Dual pathways for GTP-dependent regulation of chemoattractant-activated K+ conductance in murine J774 monocytes.

Whole-cell patch clamp recording and digital imaging microscopy were used to investigate the electrical and calcium signaling responses of murine J774 monocytes to chemoattractants and other calcium-mobilizing agonists. A latent outwardly rectifying K+ conductance, GkOR, was elicited within seconds by each of the following agonists: C5a, ATP, ADP, interleukin-8, and the adenosine analog 5'-(N-ethylcarboxamido)-adenosine. In terms of its pharmacologic profile and current-voltage (I-V) relation, GkOR was very similar to a P2 purinoceptor-activated K+ conductance previously described in rat mast cells and to a K+ conductance elicited in J774 cells by the GTP analog guanosine 5'-O-(3-thiotriphosphate). Agonist-induced elevation of calcium, primarily due to intracellular release, and the induction of GkOR both required a GTP-binding protein of the Gi family, as both events were blocked by pertussis toxin; intracellular dialysis with guanosine 5'-O-(2-thiodiphosphate) also prevented the induction of GkOR, further implicating mediation by a G protein. Induction of GkOR did not depend upon influx of Ca2+, as it occurred equally well when the concentration of external Ca2+ was 100 nM or 2 mM. We attempted to uncouple agonist-induced calcium release from induction of GkOR by dialyzing the cell cytoplasm with Ca(2+)-EGTA or Ca(2+)-1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffers before agonist application. When the concentration of free Ca2+ ([Ca2+]i) was set to approximately 15 nM with 1.1 or 11 mM EGTA, a "slow"Ca2+ buffer, 10 nM C5a induced a large GkOR (11 nS at 1.1 mM EGTA versus 13.4 nS at 11 mM EGTA). Surprisingly, when [Ca2+]i was buffered at 15 nM with 10 mM BAPTA, a "rapid" Ca2+ buffer, C5a elicited a much smaller although significant K+ conductance (approximately 3 nS). Systematic increase in cytosolic [Ca2+]i upon dialysis with a series of 10 mM BAPTA-Ca2+ buffers (15-2400 nM [Ca2+]i) revealed activation of a very large K+ conductance (maximum 17.4 nS), even in the presumed absence of receptor stimulation. This conductance had a similar I-V relationship to GkOR, and activation occurred within the range of [Ca2+]i observed in intact cells following stimulation with C5a or ADP (EC50 approximately 475 nM [Ca2+]i). Activation of Gk thus may proceed in part via the release of intracellular calcium from a source in close proximity to the channel or other calcium-binding regulatory protein.(ABSTRACT TRUNCATED AT 400 WORDS)