2017 - lnvestigation of the Ion Channels hTMEM16A/Ano1 and TRPC5 and their Modulation by Intracellular Calcium
SyncroPatch 384PE poster, BPS Meeting 2017 (1.3 MB)
hTMEM16A/Anoctamin1 is a Ca2+ activated Cl- channel (CACC) which has a broad functional spectrum in processes including trans-epithelial ion transport, olfaction, photo-transduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. The ion channel has been implicated to play a role in a number of health disorders and may be an important therapeutic target in cystic fibrosis, asthma, pain and some human cancers.
hTRPC5 is a non-selective cation channel expressed in many areas of the brain particularly the hippocampus, amygdala and cerebellum, amongst others. Although the physiological and patho-physiological role of hTRPC5 is not fully known, it does appear to be important in neuronal function, in particular during development where it is involved in hippocampal neurite outgrowth and growth cone morphology. Knockout mouse studies have also revealed that TRPC5 plays an essential role in innate fear. hTRPC5 is expressed in some regions outside of the CNS including the heart where it contributes to cardiac hypertrophy in heart failure.
hTMEM16A and hTRPC5 are activated by elevated cytosolic Ca2+ concentrations. To initiate channel activity intracellular solution must be exchanged from a Ca2+ free solution to a solution containing Ca2+. Some of the common instruments for automated patch clamp are limited to do an internal exchange at all. But also for the other devices it is challenging because they rely on fluoride in the internal solution to perform proper seals. Unfortunately fluoride has a strong tendency to precipitate with calcium, which makes it impossible to correctly adjust a precise Ca2+ concentration needed for channel activation. Furthermore current run-down or desensitization are common problems associated with recording hTMEM16A7 and hTRPC510.
On the SyncroPatch 384PE we developed two very reliable assays for hTMEM16A and hTRPC5 in the presence of fluoride-free intracellular solutions using the internal perfusion feature. The presented data show that the intracellular solution can be exchanged in a very robust manner to investigate Ca2+ sensitivity. Besides voltage dependence and pharmacology characteristics of hTMEM16A and hTRPC5 are presented on this poster. The hTMEM16A assay was established using the PE/Felix wheatear the hTRPC5 assay was run on the PE/Biomek.