Electrophysiological Research On The Offset Response Of PKDL Channels To Acid Stimulation

Background and Objective:TRPP is a very important subfamily in TRP. Since PKD2 was found to be related to Autosomal Dominant Polycystic Kidney Disease, it was named as TRPP. This subfamily includes PKD1,PKD2,PKD2L1,PKD1L3,PKD2L2 and so forth. The complex formed by PKD1L3 and PKD2L1 is tightly related to cell sensitivity to mechanical stimulation. The complex formed by PKD1 and PKD2 closely resembles PKD1L3/2L1 complex. In 2006, according to the papers published by three research groups, it was suggested that PKDL channel was the sought-after candidate for proton sensing in taste cells. In this passage, we firstly made sure that the inward current is an offset response rather than a delayed response. Then, we studied the relationship between off-response and pH. At last, we examined the Ca2+ dependency of off current. Although the mechanism of the acid code is still unclear, we try to give some explanation in this passage using the electrophysiology method.Methods:HEK293 cells are raised in DMEM culture medium (also contain fetal bovine serum, Kanamycin and Ampicillin). We used Lipofectamine 2000 (Invitrogen) to do the transfection with GFP,PKD1L3,PKD2L1.24-48 hours after the transfection, we did the whole-cell patch clamp experiment on the HEK293 cells. We used Multiclamp 700A amplifier and pCLAMP software to amplify electronic signals and analyzed signals. The data collection interface is Digidata 1322A. The sampling frequency was 10 kHz, the filter 1 kHz. Holding potential was-60 mV. We used the Rapid Solution Changer to change different ESCs.Results:1. The pH of the solution after giving acid does not have obvious effect on the off-response.2. The concentration of Ca2+ in ECS has great effect on the attenuation time of the off current. When Ca2+ free ECS was used, it took nearly 20 seconds for the off current to return down to baseline, while when Ca2+ was 10 mM, it took only less than 5 seconds.3. The concentration of Ca2+ in ICS also has a great effect on the attenuation time of the off current. When there was 0.5 mM EGTA in ICS, the off current returned down to baseline so fast within less than 1 second. The attenuation time differed significantly among three the ICSs.4. The cells transfected with PKD2L1 or PKD2 alone also exhibit the off current.Conclusion:Not only the pH of acid but also the pH of the solution after giving acid can have some kind of effect on the off response. PKDL channels seem regulated by different Ca2+ concentration in ECS and ICS.