Outside-in signal transmission by conformational changes in integrin Mac-1.

Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils.

Activation of human neutrophil Mac-1 by anion substitution.

Substituting the medium chloride with glucuronate or glutamate causes a rapid, 10 to 30-fold, increase in the binding of the monoclonal antibody, CBRM1/5, which recognizes the high-affinity conformation of the Mac-1 integrin. This change is reflected in functional adhesion assays that show increased adhesion to ICAM-1 coated beads. Blocking antibodies indicate that the increased adhesion is almost entirely due to Mac-1.

Inhibition of Na+/H+ exchanger enhances low pH-induced L-selectin shedding and beta2-integrin surface expression in human neutrophils.

Ischemia-reperfusion injury is a common pathological occurrence causing tissue damage in heart attack and stroke. Entrapment of neutrophils in the vasculature during ischemic events has been implicated in this process. In this study, we examine the effects that lactacidosis and consequent reductions in intracellular pH (pH(i)) have on surface expression of adhesion molecules on neutrophils.

Relocation of the disulfonic stilbene sites of AE1 (band 3) on the basis of fluorescence energy transfer measurements.

Previous fluorescence resonance energy transfer (FRET) measurements, using BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) as a label for the disulfonic stilbene site and FM (fluorescein-5-maleimide) as a label for the cytoplasmic SH groups on band 3 (AE1), combined with data showing that the cytoplasmic SH groups lie about 40 A from the cytoplasmic surface of the lipid bilayer, would place the BIDS sites very near the membrane's inner surface, a location that seems to be inconsistent with current models of AE1 structure and mechanism. We reinvestigated the BIDS-FM distance, using laser single photon counting techniques as well as steady-state fluorescence of AE1, in its native membrane environment. Both techniques agree that there is very little energy transfer from BIDS to FM.

Volume-sensitive chloride channels do not mediate activation-induced chloride efflux in human neutrophils.

Many agents that activate neutrophils, enabling them to adhere to venular walls at sites of inflammation, cause a rapid Cl(-) efflux. This Cl(-) efflux and the increase in the number and affinity of beta(2) integrin surface adhesion molecules (up-regulation) are all inhibited by ethacrynic acid and certain aminomethyl phenols. The effectiveness of the latter compounds correlates with their inhibition of swelling-activated Cl(-) channels (I(Clvol)), suggesting that I(Clvol) mediates the activator-induced Cl(-) efflux.

Substrate-dependent reversal of anion transport site orientation in the human red blood cell anion-exchange protein, AE1.

The tightly coupled, one-for-one exchange of anions mediated by the human red blood cell AE1 anion-exchange protein involves a ping-pong mechanism, in which AE1 alternates between a state with the anion-binding site facing inward toward the cytoplasm (Ei) and a state with the site facing outward toward the external medium (Eo). The conformational shift (Ei <--> Eo) is only permitted when a suitable substrate such as Cl(-) or HCO(3)(-) (B(-)) is bound. With no anions bound, or with Cl(-) bound, far more AE1 molecules are in the inward-facing than the outward-facing forms (Ei Eo, ECli EClo).