Novel artificial ion channels (1 and 2) based on CB[n] (n = 6 and 5, respectively) synthetic receptors with carbonyl-fringed portals (diameter 3.9 and 2.4 A, respectively) can transport proton and alkali metal ions across a lipid membrane with ion selectivity. Fluorometric experiments using large unilamellar vesicles showed that 1 mediates proton transport across the membranes, which can be blocked by a neurotransmitter, acetylcholine, reminiscent of the blocking of the K+ channels by polyamines. The alkali metal ion transport activity of 1 follows the order of Li+ > Cs+ approximately Rb+ > K+ > Na+, which is opposite to the binding affinity of CB[6] toward alkali metal ions. On the other hand, the transport activity of 2 follows the order of Li+ > Na+, which is also opposite to the binding affinity of 2 toward these metal ions, but virtually no transport was observed for K+, Rb+, and Cs+. It is presumably because the carbonyl-fringed portal size of 2 (diameter 2.4 A) is smaller than the diameters of these alkali metal ions. To determine the transport mechanism, voltage-clamp experiments on planar bilayer lipid membranes were carried out. The experiments showed that a single-channel current of 1 for Cs+ transport is approximately 5 pA, which corresponds to an ion flux of approximately 3 x 107 ions/s. These results are consistent with an ion channel mechanism. Not only the structural resemblance to the selectivity filter of K+ channels but also the remarkable ion selectivity makes this model system unique.

BACKGROUND AND PURPOSEThe Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca 2+ sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels. EXPERIMENTAL APPROACHWe examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques. KEY RESULTSThe Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on-and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca 2+ sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPgS-activated non-selective cation currents. CONCLUSIONS AND IMPLICATIONSThe Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca 2+ sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca 2+ sensitivity of the contractile apparatus.