Differential activation of cation channels and non-selective pores by macrophage P2z purinergic receptors expressed in Xenopus oocytes.
In macrophages and certain other cell types, extracellular ATP4- can increase plasma membrane permeability through activation of the P2z purinergic receptor. This permeability change involves the induction of non-selective pores which are permeable to molecules with M(r) < or = 900. Electrophysiological studies indicate that agonist occupation of P2z purinergic receptors can additionally activate cation channels which may be distinct from the non-selective pores. We have observed that mammalian P2z purinergic receptors can be expressed in Xenopus oocytes injected with mRNA from the BAC1.2F5 murine macrophage cell line. Under voltage-clamp analysis, these oocytes exhibit a multiphasic inward current in response to ATP or 3'-O-(4-benzoyl)-benzoyl-ATP (BzATP), a selective agonist for the P2z purinergic receptor. This ATP/BzATP-induced current is characterized by a rapidly activated phase which is followed by a delayed, but steady, increase in conductance. We have used two-electrode voltage-clamp analysis and ion substitution to further characterize these P2z purinergic receptor-induced currents as expressed in mRNA-injected oocytes. N,N-Hexamethylene amiloride (HMA), a potent inhibitor of various exchangers and channels, selectively and reversibly inhibited the delayed component of the BzATP-induced inward current. This delayed HMA-sensitive current can be carried by large organic cations, such as N-methyl-D-glucamine (NMG+) and Tris+, in addition to small inorganic cations including Na+, Li+, and K+. In contrast, the rapidly activated HMA-insensitive current is readily carried by Na+, Li+, and K+, but is poorly carried by NMG+ and Tris+. Additional studies characterized P2z receptor regulation of Ca2+ influx, depolarization, ethidium uptake, and fura-2 loss in native BAC1.2F5 macrophages. Reduced temperature permitted discrimination of two distinct permeability pathways which could be activated by BzATP. At 20 degrees C, BzATP did not significantly increase membrane permeability to NMG+ (M(r) 195), ethidium+ (M(r) 314), or fura-2 (M(r) 831) but did stimulate an ion conducting pathway which could be competitively permeated by Na+ or Ca2+. At 37 degrees C, BzATP treatment increased membrane permeability to NMG+, ethidium+, and fura-2, in addition to Na+ and Ca2+. These data indicate that macrophage P2z purinergic receptors can be differentially coupled to: 1) a rapidly gated cation channel, and 2) a time- and temperature-dependent formation of non-selective pores. These two permeability pathways can be distinguished by their rates of activation, ion selectivities, sensitivities to amiloride analogs, and temperature dependence.
Nuttle
L C
LC
Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106.
Dubyak
G R
GR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002412:Cations / Q000378:metabolism*; D002460:Cell Line; D005260:Female; D007473:Ion Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009865:Oocytes; D012333:RNA, Messenger; D011983:Receptors, Purinergic / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014982:Xenopus laevis
D002412:Cations; D007473:Ion Channels; D012333:RNA, Messenger; D011983:Receptors, Purinergic; D011994:Recombinant Proteins; C057360:5-(N,N-hexamethylene)amiloride; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000584:Amiloride; D000255:Adenosine Triphosphate