Mark A. Milanick

Biochemical properties of vacuolar zinc transport systems of Saccharomyces cerevisiae

The yeast vacuole plays an important role in zinc homeostasis by storing zinc for later use under deficient conditions, sequestering excess zinc for its detoxification, and buffering rapid changes in intracellular zinc levels. The mechanisms involved in vacuolar zinc sequestration are only poorly characterized. Here we describe the properties of zinc transport systems in yeast vacuolar membrane vesicles. The major zinc transport activities in these vesicles were ATP-dependent, requiring a H+ gradient generated by the V-ATPase for function. One system we identified was dependent on the ZRC1 gene, which encodes a member of the cation diffusion facilitator family of metal transporters. These data are consistent with the proposed role of Zrc1 as a vacuolar zinc transporter. Zrc1-independent activity was also observed that was not dependent on the closely related vacuolar Cot1 protein. Both Zrc1-dependent and independent activities showed a high specificity for Zn2+over other physiologically relevant substrates such as Ca2+, Fe2+, and Mn2+. Moreover, these systems had high affinities for zinc with apparentK m values in the 100–200 nm range. These results provide biochemical insight into the important role of Zrc1 and related proteins in eukaryotic zinc homeostasis.

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

This study is aimed at identifying the Na pump isoform composition of human erythroid precursor cells and mature human erythrocytes. We used purified and synchronously growing human erythroid progenitor cells cultured for 7–14 days. RNA was extracted from the progenitor cells on different days and analyzed by RT-PCR. The results showed that only the α1, α3, β2, and β3 subunit isoforms and the γ modulator were present. Northern analysis of the erythroid progenitor cells again showed that β2 but not β1 or α2 isoforms were present. The erythroid cells display a unique β subunit expression profile (called β-profiling) in that they contain the message for the β2 isoform but not β1, whereas leukocytes and platelets are known to have the message for the β1 but not for the β2 isoform. This finding is taken to indicate that our preparations are essentially purely erythroid and free from white cell contamination. Western analysis of these cultured progenitor cells confirmed the presence of α1, α3, (no α2), β2, β3, and γ together now with clear evidence that β1 protein was also present at all stages. Western analysis of the Na pump from mature human erythrocyte ghosts, purified by ouabain column chromatography, has also shown that α1, α3, β1, β2, β3, and γ are present. Thus, the Na pump isoform composition of human erythroid precursor cells and mature erythrocytes contains the α1 and α3 isoforms of the α subunit, the β1, β2, and β3 isoforms of the β subunit, and the γ modulator.

The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity

Peroxynitrite is a reactive nitrogen species produced when nitric oxide and superoxide react. In vivo studies suggest that reactive oxygen species and, perhaps, peroxynitrite can influence Na-K-ATPase function. However, the direct effects of peroxynitrite on Na-K-ATPase function remain unknown. We show that a single bolus addition of peroxynitrite inhibited purified renal Na-K-ATPase activity, with IC50 of 107+/-9 microM. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (approximately 0.85 microM/s) peroxynitrite. The inhibition of Na-K-ATPase activity induced by this treatment was similar to that induced by a single bolus addition of equal cumulative concentration. Peroxynitrite produced 3-nitrotyrosine residues on the alpha, beta, and FXYD subunits of the Na pump. Interestingly, the flavonoid epicatechin, which prevented tyrosine nitration, was unable to blunt peroxynitrite-induced ATPase inhibition, suggesting that tyrosine nitration is not required for inhibition. Peroxynitrite led to a decrease in iodoacetamidofluorescein labeling, implying that cysteine modifications were induced. Glutathione was unable to reverse ATPase inhibition. The presence of Na+ and low MgATP during peroxynitrite treatment increased the IC50 to 145+/-10 microM, while the presence of K+ and low MgATP increased the IC50 to 255+/-13 microM. This result suggests that the EPNa conformation of the pump is slightly more sensitive to peroxynitrite than the E(K) conformation. Taken together, these results show that peroxynitrite is a potent inhibitor of Na-K-ATPase activity and that peroxynitrite can induce amino acid modifications to the pump.

Mechanism of Intracellular pH Increase During Parthenogenetic Activation of In Vitro Matured Porcine Oocytes

Abstract Parthenogenetic activation of porcine oocytes by using 7% ethanol, 50 or 100 μM A23187 results in an increase in intracellular pH as does prolonged exposure to thimerosal. We attempt to specify which transporters or mechanisms are involved in the observed increase in intracellular pH during oocyte activation. Experiments were performed in the absence of sodium; the presence of 2.5 mM amiloride, a potent inhibitor of the Na+/H+ antiport; in the absence of bicarbonate; and in the presence of 4,4′-diisothiocyanatodihydrostilbene-2,2′-di-sulfonic acid, disodium salt (H2DIDS) for all three activation methods. These treatments had no effect on the increase in intracellular pH induced by the calcium ionophore or thimerosal, but all reduced the increase in pH (P < 0.001) in the 7% ethanol group. This suggests that the Na+/H+ antiport and the HCO3−/Cl− exchangers are not playing a role during treatment with calcium ionophore or thimerosal, and the pH increase observed during treatment with 7% ethanol may be dependent upon a sodium or bicarbonate flux (or both) into the oocyte. Bafilomycin A1 (500 nm), an inhibitor of vacuolar-type H+ ATPases, had no effect on 7% ethanol or thimerosal treatments, but significantly reduced the increase in intracellular pH observed during calcium ionophore treatment. This may be the result of an initial local increase in intracellular free calcium levels.

