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

Abstract

1. 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. 2. Rapid application of 5-HT (50 microM) 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 approximately 230 ms), but dramatically slowed with time by 1120 +/- 160%. 3. 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 microM) to block voltage-gated Ca2+ channels. The rate of desensitization was also dependent on membrane potential. 4. 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. 5. 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. 6. 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.

Figure 1. Increased [Ca²⁺]_i buffering prevented the slowing in the rate of 5-HT₃R desensitization

A responses elicited by a 10 s application of 50 μM 5-HT (indicated by the horizontal bar) at a holding potential of −70 mV at 0 and 8 min after break-in in the same cell when buffering internal free Ca²⁺ with EGTA (5 mM) and Ca²⁺ (0.5 mM). The values of t½, τ_fast, τ_slow and R_f/s at 0 min for this cell were 145 ms, 138 ms, 2850 ms and 4.1, respectively. The value of t½ at 8 min was 927 ms. B responses in a different cell when buffering internal free Ca²⁺ with BAPTA (20 mM). The values of t½ at 0 and 8 min for this cell were 138 and 165 ms, respectively. C plot of the mean values of t_½ with time in EGTA- (22 cells) or BAPTA-dialysed (24 cells) cells.

Figure 2. Cadmium, but not nickel, prevented the slowing in the rate of 5-HT₃R desensitization

A 5-HT responses in EGTA-dialysed cells with cadmium (100 μM) in the bathing solution prior to break-in. The values of t_½ at 0 and 8 min for this cell were 164 and 224 ms, respectively. B mean values of the maximum percentage increase in t_½ under control conditions (i.e. EGTA-dialysed cells), or with cadmium or nickel (50 μM) in the bathing solution prior to break-in. The number of cells are shown in parentheses. The asterisk indicates a significant difference (P < 0.05) using Student's t test.

Figure 3. Slowing in the rate of 5-HT₃R desensitization was dependent on membrane potential

Mean values of t_½ with time at −50 mV (6 cells) and −90 mV (9 cells).

Figure 4. The kinetics of desensitization were stable in perforated-patch recording conditions

A 5-HT responses at 0 and 8 min. The values of t_½ at 0 and 8 min for this cell were 180 and 192 ms, respectively. B mean value of t_½ (12 cells) with time. C the maximum percentage increase in t_½ in perforated-patch (PP) versus whole-cell recording (WCR) conditions.

Figure 5. Depolarization prior to 5-HT application in perforated-patch recordings slowed desensitization

A 5-HT responses at 20 min (the first time point where 5-HT application was preceded by depolarization to 0 mV; upper trace) and 60 min. The values of t_½ at 20 and 60 min for this cell were 180 and 4000 ms, respectively. B mean values of t_½ with time without (control; ▪; 5 cells) or with the depolarizing prepulse (•; 17 cells) which began at the 20 min time point (indicated by the arrow).

Figure 6. Depolarization-induced slowing of desensitization in perforated-patch recordings was blocked by BAPTA-AM pre-incubation and bath-applied cadmium

A 5-HT responses at 20 and 60 min in a BAPTA-AM-pretreated cell following a depolarizing prepulse to 0 mV (upper traces); the values of t_½ were 431 and 166 ms, respectively. B 5-HT responses at 20 and 60 min with cadmium (100 μM) added to the bathing solution; the values of t_½ were 104 and 96 ms, respectively. C mean values of t_½ with time with a depolarizing prepulse which began at the 20 min time point (indicated by the arrow). BAPTA-AM, 6 cells; cadmium, 4 cells.