC3 in which the amino acid glycine in position 652, in the transmembrane domain 6, the putative pore region of the channel, is exchanged with alanine. Whole-cell voltage clamp recordings were made from HEK293 cells, transfected stably with wild-type TRPC3 or the mutant channel TRPC3G652A. The present study shows that the exchange of G652 with alanine results in a decreased lipid-sensitivity of the channel, making G652 a decisive point for the lipid-sensitivity and therefore lipid-mediated gating of TRPC3. Furthermore, our results show that GSK is able to induce currents through wildtype TRPC3 as well as through the G652A mutant. GSK-induced currents were characterized by a change in the ion selectivity as compared to Carbachol-induced currents with both TRPC3 wildtype and TRPC3 G652A, leading us to speculate that GSK is able to influence the ion selectivity of TRPC3 channels through a “pore dilation”-process.