Encapsulation of FITC to monitor extracellular pH: a step towards the development of red blood cells as circulating blood analyte biosensors

A need exists for a long-term, minimally-invasive system to monitor blood analytes. For certain analytes, such as glucose in the case of diabetics, a continuous system would help reduce complications. Current methods suffer significant drawbacks, such as low patient compliance for the finger stick test or short lifetime (i.e., 3–7 days) and required calibrations for continuous glucose monitors. Red blood cells (RBCs) are potential biocompatible carriers of sensing assays for long-term monitoring. We demonstrate that RBCs can be loaded with an analyte-sensitive fluorescent dye. In the current study, FITC, a pH-sensitive fluorescent dye, is encapsulated within resealed red cell ghosts. Intracellular FITC reports on extracellular pH: fluorescence intensity increases as extracellular pH increases because the RBC rapidly equilibrates to the pH of the external environment through the chloride-bicarbonate exchanger. The resealed ghost sensors exhibit an excellent ability to reversibly track pH over the physiological pH range with a resolution down to 0.014 pH unit. Dye loading efficiency varies from 30% to 80%. Although complete loading is ideal, it is not necessary, as the fluorescence signal is an integration of all resealed ghosts within the excitation volume. The resealed ghosts could serve as a long-term (>1 to 2 months), continuous, circulating biosensor for the management of diseases, such as diabetes.

Extracellular Protons Regulate the Extracellular Cation Selectivity of the Sodium Pump

The effects of 0.3–10 nM extracellular protons (pH 9.5–8.0) on ouabain-sensitive rubidium influx were determined in 4,4′-diisocyanostilbene-2, 2′-disulfonate (DIDS)-treated human and rat erythrocytes. This treatment clamps the intracellular H. We found that rubidium binds much better to the protonated pump than the unprotonated pump; 13-fold better in rat and 34-fold better in human erythrocytes. This clearly shows that protons are not competing with rubidium in this proton concentration range. Bretylium and tetrapropylammonium also bind much better to the protonated pump than the unprotonated pump in human erythrocytes and in this sense they are potassium-like ions. In contrast, guanidinium and sodium bind about equally well to protonated and unprotonated pump in human red cells. In rat red cells, protons actually make sodium bind less well (about sevenfold). Thus, protons have substantially different effects on the binding of rubidium and sodium. The effect of protons on ouabain binding in rat red cells was intermediate between the effects of protons on rubidium binding and on sodium binding. Remarkably, all four cationic inhibitors (bretylium, guanidinium, sodium, and tetrapropylammonium) had similar apparent inhibitory constants for the unprotonated pump (∼5–10 mM). The K d for proton binding to the human pump, with the empty transport site facing extracellularly is 13 nM, whereas the extracellular transport site loaded with sodium is 9.5 nM, and with rubidium is 0.38 nM. In rat red cells there is also a substantial difference in the K d for proton binding to the sodium-loaded pump (14.5 nM) and the rubidium-loaded pump (0.158 nM). These data suggest that important rearrangements occur at the extracellular pump surface as the pump moves between conformations in which the outward facing transport site has sodium bound, is empty, or has rubidium bound and that guanidinium is sodium-like and bretylium and tetrapropylammonium are rubidium-like.

Kinetic Models of Na-Ca Exchange in Ferret Red Blood Cells

The kinetic equation that best describes the intracellular Na dependence of Ca influx into ferret red cells is sequential; whether this implies that there is a conformation of the protein that has both Na and Ca ions bound remains to be determined. Cd and Mn substitute very well for Ca on the exchanger in ferret red cells; this suggests that the Ca-binding site does not contain an important thiol and that the one of the Na steps may be rate limiting.

Divalent cation interactions with Na,K-ATPase cytoplasmic cation sites: implications for the para-nitrophenyl phosphatase reaction mechanism.

