CHARACTERIZATION OF LARGE-CONDUCTANCE CHLORIDE CHANNELS IN RABBIT COLONIC SMOOTH-MUSCLE

被引:54
作者
SUN, XP
SUPPLISSON, S
TORRES, R
SACHS, G
MAYER, E
机构
[1] UNIV CALIF LOS ANGELES,DEPT MED,LOS ANGELES,CA 90024
[2] UNIV CALIF LOS ANGELES,DEPT PHYSIOL,LOS ANGELES,CA 90024
来源
JOURNAL OF PHYSIOLOGY-LONDON | 1992年 / 448卷
关键词
D O I
10.1113/jphysiol.1992.sp019046
中图分类号
Q189 [神经科学];
学科分类号
071006 ;
摘要
1. A large-conductance Cl- channel was characterized in cell-free membrane patches from the rabbit longitudinal colonic smooth muscle using the patch clamp technique. In addition, the regulation of these channels by neurokinin-1 (NK-1) receptor agonists and G proteins was studied. 2. No spontaneous channel activity was observed in cell-attached patches at the cell resting potential, or in excised patches at pipette potentials (V(p)) between -20 and 20 mV. In excised patches, channel activity could be induced in thirty-six out of ninety-six patches by holding the patch at V(p) values more negative than -60 mV or more positive than 60 mV. Once induced, the channel showed a bell-shaped voltage activation curve in high symmetric [Cl-], with maximal open probability between 20 and -5 mV. Varying cytosolic calcium concentration ([Ca2+]) between 5 x 10(-8) M and 1.0 mM had no effect on the voltage activation of the channel. 3. In inside-out and outside-out patches, when pipette and bath solutions contained equal [Cl-] (130 mM), the anion channel showed a linear current-voltage (I-V) relationship between -60 and 60 mV with a slope conductance of 309 +/- 20 pS (n = 13). Reversal potential measurements indicated that the channel was selective for Cl- over Na+ and K+ (P(Cl)/P(Na) = 6:1). 4. Channel openings from the closed state to the full open state as well as transitions through smaller conductance states were observed. The smallest detectable substate had a conductance of 15.6 pS. Based on the similarities in selectivity and linearity of the I-V curve of the smaller conductances with the full open state, and kinetic analysis of channel activity, it is concluded that the large conductance channel is composed of multiple substates which can either open and close independently, or simultaneously via a main gate. 5. The stilbene derivative diiso-thiocyanato-stilbene-disulphonic acid (DIDS) and the diphenylamine-2-carboxylate analogue 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) caused a dose-dependent, reversible flicker block of the small conductance and significantly reduced the macroscopic current flow through the channel. 6. In quiescent outside-out patches, when the pipette contained a 140 mM-CsCl solution with 10(-6) M-CaCl2, 1.2 mM-MgCl2 and 1 mM-GTP, and the bath contained Ringer solution, addition of the NK-1 receptor antagonists substance P methylester resulted in activation of the full conductance state and of smaller substates. 7. In inside-out patches, when the NK-1 agonist (10(-10) M) was added to the pipette solution (Ringer) and the bath contained 100 mM-KCl with 10(-6) M-CaCl2, 1.2 mM-MgCl2, 30 mM-TEA+ and 1 mM-GTP, channel activity in the form of substates was seen in eight out of ten patches. The full open state was only seen after additional voltage induction. 8. In quiescent inside-out patches at a V(p) of 20 mV, when the pipette contained 90 mM-N-methyl glucosamine chloride (NMGCl), 30 mM-TEACl, 1 mM-CaCl2 and 1.2 mM-MgCl2, and the bath was a 130 mM-NaCl solution with 1 mM-CaCl2, addition of the non-hydrolysable GTP analogue guanosine 5'-O-(3-thiophosphate) (GTP-gamma-S) resulted in channel activation with both full and smaller conductances observed. When channel activity had been induced by voltage, addition of guanosine 5'-O-(thio)bisphosphate (GDP-beta-S) resulted in reversible inhibition of channel activity.
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页码:355 / 382
页数:28
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