Activation of recombinant h 5-HT1B and h 5-HT1D receptors stably expressed in C6 glioma cells produces increases in Ca2+-dependent K+ current.

The putative coupling between stably expressed recombinant h 5-HT1B or h 5-HT1D receptors and K+ channels which regulate excitability was investigated in C6 glioma cells. Outward K+ currents (IK) were examined in nontransfected C6 glioma cells and in cells expressing cloned h 5-HT1B or h 5-HT1D receptors using the patch-clamp technique in the whole-cell configuration. IK was elicited by a depolarizing step from a holding potential of -60 mV. In C6 glioma cells expressing either recombinant h 5-HT1B or h 5-HT1D receptors, sumatriptan similarly increased IK in a concentration-dependent manner (maximum increase 19.4+/-7.2%, n=8, P<0.05 and 25.1+/-3.9%, n=6, P<0.001, respectively) with EC50 values (geometric mean with 95% confidence intervals in parentheses) of 56.3 nM (7.9-140 nM) and 68.7 nM (16-120 nM), respectively. Sumatriptan failed to elicit increases in IK in non-transfected cells, confirming a specific involvement of the respective membrane h 5-HT1B and h 5-HT1D receptors in transfected C6 cells. In the presence of the mixed 5-HT1B/D receptor antagonist GR 127935 (0.1 microM), sumatriptan (1 microM) failed to significantly increase IK in C6 cells expressing h 5-HT1B receptors (-7.5+/-3.5%, P=NS, n=6), although a higher concentration of GR 127935 (1 microM) was required to significantly inhibit sumatriptan-evoked increases in IK in C6 cells expressing h 5-HT1D receptors (-1.8+/-3.5%, P=NS, n=6), confirming that sumatriptan-evoked responses were indeed mediated by h 5-HT1B and h 5-HT1D receptors, respectively. In C6 cells expressing either cloned h 5-HT1B or h 5-HT1D receptors, sumatriptan-induced increases in IK were prevented by the calcium chelator EGTA (5 mM) when included in the patch pipette (maximum increase 0.57+/-0.6%, n=3, P=NS and -2.8+/-1.6%, n=5, P=NS, respectively). In C6 cells expressing cloned h 5-HT1B receptors, sumatriptan (1 microM) similarly failed to significantly increase IK in the presence of dibutyryl cAMP (10 microM) or when a nominally Ca2+-free medium was included in the patch pipette (-19.4+/-5.1%, n=5 and -5.2+/-4.3%, n=5, respectively, P=NS in each case). In addition, the Ca2+-dependent K+ channel blockers iberiotoxin (0.1 microM) and tetraethylammonium (TEA, 1 mM) abolished sumatriptan-induced increases in IK (-0.5+/-1.0%, n=4 and -3.9+/-3.1%, n=4, respectively, P=NS in each case) in C6 cells expressing h 5-HT1B receptors, confirming the involvement of Ca2+-dependent K+ channels. In C6 cells expressing cloned h 5-HT1B receptors, sumatriptan (1 microM) similarly failed to significantly increase IK after 30-min incubation with thapsigargin (1 microM) or when heparin (2 mg/ml) was included in the patch pipette (1.1+/-0.4%, n=5 and 1.2+/-2.4%, n=5, respectively, P=NS). In conclusion, evidence is provided that both recombinant h 5-HT1B and h 5-HT1D receptors stably transfected in C6 glioma cells are positively coupled to Ca2+-dependent K+ channels, and the outward hyperpolarizing current mediated by these channels is dependent upon IP3 receptor-mediated intracellular Ca2+ release.