Perforated patch recording of L-type calcium current with beta-escin in guinea pig ventricular myocytes.
OBJECTIVE
To establish a perforated patch recording (PPR) mode with beta-escin and compare L-type calcium current (I(Ca,L)) recorded under PPR and normal whole-cell recording (WCR) condition in isolated guinea-pig ventricular myocytes.


METHODS
Single myocytes were dissociated by enzymatic dissociation method. beta-escin was added to the pipette solution to perforate the cell membrane and obtain PPR mode. I(Ca,L) was recorded using PPR and WCR techniques.


RESULTS
beta-Escin 20, 25, and 30 micromol/L could permeabilize the cell membrane and obtain PPR mode. With beta-escin 25 micromol/L, the success rate was highest (16/17, 94 %) and the time required for permibilization was 2-15 (8+/-4) min. Run-down of I(Ca,L) was considerably slower in PPR than in WCR condition. The amplitude of I(Ca,L) was decreased by 36 % at 20 min after the formation of WCR, while it was slowly decreased by 8 % at 30 min after the formation of PPR. The current-voltage relation (I-V) curves, activation and inactivation curves of I(Ca,L) were not significantly different between WCR and PPR. The inactivation rate of ICa,L was slower in PPR than in WCR, the faster inactivation time constant (tau(f)) was longer in PPR than in WCR at membrane potentials of -20 mV -- +10 mV (n=6, P<0.05), and the slower time constant (tau(s)) was also longer in PPR than in WCR at membrane potentials of -10 mV to +10 mV (n=6, P<0.05). There was no significant difference between the activation rate in WCR and PPR.


CONCLUSIONS
Using beta-escin 25 micromol/L can easily obtain stable PPR in isolated guinea-pig ventricular myocytes, and this method is useful in dealing with channels, which show run-down under normal WCR such as L-type Ca channel.
Fu
Li-Ying
LY
Department of Pharmacology, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China. liyingfu215@yahoo.com.cn
Wang
Fang
F
Chen
Xue-Song
XS
Zhou
Hong-Yi
HY
Yao
Wei-Xing
WX
Xia
Guo-Jin
GJ
Jiang
Ming-Xing
MX
D000818:Animals; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002469:Cell Separation; D004928:Escin; D005260:Female; D006168:Guinea Pigs; D006352:Heart Ventricles; D008297:Male; D032383:Myocytes, Cardiac / Q000166:cytology*; D018408:Patch-Clamp Techniques / Q000379:methods
D020746:Calcium Channels, L-Type; D004928:Escin