The interactions of divalent cations with the adenosine triphosphatase (ATPase) and para-nitrophenyl phosphatase (pNPPase) activity of the purified dog kidney Na pump and the fluorescence of fluorescein isothiocyanate (FITC)-labeled pump were determined. Sr2+ and Ba2+ did not compete with K+ for ATPase (an extracellular K+ effect). Sr2+ and Ba2+ did compete with Na+ for ATPase (an intracellular Na+ effect) and with K+ for pNPPase (an intracellular K+ effect). These results suggest that Ba2+ or Sr2+ can bind to the intracellular transport site, yet neither Ba2+ nor Sr2+ was able to activate pNPPase activity; we confirmed that Ca2+ and Mn2+ did activate. As another measure of cation binding, we observed that Ca2+ and Mn2+, but not Ba2+, decreased the fluorescence of the FITC-labeled pump; we confirmed that K+ substantially decreased the fluorescence. Interestingly, Ba2+ did shift the K+ dose-response curve. Ethane diamine inhibited Mn2+ stimulation of pNPPase (as well as K+ and Mg2+ stimulation) but did not shift the 50% inhibitory concentration (IC50) for the Mn2+-induced fluorescence change of FITC, though it did shift the IC50 for the K+-induced change. These results suggest that the Mn2+-induced fluorescence change is not due to Mn2+ binding at the transport site. The drawbacks of models in which Mn2+ stimulates pNPPase by binding solely to the catalytic site vs. those in which Mn2+ stimulates by binding to both the catalytic and transport sites are presented. Our results provide new insights into the pNPPase kinetic mechanism as well as how divalent cations interact with the Na pump.

Changes of membrane potential demonstrated by changes in solution color

Since membrane potential regulates the activity of nerves, muscle, and endocrine cells, and many drugs alter the membrane potential, an understanding of the general concepts for the regulation of membrane potential is important. Yet, membrane potentials and ion gradients are often tough areas for students to understand. Analogies can be helpful; for example, Cardozo (2) recently provided a spring analogy for membrane potential. However, in my experience, part of the difficulty probably relates to an adverse reaction to physics and math terms; another part of the problem is just the new technical terms that make it difficult for the students to decipher the concepts. For some of these students, springs may not be enough of an improvement. I have found that a qualitative understanding is facilitated by providing a demonstration and a series of analogies. These analogies will quickly help the students understand the different ways in which a cell can be depolarized (opening Na channels or closing K channels) and hyperpolarized (opening additional K channels or closing Na channels). The analogy is to consider the color of solution in a clear bucket as representing the membrane potential. Two carboys, one filled with blue solution and one with red solution, have tubes that lead to the bucket. The blue solution represents the K gradient, and the red solution represents the Na gradient. The rate of flow of fluid, governed by the valve, reflects the conductance. For most cells, the resting potential is pretty close to the K equilibrium potential, so the flow from the blue solution into the bucket is much greater than that from the red solution and the bucket is slightly purple‐but mostly blue. I then ask the class how I can depolarize the bucket, that is, how can I make the solution more purple (or even red). They all immediately see that opening up the valve from the red solution (opening Na channels) will do it. Then, I ask “what other way is there?” After a few moments of thought, they realize that closing K channels (reducing the flow from the blue solution) also works, which is how pancreatic -cells depolarize. One can extend the analogy to cover Cl channels. It is probably worthwhile to remind the students that this analogy is just an approximation and not to overinterpret this color analogy. To extend the analogy, a third carboy (the Cl gradient) is introduced; it can be merely a virtual carboy because the students can quickly pick this up after having seen the original demonstration. It is important to stress at this point that the Cl gradient differs in each cell, and so the color of this third carboy is cell dependent. For skeletal muscle cells, it is the same color purple as the resting membrane potential (which is mostly blue and a bit of red). At this point, one may want to talk about fainting goats (1), including showing a short YouTube video. These goats have reduced Cl channel activity in their muscle cells, and thus the muscle membrane potential is less stable. In wild-type goat muscle cells, the valve is substantially open. This does not change the color of the solution in the bucket. But, the class will quickly realize that one has to really open the red carboy valve (Na channels) to get the bucket solution to change color. However, in the fainting goats, the valve from the third carboy (Cl channels) is almost closed, and so a small change in Na conductance can drastically change the color of the bucket solution. In other cells, the Cl gradient can be either more blue than the resting purple membrane potential or more red. Thus, when the Cl channel opens, the direction of the change of membrane potential (more blue, more hyperpolarized; more red, more depolarized) depends on the color of the Cl gradient. One can even push the analogy further and talk about cells or conditions where the Na and K gradients change. A smaller K gradient can be represented by a lighter blue solution in the carboy. This means that, even when the valves on the Na and K carboys are not changed, the resting potential is more reddish than when the K gradient was larger (the carboy had a darker blue solution).

Soymilk: an effective and inexpensive blocking agent for immunoblotting.

Blocking efficacy of whole soymilk, nonfat soymilk, SuperBlock, and nonfat milk was evaluated by performing standard protein immunoblotting procedures on both purified protein and crude nuclear extracts from HEK 293 cells. Nonfat soymilk was found to have superior blocking efficacy compared with other blocking agents in terms of high signal-to-noise ratio with the shortest blocking times. In addition, the presence of low concentrations of the detergent Tween 20 (0.05-0.1%, v/v) in the wash buffer as well as antibody incubations significantly lessened the background compared with including only the detergent during wash steps.