ATP‐Activated Nonselective Cation Current in NG108‐15 Cells

Abstract

ATP (1 mM) induced a biphasic increase in intracellular Ca 2~concentration ([Ca2~]), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[17~-3methoxyestra-1,3, 5(10)-trien -1 7-yl]amino]hexyl]-1Hpyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca2~.We examined the mechanism of the AlP-elicited sustained [Ca2~], increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC 50 of ATP was -~0.75mM. The permeability ratio of various cations carrying this current was Na(defined as 1)> Li(0.92 ± 0.01; n = 5)> K(0.89 ± 0.03; n = 6) > Rb(0.55 ± 0.02; n = 6) > Cs~(0.51 ± 0.01; n = 5) > Ca~(0.22 ± 0.03; n = 3) > N-methyl-n-glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg Abbreviations used: ADP~3S, adenosine 5 '-O-(2-thiodiphosphate); AMPCPP, a~8-methylene-ATP;AMPPCP,~3,y-methylene-ATP; ATPyS, adenosine 5 '-O-(3-thiotriphosphate); BzATP, benzoylbenzoic-ATP; [Ca 2~]~, intracellular Ca2~concentration; I-V, current-voltage; 1P3, inositol 1 ,4,5-trisphosphate; 2MeSATP, 2methylthio-ATP; MS -, methanesulfonic acid; NMG N-methyl-Dglucamine; PLC, phosphoinositide-specific phospholipase C; U73122, 1-[6-[17~3-3-methoxyestra-l,3,5(10)-trien-l7-yl]amino]hexyl ] -lH-pyrrole-2,5-dione.

Identification of P2X7 (P2Z) Receptor in N18TG‐2 Cells and NG108‐15 Cells

“…The rank order of agonist potency to induce this current was BzATP 2-methylthio-ATP > ATP > adenosine 5'-O-(3-thiotriphosphate)~°ADP. These properties of the P2X7 receptor are similar to those of the P2Z receptor in NG1O8-i5 cells (Kaiho et a!., 1996).…”

“…Whole-cell voltage clamp analyses revealed that 1 mM ATP induced a nonselective cation current with an Erev of 9.3 ±1.2 mY (n = 22) in N18TG-2 cells, which was similar to that with an~of 12.5 ±0.8 mV (n = 10) in NG1O8-15 cells (Kaiho et al, 1996). In contrast, ATP at the same concentration did not induce the current in C6Bu-1 cells.…”

Inhibitory Effects of Metals on ATP-Induced Current Through P2X7 Receptor in NG108-15 Cells

“…INS·P2X7 was measured using the whole-cell configuration of the patch-clamp technique (20) as descried previously (7,10,21). Patch pipettes had a tip resistance of about 2 MW when filled with an intracellular solution containing 110 mM CsOH, 30 mM CsCl, 50 mM DL-aspartic acid, 5 mM MgATP, 3 mM MgCl 2 , 5 mM potassium creatine phosphate, 10 mM EGTA and 20 mM HEPES (pH 7.2 with DL-aspartic acid).…”

“…10; I. Matsuoka, unpubished result of the full length sequence) to that of mouse P2X7 receptor first cloned by Chessell et al (11) in NTW8 mouse microglial cells. A high concentration (>0.3 mM), but not a low concentration, of ATP activates a nonselective cation current, indicating that the only functional P2X receptor is the P2X7 receptor in NG108-15 cells (7,12,13). Rat and human P2X7 receptors transform themselves from channels to large pores by prolonged application of ATP (8,14), but this does not occur in mouse P2X7 receptors in NG108-15 cells (15).…”

“…Rat P2X7 receptor currents were inhibited by various divalent cations such as Ca 2+ , Mg 2+ (8,14), Cu 2+ , Cd 2+ , Zn 2+ , Ni 2+ , Co 2+ , Mn 2+ , Ba 2+ and Sr 2+ (18) in HEK293 cells that stably express the receptor. The effects of metal ions on the mouse P2X 7 receptor current have not been investigated in NG108-15 cells except for Mg 2+ (7). Therefore, we examined the effects of metal ions such as Cu 2+ , Ni 2+ , Cd 2+ , Zn 2+ , Co 2+ , Mn 2+ and Fe 3+ on the mouse P2X 7 receptor-mediated current in NG108-15 cells using the whole-cell voltage clamp.…”