Effects And Mechanisms Of Docosahexenoic Acid On Large Conductance Ca+-activated K+ Channels In Retinal Smooth Muscle Cells

Objective Using single-channel patch-clamp techniques,we sought to investigate the characteristics of large conductance Ca2+-activated K+(BK)channels.This study was using single-channel patch-clamp techniques and Western Blot and reverse transcription-polymerase chain reaction(RT-PCR)to investigate the effects of docosahexenoic acid(DHA)on BK channels in normal retinal artery smooth muscle cells(RASMCs).Methods(1)The BK currents in RASMCs were recorded by inside-out single channel patch-clamp technique;(2)Effects of DHA on BK channels were studied by patch-clamp in inside-out single channel configuration.(3)The protein and mRNA expressions of ?1 subunit of BK channels in RASMCs from control group,low and high doses of DHA groups were measured by Western Blot and RT-PCR.Results(1)At 1 mol/L calcium concentration in external solution and testing potentials at OmV,20mV,40mV,60mV,80mV and 100mV,the open probabilities(NPo)of BK channels were respectively(0.0001±0)?(0.0041±0)?(0.0386±0.0002)?(0.0625±0.0003)?(0.4577±0.0032)and(1.7040±0.0383)(F=45.6825,P<0.05);At Omol/L,0.01mol/L,0.1mol/L,1 mol/L,10mol/L and 100mol/L calcium concentration in external solution,the NP0 of BK channels at 60 mV testing potentials were respectively 0?(0.0030±0)?(0.0100±0.0032)?(0.0432±0.0030?(0.6363±0.0251)and(3.0847±0.1187)(F=43.7799,P<0.05);(2)At test potential 60 mV,1?mol/L calcium,the NPo of BK channels were(0.0444±0.0012),(0.0812±0.0042),(0.20900±0.0061),(0.3105±0.0053),(0.4650±0.0078)and(0.4977±0.0145)with perfusate of 0?mol/L,1?mol/L,3?mol/L,5?mol/L,7.5?mol/L and 10 ?mol/L DHA,DHA activated BK channels in a dose-dependent manner(F=252.6885,P<0.05).(3)There was no significant difference in the protein and mRNA expression of BK-?1 subunit in control group,low-dose DHA group and high-dose DHA group(F=1.172,P>0.05)(F=0.3797,P>0.05).Conclusions(1)BK channels can be activated by increased testing potentials and calcium concentrations in external solution.(2)DHA can directly activate BK channels,no increasing in subunit expression of BK channels.