Abteilung für Physiologie

Research

Immune cells are part of nearly every defence against illnesses in the human body.  We investigate the ion channels and ion transporters of these essential cells. Here, the voltage-gated proton channel Hv1 is a key protein.  Our research integrates electrophysiology, structure biology, and immunology.

Phagocytosis and respiratory burst

The innate immune system prevents most of the infections we are confronted with before they inflict harm.   We research the biophysics of immune cells and focus on ion channels and ion transporters. Ion channels play a decisive role in immune cell maturation, chemotaxis, reactive oxygen release, production of cytokines- and volume regulation, just to mention a few.  One of the most important ion channels is the voltage-gated proton channel Hv1 (1). This channel is expressed in all phagocytes. Phagocytes are the main defence against bacterial, parasite and fungal attacks. “Phago” stands for eater and “cyt” in biology is a suffix for cell. Phagocytes are “eating cells” or “scavenger cells” devouring e.g. bacteria.  The phagocyte encloses the bacterium with plasma membrane and takes the bacterium up into its cell body. This is called phagocytosis. The intracellular vesicle is called phagosome, by fusion with lysosomes a phagolysosome is created in this enclosed space (0.2-1 fl), destroying the bacterium (Figure 1). 

 

Figure 1. Schema of the respiratory burst during phagocytosis. The phagocyte is engulfing the bacterium and the respiratory burst starts by generating electron current across the plasma membrane (NADPH oxidase, red diamond). Superoxide (O2-) is converted into hydrogen peroxide (H2O2) and enzymatically hypochlorous acid (HOCl) is generated. Charge is balanced by conducting protons (H+) via Hv1 (blue ovals) to the phagosome. The internal pH drops by releasing protons from NADPH. The cytosolic pH is regulated by Hv1 conducting protons out of the cell.

 

One of the tasks of Hv1 is to prevent a high potential difference across the membrane of the phagocytes. This change in potential in positive direction is called depolarization. In phagocytes, this is due to the activity of the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase).  This enzyme oxidises NADPH by taking electrons from NADPH and translocating them across the plasma membrane or phagosome. Molecular oxygen (O2) is reduced and takes up the electrons. Thus, two molecules of O2- are formed for each molecule of NADPH. O2- is an oxygen-radical that will transform in the phagosome from hydrogen peroxide (H2O2) to hypochlorous acid (HOCl). Hypochlorous acid is a strong bacteriocide. Together with the lysosomal enzymes, HOCl is responsible for the destruction of bacteria.  Oxidation/Reduction processes are connected to a redox potential. The redox potential describes the voltage that would be generated or consumed during the redox process. The redox potential of NADPH/NADP+ is -320 mV. The redox potential of O2/O2- is -160 mV. Resulting from these numbers, the so-called redox potential of the NADPH oxidase is around +160 mV. This value is additionally the expected quasi reversal potential of pure electron conductance through the phagocyte membrane. The reversal potential is the value where the current changes its direction.

Figure 2: Proton and electron current in a human phagocyte. A) Proton current under control conditions. B) Proton currents after activation by the phorbol ester PMA. C) Proton currents after electron current inhibition by diphenyliodonium (DPI).  D) Electron current activates over time. E) Electron current deactivated by DPI.

Hv1 opens due to depolarization and selectively conducts protons (H+) out of the cytosol. The driving force for the proton conduction is the electrochemical gradient. The electro-chemical gradient for protons is dependent on the activity of H+ inside and outside the cell. Furthermore it depends of the electrical potential across the membrane. Under resting conditions (pH 0.1 units lower in cytosol than outside (blood)), the chemical potential will drive protons out of the cell. However, the electrical potential (cell-dependent in phagocytes around -60 mV) will hold the protons back. During the activity of the NADPH oxidase vast amounts of H+ are generated due to the oxidation of NADPH. Additional amounts of protons are generated by the pentose phosphate pathway needed to generate and replenish NADPH.  The chemical gradient would force the H+ out of the cell. However, the electrical force is increased too. While electrons cross the focus of the electric field of the membrane, the membrane is charged, leading to a massive depolarization in the direction of +160 mV.  The electrical force will drive the H+ out of the cell too. An important detail left apart is that HV1 is voltage-gated.  Hv1 opens exclusively with depolarization and is closed as long as the electrical force is negative. Still, this voltage-dependence is dependent on the pH inside and outside the cell. The voltage-gated proton channel is able to adjust its voltage-dependent gating in a way, that it works comparable to an over pressure valve, and exclusively conducts H+ outward. Therefore, HV1 is the perfect partner for the NADPH oxidase, as while it is open, it conducts H+ outside the cell preventing an acidification of the cytosol (Figure 2). While it is open, the permeation of protons will balance the charge of the moving electrons and the membrane potential will be less positive.  One more detail to mention is that the electron current of a phagocyte is voltage dependent. Depolarizations that will reach the quasi reversal potential of the NADPH oxidase will cease its function. Finally, NADPH oxidase is pH dependent. The electron current decreases drastically in low pH.

 

Structure and Function of Hv1

We are investigating the structure and function interdependence of voltage-gated proton channels. The human Hv1 is a homodimer. Each of its monomers consists of 273 amino acids (Figure 3).  Each monomer has 4 transmembrane domains, both N- and C- termini are cytosolic. Comparable to the classical voltage-gated ion channels, the fourth transmembrane domain has several positively charged arginines in an α-helix.  This structure is also known as the voltage sensor. The mechanic of the voltage sensor is imagined as being a ratchet that moves one arginine after another through the membrane. This movement is still under scientific discussion but its goal would be to open a pore enabling ionic current to be conducted.

