Band3

This post started out as an exploration of whether Band 3, aka the anion exchanger, aka the SLC4a1 gene product can transport sulfate and pyidoxal 5′-phosphate and other vitamers in a way that impacts autism and other conditions. We have about half a century worth of experiments with red blood cell (RBC) membrane Band 3 transporting many different anion combinations. We have known of these capabilities for so long because the experiments are inexpensive and do not require sophisticated instruments. Ironically we do not fully the medical relevance of these capabilities. The good news is that is is easy to process RBC and measure these transported anions, particularly sulfate, inside the RBC.

The Jennings Review and other work [1]

Wikipedia authors list renal and red blood cell isoforms of band 3, also known as the bicarbonate/chloride exchanger and the ALC4A1 gene product.

The major trans membrane protein of the red blood cell, known as band 3, AE1, and SLC4A1, has two main functions:

1. catalysis of Cl⁻/HCO⁻³ exchange, one of the steps in CO₂ excretion,
2. anchoring the membrane skeleton. [1] In the kidney Band 3 is transcribed by an alternate promoter resulted in an N-terminus that is 65 amino acids shorter than the RBC variety. The first 400 amino acids are in the cytoplasmic domain of Band 3 according to UniProt. Residues 1-29 are acidic.

Table of band 3 substrates

This table is from the Jennings 2021 review. [1] The references links work fairly well.

Relative Rate	Anion (References)	Additional Information
1	Cl−, HCO−3 (76, 224, 352)	Transported at similar rates; HCO−3 has higher affinity for transport site.
0.3–0.7	NO−3 (493)	Fast, similar to HCO−3.
	Formate (HCOO−) (108, 225, 227)	Slightly slower than HCO−3. Also transported as free acid.
	NO−2 (494, 495, 513, 514)	Rate not known exactly because of parallel HNO2 transport. Could be similar to formate.
	Br− (83)	Cl−/Br− exchange is faster than Br−/Br− exchange, as predicted by ping-pong mechanism.
	HS− (515)	Measured as Jacobs–Stewart cycle of rapid transport of both HS− and H2S.
0.1–0.3	Oxalate (−OOCCOO−) (227, 516, 517)	Fastest divalent anion transported by band 3.
	Superoxide (O−2) (518)	Transport rate not clear but probably fast.
	Peroxynitrite (OONO−) (375, 496, 497)	Causes oxidative damage of band 3 and reduced transport. Undissociated acid also transported.
	F− (43)	Slower than Br−.
	OH− (90)	Detectable but hard to quantify because very high pH inhibits monovalent anion transport.
	Selenite (HSeO−3) (107, 519)	Possible connection with arsenite toxicity.
0.03–0.1	Malonate (−OOCCH2COO−) (516, 520)	Almost as fast as oxalate; larger dicarboxylates are slower.
	I− (493)	Slow, but has high affinity for self-inhibitory site, so some of slow rate could be self-inhibition.
	Thiocyanate (SCN−) (51)	Inhibits Cl− transport strongly.
	Bisulfite (HSO−3) (521)	Much faster than SO2−4.
	Phosphite (H2PO2−3) (106, 109)	Much faster than H2PO−4.
	Borohydride (BH−4) (522)	Enters cells in <1 min at 3°C, but rate not quantified.
0.01–0.03	Hypophosphite (PO−2) (106, 109)	Slower than PO2−3.
	Glyoxylate (HCOCOO−) (227)	Much slower than HCO−3.
	Glycolate (HOCH2COO−) (227)	Much slower than HCO−3.
	Fluorophosphate (FPO−3) (109)	Slower than planar oxyanions of phosphorus.
	Acetate (H3CCOO−) (225, 493)	Hard to quantify because of rapid free acid transport. Used as spectator anion.
0.003–0.01	Selenate (SeO2−4) (107, 519)	Much slower than selenite.
	Vanadate (523)	Rate not known precisely; inhibits ATPases and PTPs.
0.001–0.003	Dithionite (S2O2−4) (91)	Measured as exchange with SO2−4.
	Pyruvate (225, 524)	Also transported by monocarboxylate transporter.
	Sulfate (SO2−4) (118)	Measured at very low extracellular pH; much slower at neutral pH.
<0.001	Chromate (CrO2−4) (525, 526)	Influx facilitates labeling red cells with 51Cr for red cell lifetime measurements.
	Glycine anion (H2NCH2COO−) (527)	Slower than glycolate.
	H2PO−4/HPO2−4 (50, 106, 109, 528, 529)	Also transported by Na+-coupled cotransporter.
	Phosphoenolpyruvate (240, 530)	Only known glycolytic intermediate transported across red cell membrane.
	Lithium carbonate (LiCO−3) (531, 532)	Under physiological conditions represents over half the lithium flux in red cells.
	Pyridoxal phosphate (241)	Also reacts with K851.
	NBD-taurine (242)	Used to measure transport by fluorescence
	Taurine monochloramine (533)	Produced from taurine by neutrophil myeloperoxidase.

