Does the carrier of chromaffin granules transport the protonated or the uncharged species of catecholamines?

Summary

By osmotic lysis in the presence of urea ghosts (60–100 nmol catecholamine/mg prot.) were prepared from chromaffin granules (4–6 μmol catecholamine/mg prot.) of the bovine adrenal medulla. In the presence of 1–300 μmol/l³H-catecholamine and ATP-Mg²⁺, ghosts show a net uptake of catecholamine. The net uptake is sensitive to reserpine or agents (uncouplers and ammonium) which diminish the electrochemical potential difference for protons at the granule membrane (Δp). The same uptake was found by³H-counting or by fluorimetric measurements. At various pH-values (pH 6.2–82.) theK _m andV _max of the ATP-stimulated rate of uptake of³H-catecholamine into ghosts was determined (at 30°C) to identify the species of catecholamine (protonated, uncharged, or anionic) which is the substrate for the granule carrier. The pH difference (ΔpH=pH_out-pH_in) and the electrical potential difference (ΔΨ) were determined to calculate Δp under conditions of³H-catecholamine uptake. When the pH_out was increased (pH 6.2, 7.4, 8.2), the apparentK _m of uptake decreased (50, 5, 1–2 μmol/l), showing a linear relation between pH and logarithm ofK _m. TheK _m was calculated for the uncharged catecholamine (with pK₁=8.8 and pK₂=10.0); it was nearly pH-independent and amounted to about 0.2 μmol/l. TheV _max declined only in the extreme pH-range. Between pH 6.6 and 7.8V _max and Δp showed a slight increase from 16 to 20 nmoles/(mg prot.·min) and from 110 to 140 mV, resp. In the same pH-range the pH_in inside ghosts increased from pH 5.2 to 5.7, whereas ΔΨ was constant (30 mV). At constant pH_out (=7.3) ammonium (0–30 mmol/l) caused an increase of pH_in from 5.5 to 6.6. The increase of pH_in was accompanied by an increase ofK _m from 5 to 20 μmol/l³H-catecholamine and by a decrease of bothV _max and Δp from 20 to 5 nmoles/(mg prot.·min) and from 123 to 85mV, respectively. From the dependence of theK _m of uptake on pH_out is concluded that the uncharged species of catecholamine is transported, whereas the dependence ofK _m on pH_in suggests that the translocation of the catecholamine-carrier complex across the granule membrane is not the rate-limiting step of catecholamine uptake.