Evaluation of the functional roles of canonical TRPC1/4/5 channels in native cells has for a long time been hampered by the lack of highly potent, subtype-selective modulators of these channels. However, with the recent discovery of the direct and potent agonist of these channels, (-)-englerin A (EA) [1], as well as their subtype-specific small-molecule blocker termed Pico145 [2], there is now a good prospect for our better understanding of both the physiological significance and therapeutic potential of these receptor-operated channels, which are widely expressed in various cell types. We have previously extensively characterised biophysical and pharmacological properties of the muscarinic cation current (mICAT), which is activated through synergy between M2 and M3 muscarinic receptors in gastrointestinal myocytes, whereby it underlines cholinergic excitation-contraction coupling [3]. Since mICAT is predominantly mediated by TRPC4 channels [4] we now aim to characterise its inhibition by Pico145. Ileal myocytes that were freshly isolated from two month-old male BALB/c mice were used for patch-clamp recordings of whole-cell currents. These were carried out at room temperature (20-22 0C) using symmetrical Cs+ containing (125 mM) solutions with [Ca2+]i ‘clamped’ at 100 nM using 10 mM BAPTA/4.6 mM CaCl2 mixture, as necessary for mICAT isolation [4]. The steady-state I-V relationships of mICAT were measured by slow (6 s duration) voltage ramps from 80 to ‑120 mV, which were applied every 30 s. Values are means±S.E.M. Carbachol (50 µM)-induced mICAT was already strongly inhibited by Pico145 at 1 pM. However, considering that the inhibition developed rather slowly, thus overlapping with current desensitisation, for a more reliable quantitative characterisation of this effect we used intracellularly applied GTPγS (200 µM) to induce mICAT bypassing muscarinic receptors, in which case no or very little desensitisation was present. The IC50 value for the inhibitory effect of Pico145 on this current was found to be 3.1 pM. The inhibition developed rather slowly, with the mean time constant of 140±6 s (n=9), which was surprising for such a high affinity blocker. Moreover, voltage steps to -120 mV relieved the inhibition, while it was rapidly re-established upon stepping to 80 mV with the mean time constant of 108.0±10.3 ms (n=6). Notably, EA (10 nM)-induced current was much more resistant to the inhibitory action, with 100 pM Pico145 causing current inhibition by only 43±15 % (n=6). Finally, functional assessment of the Pico145 effect using in vitro tensiometry showed significant concentration-dependent suppression of 50 µM carbachol-induced ileal contractions by Pico145 applied cumulatively at 0.1-100 pM.  These properties of Pico145 are generally consistent with those reported for overexpressed TRPC4 channels (including its reduced potency at elevated EA concentration as well as some voltage dependence) [2], yet we note substantially higher (by 1-2 orders) affinity of Pico145 in the case of native TRPC4-mediated mICAT. All animal studies using  BALB/c mice were carried out in accordance with the recommendations of the EU Directive 2010/63 on the protection of animals used for scientific purposes and approved by the Institutional Ethics Committee (No. 04/20).