the Cl⁻/HS⁻ exchange rate is about one-third that of Cl⁻ and HCO₃⁻ [2]

The Jacobs-Stewart cycle consists of the following four steps:

1. Cl⁻/HCO₃⁻ exchange Cl⁻/HS⁻ exchange
2. intracellular HCO₃⁻ protonation and dehydration intracellular HS^– protonation
3. CO₂ efflux, H₂S efflux
4. extracellular CO₂ hydration and deprotonation.extracellular H₂S deprotonation

We just do not know how to interpret these data. We have these data because RBC are so easy to work with. Do the H₂S equilibria matter? Sulfite/ sulfate equilibrium might be interesting. B6 vitamers would be interesting.

Pyridoxal 5′-Phosphate [3]

Central to this narative is that PLP contains an aldehyde group that can form Schiff basesa with lysine side chains.

Transport of pyridoxal 5-phosphate(PLP) into erythrocytes was inhibited by inhibitors of anion transport including stilbene disulfonate compounds,indicating that it is mediated by Band 3 protein. The purple arrow int the figure below points to the reactive alkdehyde of PLP.

1. Erythrocytes were irreversibly labeled when the cells were treated with PLP in the buffered saline, pH 7.2,
2. The Schiff base linkage between PLP and the amino group of themembrane by [³H]NaBH₄. leaving a radioactive label on the formerly labeled protein
3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the membrane from the labeled cells showed that most of the radioactivity was recovered in Band 3 protein except for minor peaks in PAS-positive proteins (Fig. 1).
4. The addition of free lysine prevented PAS-positive proteins from being labeled, probably because of their low affinity to the labeling probe (Fig. 1).The labeling of Band 3 decreased to about40%
5. of the control (labeled with PLP, lysine, and NaBH₄ by the treatment of the cells with DIDS, which inhibited the

When erythrocytes were treated with PLP and large amounts of free lysine and NaBH.,, two membrane-spanning fragments of Band 3 (Mr = 17,000 and 35,000) were specifically labeled.

When the cells were pretreated with 4,4‘-dinitrostilbene 2,2’-disulfonate, the labeling in the 35,000-dalton fragment was inhibited.

Erythrocytes labeled by PLP in both the 17,000- and 35,000-dalton fragments transported PLP at a decreased rate, whereas the cells labeled in only the 17,000-dalton fragment had essentially the same transport activity as the control when 4,4’-dinitrostilbene 2,2’-disulfonate was removed.

The extent of inhibition of transport of inorganic phosphate in the labeled cells was similar to that of PLP.The results indicate that the 35,000-dalton fragment participates in the anion transport of the cell membrane.

Marino Lab insights [4,5]

The Marino Laboratory is almost as prolific as the Jennings laboratory when it comes to RBC publications. Their interest is more in terms of using these ghosts membrane proteins to measure the effect of oxidative stress and dietary antioxidants. This is a rabbit hole to keep us from our ultimate goal of making use of decades of Band 3 research.

data suggesting but not proving GSH adducts[4]

These authors do have a very interesting figure that is very suggestive of a process called gluathionylation. [4] It should be noted that they really do not favor this explanation for GSH induced reduction in Band 3/anion exchanger activity. They did note that GSH cannot get into RBC.

