Ca2+ influx through voltage‐gated Ca2+ channels regulates 5‐HT3 receptor channel desensitization in rat glioma × mouse neuroblastoma hybrid NG108–15 cells

The kinetics of desensitization of the 5-HT3 receptor (5-HT3R)-gated ion channel were investigated using whole-cell and perforated-patch recording techniques in NG108-15 cells. Rapid application of 5-HT (50 μM) elicited a 5-HT3R-mediated inward current response that desensitized completely in the continued presence of agonist. In the whole-cell recording configuration (holding potential of −70 mV) while buffering internal calcium (Cai2+) with 5 mM EGTA (0.5 mM added Ca2+; with an estimated free [Ca2+] of 30 nM), the rate of desensitization was initially rapid (with a half-time of ≈230 ms), but dramatically slowed with time by 1120 ± 160 %. This slowing in the rate of desensitization was reduced by stronger Ca2+ buffering (20 mM BAPTA, without added Ca2+), or by the bath application of cadmium (100 μM) to block voltage-gated Ca2+ channels. The rate of desensitization was also dependent on membrane potential. In perforated-patch recordings, the rate of desensitization remained constant. However, a slowing in the desensitization rate could be induced by depolarizing cells immediately prior to the application of 5-HT. The depolarization-induced slowing was blocked by incubating cells with BAPTA-AM (a membrane-permeant analogue of BAPTA) or by the bath application of cadmium. These data suggest that Ca2+ influx through a cadmium-sensitive voltage-gated Ca2+ channel increases the cytoplasmic Ca2+ concentration ([Ca2+]i) and induces a dramatic slowing in the kinetics of desensitization of the 5-HT3R channel. These data provide evidence for cross-talk between voltage-gated Ca2+ channels and 5-HT3Rs in NG108-15 cells. The 5-HT3 receptor (5-HT3R) is a ligand-gated ion channel found in the central and peripheral nervous systems where it can mediate fast synaptic transmission and neurotransmitter release (see review by Jackson & Yakel, 1995). Like other ligand-gated ion channels, the 5-HT3R channel undergoes desensitization (i.e. a closed and/or inactivated state) in the continued presence of agonist (Katz & Thesleff, 1957). For excitatory and inhibitory synaptic responses in the CNS, desensitization of glutamatergic and GABAergic receptor channels is thought to significantly affect the shape of the synaptic responses (see review by Jones & Westbrook, 1996). The molecular mechanism of desensitization is unknown and controversial (e.g. see Lin & Stevens, 1994; Colquhoun & Hawkes, 1995), but several factors are known to regulate the kinetics of desensitization. For many ligand-gated ion channels including the 5-HT3R channel, changes in the cytoplasmic Ca2+ concentration ([Ca2+]i) and/or Ca2+-dependent signal transduction cascades are known to play a role. Conditions that alter [Ca2+]i modulate desensitization of the NMDA glutamate receptors (Legendre, Rosenmund & Westbrook, 1993; Vyklick´y, 1993; Medina et al. 1995; Kyrozis, Goldstein, Heath & MacDermott, 1995; Kyrozis, Albuquerque, Gu & MacDermott, 1996), P2X ATP receptors (Khiroug, Giniatullin, Talantova & Nistri, 1997b), nicotinic ACh receptors (Khiroug, Giniatullin, Sokolova, Talantova & Nistri, 1997a) and vanilloid or capsaicin receptors (Docherty, Yeats, Bevan & Boddeke, 1996; Koplas, Rosenberg & Oxford, 1997). In addition, a dependence of desensitization kinetics on the Ca2+-calmodulin-regulated protein phosphatase, calcineurin, has been reported for the 5-HT3, NMDA, GABAA, nicotinic ACh and capsaicin receptors (see review by Yakel, 1997). The present study has investigated the role of [Ca2+]i in regulating desensitization of the 5-HT3R channel. The NG108-15 rat glioma × mouse neuroblastoma hybrid cell line was used as a model system to study 5-HT3R channel desensitization (Yakel, Shao & Jackson, 1991). NG108-15 cells have a number of mechanisms for increasing [Ca2+]i, including voltage-gated Ca2+ channels, release from internal Ca2+ stores, Ca2+-permeable ligand-gated ion channels, and a Na+–Ca2+ exchange pump (Freedman, Dawson, Villereal & Miller, 1984; Caulfield, Robbins & Brown, 1992; Kasai & Neher, 1992; Chueh & Kao, 1993; Lo & Thayer, 1993, 1995; Hsu, Chou & Chueh, 1995). In this report, utilizing whole-cell and perforated-patch recording techniques, we have demonstrated that an increase in [Ca2+]i, via the influx of Ca2+ through cadmium-sensitive voltage-gated Ca2+ channels, can dramatically decrease the rate of 5-HT3R channel desensitization. These data provide evidence for a novel mechanism of regulation of the 5-HT3R, and represent a new example of regulation of ligand-gated ion channels by voltage-gated Ca2+ channels.