Electrophysiology of parasympathetic neurones isolated from the interatrial septum of bull-frog heart.
1. Whole-cell voltage-clamp techniques were used to study the voltage-dependent membrane conductances in parasympathetic neurones enzymatically isolated from the interatrial septum of bull-frog heart and maintained in short-term (1-10 day) tissue culture. 2. The resting potential of the isolated neurones averaged -55.4 +/- 1.1 mV (+/- S.E.M., n = 11). Action potentials evoked in the isolated cells by brief (1-2 ms) current injections were similar to those recorded from neurones in the 'intact' septum. The amplitude of action potentials of isolated neurones averaged about 113 mV, with a peak depolarization of +32.8 +/- 2.8 mV and after-hyperpolarization of -80.0 +/- 2.8 mV. 3. The pattern of membrane currents recorded using voltage clamp with 'normal' external (containing 110 mM-Na+) and internal (110 mM-K+) solutions consisted of a rapidly activating and inactivating inward current followed by a slower, sustained outward current. 4. The inward components of current were isolated by using an internal solution in which Cs+ and TEA+ (tetrathylammonium) ions replaced K+. Depolarizations from holding potentials of -50 to -70 mV produced inward currents which had an initial transient phase followed by a maintained, or very slowly inactivating, component. The current-voltage relation for the initial transient phase reached a peak at membrane potentials near 0 mV, while the maintained phase, measured, for example, at the end of 50 ms voltage-clamp steps, had its peak near +10 mV. 5. The transient component of inward current was carried primarily by Na+ ions, as replacement of Na+ by TEA+ in the external solution abolished the transient. This current was thus identified as a voltage-dependent Na+ current, INa. The maintained component was greatly attenuated by removing 80-90% of the external Ca2+ ions, and it was abolished by divalent cations such as Cd2+ (0.2-0.4 mM), Ni2+ (0.5 mM) and La3+ (10-100 microM). This maintained component was thus a voltage-dependent calcium current, ICa. 6. About 80% of INa recorded in the presence of low (0.2-0.5 mM) external Ca2+ and 2 microM-LaCl3 was blocked by tetrodotoxin (TTX) with an apparent Kd of about 8 nM. The remaining 20% of INa was resistant to block by 2-10 microM-TTX. However, the 'TTX-resistant' component of INa was blocked by Cd2+ (0.2-0.4 mM). 7. The voltage-dependent calcium current, ICa, measured in saline in which Na+ was replaced by N-methyl-D-glucamine, activated near -40 mV and reached a peak near +10 to +15 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Clark
R B
RB
Department of Medical Physiology, University of Calgary, Canada.
Tse
A
A
Giles
W R
WR
D000200:Action Potentials; D000818:Animals; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D004594:Electrophysiology; D006346:Heart Septum / Q000294:innervation*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D010275:Parasympathetic Nervous System / Q000187:drug effects / Q000502:physiology*; D011188:Potassium / Q000502:physiology; D011892:Rana catesbeiana; D012964:Sodium / Q000502:physiology; D013779:Tetrodotoxin / Q000494:pharmacology
D013779:Tetrodotoxin; D012964:Sodium; D011188:Potassium; D002118:Calcium