It should be noted that these authors did not observe any long term change in free thiols iwth just H₂O₂ treatment. The Jennings review states, “Despite the fact that band 3 has no cysteine residues that are necessary for transport, treatment with N-ethylmaleimide (NEM) or oxidative stress inhibits red cell anion transport to varying degrees” NEM is a thiol labeling molecule.

a cartoon of an almost clinic ready protocol [5]

A year after the Jennings review [1] the Marino group came up with a group that included Band3 and Sat1[5] It’s a nice review. What this post will focus on is the pictorial diagram of a Band3 protocol that can be used in a clinic. The authors have generously supplied readers with a pictorial protocol that

The almost clinic ready protocol in words [4]

Comments in purple

1. After the last sample withdrawal, rBC were washed three times in cold isotonic medium (4°C, 1200 g, 5 min) to remove SO₄⁼ from the external medium.
2. Cells were then hemolysed by 1 ml distilled water The authors are not telling us how big the RBC pellet is that they are lysing with 1 mL of distilled water.
3. and proteins were hydrolysed by 4% v/v Perchloric acid. oWe can hydrolyze them or just precipitate all of the proteins wtih TCA.
4. Cell membranes were discarded by centrifugation (4°C, 2700 g, 10 min) oIf we used TCA to precipitate soluble proteins, we’d be getting rid of them and the RBC membranes at the same time.
5. and SO₄⁼ in the supernatant was precipitated by sequentially adding 1 ml glycerol and distilled water solution (1:1), 1 ml 4 M NaCl plus HCl (hydrochloric acid 37% v/v) solution (12:1) and 500 μl 1.24 M BaCl₂•2H₂O to 500 μl supernatant from each sample. They are still not telling us what the supernatant volume really is
6. Levels of SO₄⁼ were spectrophotometrically measured at 425 nm wavelength (Beckman DU 640). Using a calibrated standard curve obtained by precipitating known SO₄⁼ concentrations, the absorption was converted to mM of intracellular SO₄⁼, necessary to calculate the rate constant in min^-1, This will have been done for us.
7. derived from the following equation: C_t = C_∞ (1-e^-rt) + C₀, where C_t, C_∞ and C₀ represent the intracellular SO₄⁼ concentrations measured at time, t 0 and ∞ respectively; e indicates the Neper number (2.7182818), r is a constant accounting for specific velocity of the process and t is time at which intracellular SO₄⁼ concentration is measured. SO₄⁼ uptake was measured as [SO₄⁼] L cells x10^-2 This equation is historically interesting from the standpoint of the protocol being inexpensive and simple.

The journey of this post has taken through a time warp of decades of fantastic research that has been forgotten for information gathered by flashier technology. Do RBC transport sulfate/fulfite physiologically? It should be pretty easy to find out.

References

1. Jennings ML. Cell physiology and molecular mechanism of anion transport by erythrocyte band 3/AE1. Am J Physiol Cell Physiol. 2021 Dec 1;321(6):C1028-C1059. PMC free article
2. Jennings ML. Transport of H2S and HS(-) across the human red blood cell membrane: rapid H2S diffusion and AE1-mediated Cl(-)/HS(-) exchange. Am J Physiol Cell Physiol. 2013 Nov 1;305(9):C941-50. PMC free article
3. Nanri H, Hamasaki N, Minakami S. Affinity labeling of erythrocyte band 3 protein with pyridoxal 5-phosphate. Involvement of the 35,000-dalton fragment in anion transport. J Biol Chem 258: 5985–5989, 1983. free paper
4. Morabito R, Romano O, La Spada G, Marino A. H2O2-Induced Oxidative Stress Affects SO4= Transport in Human Erythrocytes. PLoS One. 2016 Jan 8;11(1):e0146485. PMC free article
5. Remigante A, Spinelli S, Pusch M, Sarikas A, Morabito R, Marino A, Dossena S. Role of SLC4 and SLC26 solute carriers during oxidative stress. Acta Physiol (Oxf). 2022 May;235(1):e13796. PMC free paper