Regulation of the electrogenic H+ channel in the plasma membrane of neutrophils: Possible role of phospholipase A2, internal and external protons

Abstract

Possible factors regulating the opening of and the rate of H+ flux through a recently described, Cd2+-sensitive, phorbol ester- and arachidonic acid (AA)-activatable H+-conducting pathway in the plasma membrane of neutrophil granulocytes were investigated. (1) The phospholipase A2 blocker p-bromophenacyl bromide (BPB) inhibited the phorbol 12-myristate 13-acetate (PMA)induced activation of this channel in a concentration-dependent manner (IC50, 4 μM). (2) Neither BPB nor the protein kinase C (PKC) inhibitor staurosporine influenced the AA-elicited stimulation of this route. (3) Intracellular acidification (cytoplasmic pH below 6.9) itself is capable of activating an electrogenic, Cd2+-sensitive H+ efflux indicating that protons can open up this route in the absence of any other stimulator. (4) PMA significantly decreases the intracellular H+ concentration ([H+](i)) threshold for the opening of the channel, thus providing a conductive state at resting pH values, and elevates the rate of H+ efflux at any [H+](i). (5) Changes in external pH also modify the operation of the channel: above an extracellular pH (pH(o)) value of 7.4, the H+-flux/driving force relationship is approx. 5-fold greater than below this value. Our results suggest a multifactorial regulation of the electrogenic H+ channel: most probably PKC activates the channel indirectly, via stimulation of phospholipase A2 that subsequently liberates AA. In addition to this, the channel conductance seems to be promoted by internal H+ and inhibited by external H+.