Activation of recombinant h 5-HT(1B) and h 5-HT(1D) receptors stably expressed in C6 glioma cells produces increases in Ca²⁺-dependent K⁺ current

The putative coupling between stably expressed recombinant h 5-HT(1B) or h 5-HT(1D) receptors and K⁺ channels which regulate excitability was investigated in C6 glioma cells. Outward K⁺ currents (I(K)) were examined in nontransfected C6 glioma cells and in cells expressing cloned h 5-HT(1B) or h 5-HT(1D) receptors using the patch-clamp technique in the whole-cell configuration. I(K) was elicited by a depolarizing step from a holding potential of -60 mV. In C6 glioma cells expressing either recombinant h 5- HT(1B) or h 5HT(1D) receptors, sumatriptan similarly increased I(K) 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 I(K) in non-transfected cells, confirming a specific involvement of the respective membrane h 5-HT(1B) and h 5-HT(1D) receptors in transfected C6 cells. In the presence of the mixed 5-HT(1B/D) receptor antagonist GR 127935 (0.1 μM), sumatriptan (1 μM) failed to significantly increase I(K) in C6 cells expressing h 5-HT(1B) receptors (-7.5±3.5%, P=NS, n=6), although a higher concentration of GR 127935 (1 μM) was required to significantly inhibit sumatriptan-evoked increases in I(K) in C6 cells expressing h 5- HT(1D) receptors (-1.8±3.5%, P=NS, n=6), confirming that sumatriptan-evoked responses were indeed mediated by h 5-HT(1B) and h 5-HT(1D) receptors, respectively. In C6 cells expressing either cloned h 5-HT(1B) or h 5-HT(1D) receptors, sumatriptan-induced increases in I(K) 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-HT(1B) receptors, sumatriptan (1 μM) similarly failed to significantly increase I(K) in the presence of dibutyryl cAMP (10 μM) 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 μM) and tetraethylammonium (TEA, 1 mM) abolished sumatriptan-induced increases in I(K) (-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-HT(1B) receptors, confirming the involvement of Ca²⁺-dependent K⁺ channels. In C6 cells expressing cloned h 5-HT(1B) receptors, sumatriptan (1 μM) similarly failed to significantly increase I(K) after 30-min incubation with thapsigargin (1 μM) 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-HT(1B) and h 5-HT(1D) 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.