The development of automated patch clamp assays for canonical transient receptor potential channels TRPC3, 6, and 7.

The canonical transient receptor potential channel subfamily (TRPC3, TRPC6, and TRPC7) contains Ca(2+) permeable non-selective cation channels that are widely expressed in a variety of tissues. There is increasing evidence implicating TRPC channels, particularly TRPC3 and 6, in physiological and pathophysiological processes, eliciting interest in these channels as novel drug targets. Electrophysiology remains a benchmark technique for measuring ion channel function and accurately determining the pharmacological effects of compounds. In this report we describe the development of TRPC inhibitor assays on 2 automated planar patch clamp platforms-the IonWorks(®) Quattro™ and QPatch(®) systems. To enable activation of TRPC channels by carbachol, Chinese Hamster Ovary-K1 cells stably expressing the muscarinic M3 receptor were transduced with human TRPC3, TRPC6, or TRPC7 using BacMam viruses. TRPC3, 6, and 7 currents could be recorded on both platforms. However, the design of each platform limits which assay parameters can be recorded. Due to its continuous recording capabilities, the QPatch can capture both the activation and decay of the response. However, the transient nature of TRPC channels, the inability to reactivate and the large variation in peak currents limits the ability to develop assays for compound screening. The IonWorks Quattro, due to its discontinuous sampling, did not fully capture the peak of TRPC currents. However, due to the ability of the IonWorks Quattro to record from 64 cells per well, the variation from well to well was sufficiently reduced allowing for the development of medium-throughput screening assays.

Synthetic 6-aryl-2-hydroxy-6-ketohexa-2,4-dienoic acid substrates for C-C hydrolase BphD: investigation of a general base catalytic mechanism.

A chemical synthesis of the 2-hydroxy-6-ketohexa-2,4-dienoic acid intermediates on bacterial meta-cleavage pathways has been established, using a Heck coupling strategy. Coupling of ethyl 3-bromo-2-acetoxyacrylate with 1-aryl vinyl ketals or 1-aryl allylic alcohols proceeded in 70-90% yield. Heck coupling with an alkyl vinyl ketal was also successful, allowing the synthesis of an alkyl-substituted ring fission intermediate. The synthetic ring fission intermediates were used to investigate the enzymatic reaction catalysed by C-C hydrolase BphD from Pseudomonas LB400. A reduced substrate analogue 2,6-dihydroxy-6-phenylhexa-2,4-dienoic acid was processed enzymatically to benzaldehyde by C-C hydrolase BphD, consistent with a catalytic mechanism involving general base-catalysed attack of water to give a gem-diol intermediate, and not consistent with a nucleophilic mechanism. A series of para-substituted 2-hydroxy-6-keto-6-phenylhexa-2,4-dienoic acid substrates were assayed against BphD, and the derived Hammett plot (rho=-0.71) is consistent with a departing carbanion in the transition state for C-C cleavage.