Comparative electrophysiological study of reconstituted giant vesicle preparations of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel.
Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were reconstituted into two types of giant vesicles: (1) Giant proteoliposomes prepared by freeze-thawing of a mixture of SR vesicles and sonicated phospholipid vesicles without the use of detergent. (2) Giant SR vesicles prepared by fusion of SR vesicles using poly(ethylene glycol) (PEG) as a fusogen and without the addition of exogenous lipid. These giant vesicles were patch-clamped and properties of the single voltage-dependent potassium channel in the excised patch were studied. Single-channel conductance in a symmetrical solution of 0.1 M KCl and 1 mM CaCl2 was 140.0 +/- 10 pS (n = 5) for freeze-thawed vesicles and 136.4 +/- 15 pS (n = 7) for PEG vesicles. Both types of vesicles exhibited a sub-conductance state having 55% of the fully open state conductance. The voltage-dependence of open-channel probability could be expressed in terms of thermodynamic parameters of delta Gi = 0.95 kcal/mol and z = -0.77 for freeze-thawed vesicles and delta Gi = 0.92 kcal/mol and z = -0.87 for PEG vesicles. These values correlated well with previous data obtained by fusion of native SR vesicles with a planar lipid membrane. Channel orientation was found to be conserved in both types of vesicles used in the present study.
Hirashima
N
N
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Ishibashi
H
H
Kirino
Y
Y
D000818:Animals; D004594:Electrophysiology; D015640:Ion Channel Gating; D008081:Liposomes; D008564:Membrane Potentials; D009132:Muscles / Q000737:chemistry*; D010743:Phospholipids / Q000378:metabolism; D015221:Potassium Channels / Q000502:physiology*; D011510:Proteolipids / Q000502:physiology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000502:physiology*
D008081:Liposomes; D010743:Phospholipids; D015221:Potassium Channels; D011510:Proteolipids; C020485:proteoliposomes