Ca2+ influx through carbachol-activated non-selective cation channels in guinea-pig gastric myocytes.
1. Ca2+ microfluorometry (100 microM K5 fura-2) and the voltage-clamp technique were combined to study the effect of carbachol (CCh, 50 microM) in inducing currents (ICCh) through non-selective cation channels (NSCCCh) and increments in global cytosolic Ca2+ concentration (Delta[Ca2+]c). 2. In Na+-containing bath solution, ICCh fell from an initial phasic to a subsequent small (5 %) tonic component; Delta[Ca2+]c fell to zero. Tonic ICCh and [Ca2+]c became prominent after substitution of extracellular 140 mM Na+ by 140 mM Cs+. Tonic ICCh and Delta[Ca2+]c were insensitive to intracellular heparin (3 mg ml-1) and ryanodine (4 microM), i.e. they did not depend on Ca2+ release from sarcoplasmic reticulum (SR). 3. Single channel currents of NSCCCh could be resolved in whole-cell recordings. Substitution of Na+ by Cs+ increased NSCCCh activity by one order of magnitude and slope conductance from 22 to 30 pS. Extracellular quinidine (3 microM) reversibly blocked the NSCCCh activity. 4. Both tonic ICCh and tonic Delta[Ca2+]c (a) followed a similar time course of activation, desensitization and facilitation, (b) were reversibly blocked by 3 microM quinidine, and (c) persisted upon block of SR Ca2+ release. 5. A Ca2+ fractional current of tonic ICCh (fCa) of 0.009 was calculated by comparing the ratio Delta[Ca2+]c (corrected for simultaneous Ca2+ redistribution) over ICCh with depolarization-induced *Delta[Ca2+]c (Delta[Ca2+]c calculated from ICa induced by a 400 ms depolarization from -60 to 0 mV at 2 mM [Ca2+]o, 145 mM [Cs+]o) over ICa. fCa was 0.023 at [Ca2+]o = 4 mM. 6. With 110 mM extracellular CaCl2 and 145 mM intracellular CsCl, ICCh reversed at +19.5 mV suggesting a permeability ratio PCa/PCs of 2.8. 7. We conclude that Ca2+ influx through NSCCCh under physiological [Ca2+]o could induce Delta[Ca2+]c. The fCa was, however, much smaller than the one calculated from the reversal potential.
Kim
S J
SJ
Department of Physiology and Biophysics, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Korea.
Koh
E M
EM
Kang
T M
TM
Kim
Y C
YC
So
I
I
Isenberg
G
G
Kim
K W
KW
D000465:Algorithms; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002586:Cesium / Q000378:metabolism / Q000494:pharmacology; D003592:Cytophotometry; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004558:Electric Stimulation; D004594:Electrophysiology; D005260:Female; D005753:Gastric Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007473:Ion Channels / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D018721:Muscarinic Agonists / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011802:Quinidine / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology
D007473:Ion Channels; D018721:Muscarinic Agonists; D002586:Cesium; D002217:Carbachol; D012964:Sodium; D011802:Quinidine; D002118:Calcium