Kinetics of the membrane current mediated by serotonin 5‐HT3 receptors in cultured mouse neuroblastoma cells.

Abstract

SUMMARY1. Ionic currents mediated by serotonin 5-HT3 receptors were studied in the mouse neuroblastoma cell line NIE-115, using suction pipettes for intracellular perfusion and voltage clamp recording. The dependence of the kinetics of the membrane current on serotonin concentration was investigated.2. At a holding potential of -70 mV application of 5-HT (5-hydroxytryptamine creatinine sulphate) causes a transient inward current. The i-V curve of the peak amplitude is linear between -80 and 60 mV. The reversal potential is 20 + 4 mV (mean+ S.D.). The kinetics of the transient ionic current are independent of the holding potential.3. In the presence of 5-HT the membrane current decays to a small steady-state level with a single-exponential time course. The time constant of decay decreases with increasing concentration of the agonist, to a minimum value of 6-5 + 1-5 s for concentrations of 5-HT . 3 ,/M. 4. When the agonist is rapidly removed, single-exponential decay of the ionic current is observed. The time constant of this decay in the absence of 5-HT amounts to 6-9 + 1-5 s and is independent of the membrane potential and of the concentration of 5-HT used.5. In the presence of low concentrations of 5-HT the peak amplitude of the inward current evoked with a high concentration of agonist is gradually reduced. The onset of this desensitization follows the same time course as the decay of the membrane current. In the range from 0 7 to 1-5 ftM-5-HT both kinetic processes show the same steep concentration dependence.6. Recovery from desensitization, measured at variable intervals after removal of the agonist, can be fitted by a single-exponential function with a time constant of 18+4 s.7. The results show that the kinetic properties of the 5-HT3 receptor-mediated ionic current can only be described by a complex, co-operative model.

Selection of distinct conformational states of the 5‐HT3 receptor by full and partial agonists

“…Application of the agonist 5-HT to whole-cell voltage clamped NlE-115 cells induces a rapidly activating cation current, which desensitizes in the continuous presence of the agonist (Neijt et al, 1989). Similar transient ion currents are evoked by the application of other 5-HT3 receptor agonists, e.g., dopamine (Neijt et al, 1986), 2-methyl-5-HT and metachlorophenylbiguanide (mCPBG) (Sepulveda et al, 1991).…”

“…Mouse neuroblastoma cells of the clone N1E-115 (Amano et al, 1972) were grown under conditions identical to those described before by Neijt et al (1989). Differentiation of subcultures of passage numbers 32-42 was initiated on day 2 in subculture by the addition of 1 mM dibutyryl-cyclic-AMP and 1 mM 3-isobutyl-1-methylxanthine to the culture medium.…”

“…Membrane currents were recorded by a suction pipette technique for whole-cell voltage clamp as described by Lee et al (1978) and Neijt et al (1989) Single channel patch clamp experiments 5-HT3 receptor-gated single channels were recorded in the cellattached patch configuration with an EPC-7 patch clamp circuit (List-Electronic, Darmstadt, Germany) as described previously (van Hooft & Vijverberg, 1995). Borosilicate glass pipettes (10-20 MQ) filled with agonist-containing external solution were positioned against the cell membrane.…”

“…These properties include molecular structure (Maricq et al, 1991) and agonist-induced activation and desensitization of ion current (Neijt et al, 1989;Eisele et al, 1993;Yakel et al, 1993), which is mediated by cation channels (Yakel et al, 1990;Yang, 1990). In mouse NIE-115 neuroblastoma cells, 5-HT3 receptors have been investigated in detail.…”

Metal interactions with voltage- and receptor-activated ion channels.

“…These cells express voltage-activated sodium channels, at least two types of voltage-activated Ca channels and both voltage-activated and Ca-activated K channels (4)(5)(6)(7). Recently, serotonin 5-HT3 receptors and neuronal type nicotinic acetylcholine (ACh) receptors, which are directly coupled to distinct cation channels, have been characterized in NlE-115 cells (8,9). The detailed knowledge of the properties of ion channels in neuroblastoma cells and the suitability of these cultured cells for intracellular electrophysiology permit the separation of the various types of ion channels in voltage clamp experiments and thereby the investigation of effects of metal ions on each type of ion channel.…”