Figure 3. Transmembrane domains of a classical voltage-gated potassium channel (left) vs. the voltage-gated proton channel HV1 (right). S1-S3 in yellow, S4 containing arginine in orange, pore domain S5-S6 in red.  Hv1 is missing the pore domains.

In contrast to the classical voltage-gated ion channels Hv1 does not have a classical pore. In potassium channels, the selectivity of the pore is achieved by a pore loop Glycine-Tyrosine- Glycine (GYG) motive that forms the selectivity filter. Dehydration of the ion, charge, and ion size are the parameters of selectivity. In sodium channels a net negative charge ensures selectivity while in calcium channels higher net charges are necessary to provide selectivity. Anyhow, size of the pore is an essential criterion of selection in all the selectivity filters. However, how is the selectivity for H+ realized if size exclusion might be challenging?

Comparison to the closest non-conducting Hv1 homolog suggested an aspartate as the selectivity filter (Figure 4) (2). Mechanistically, the aspartate is protonated and deprotonated during conduction, while without proton the opposing arginine closes the salt bridge (3,4). 

Figure 4: Snapshot from a MD simulation of a Hv1 single monomer in the open state. Red: three arginines of the S4 helix, blue: aspartate D112 (selectivity filter) in S1, purple: charge transfer center F150 in S2. Yellow sticks show histidines at the external side of the channel. White: trypthophan in S4. The aspartate D112 and arginine R208 sidechains are connected via a salt bridge.

 

We investigate the architecture of Hv1 by using zinc (Zn2+) as an inhibitor of proton current (5-7). The electrophysiological data combined with molecular dynamics simulations present a funnel-like corridor in the dimer. The funnel is built by a negative electrostatic potential that guides Zn2+ to its binding histidines between the homo monomers (Figure 5).

Figure 5: Electrostatic potential of the voltage-gated proton channel. Left: the dimeric proton channel embedded in the membrane (grey lines). The electrostatic potential is shown in 7 layers from the external bulk solution (layer 1), via the region containing the histidines H193 (layer 2), down to the region containing the two histidines H140 from each Hv1 monomer (layer 7). In each layer, the black traces represent the contours of the Hv1 monomers. A negative electrostatic potential is attracting Zn2+ cations from the bulk solution down to the histidine residues.

Hv1 is not exclusively expressed in phagocytes, but also in human lung cells, B-cells (8), sperm (9), muscle, and more. Furthermore, Hv1 seems to be expressed in some malignant cancers such as breast and colorectal cancer.  In cancer cells, knock down of the proton channel results into less invasiveness and cell proliferation.

Several questions still unanswered.

  1. Where is the conduction pathway of the H+?
  2. Is the pore water filled or dry?
  3. How is the pH sensed by Hv1?
  4. What is the mechanism of zinc inhibition?
  5. Are there more proton channels?
  6. Where is the dimerization interface?

Hv1 is not only expressed in humans but also in other eukaryotes. As far as we know today there is no Hv1 in prokaryotes. Table 1 shows proton channels which were electrophysiologically investigated. The spectrum of species ranges from protists (10) over algae and insect (11) to mouse and human (12). Several tissues and species haven’t been investigated for proton channels. There is still much to research.

 

Table 1:

NameOrganismMolecular Mass (Da)Amino acids
hHv1Homo sapiens (human)31.683273
hHv1sShort Isoform (human)29.415253
mHv1Mus musculus (mouse)31.242269
CiHv1Ciona intestinalis (sea squirt)38.501342
SpHv1Strongylocentrotus purpuratus (sea urchin)37.483328
EhHv1Emiliania huxleyi (coccolithophore)37.389339
PtHv1Phaeodactylum tricornutum (diatom)38.582338
CpHv1Coccolithus pelagicus (coccolithophore)36.281325
kHv1Karlodinium veneficum (dinoflagellat)27.624248
NpHv1Nicoletia phytophila insect)27.680239
DrHv1Danio rerio (zebrafish)27.110235

 

References

1. Musset B, DeCoursey T (2012) Biophysical properties of the voltage gated proton channel Hv1 Wiley Interdiscip Rev Membr Transp Signal 1:605-620

2. Musset B, Smith SM, Rajan S, Morgan D, Cherny VV, DeCoursey TE (2011) Aspartate 112 is the selectivity filter of the human voltage-gated proton channel Nature 480:273-277 doi:10.1038/nature10557

3. Banh R et al. (2019) Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels Proc Natl Acad Sci U S A doi:10.1073/pnas.1905462116

4. Morgan D et al. (2013) Peregrination of the selectivity filter delineates the pore of the human voltage-gated proton channel hHv1 J Gen Physiol 142:625-640 doi:10.1085/jgp.201311045

5. Chaves G, Bungert-Plumke S, Franzen A, Mahorivska I, Musset B (2020) Zinc modulation of proton currents in a new voltage-gated proton channel suggests a mechanism of inhibition FEBS J doi:10.1111/febs.15291

6. Jardin C, Chaves G, Musset B (2020) Assessing Structural Determinants of Zn(2+) Binding to Human Hv1 via Multiple MD Simulations Biophys J doi:10.1016/j.bpj.2019.12.035

7. Musset B, Smith SM, Rajan S, Cherny VV, Sujai S, Morgan D, DeCoursey TE (2010) Zinc inhibition of monomeric and dimeric proton channels suggests cooperative gating J Physiol 588:1435-1449 doi:10.1113/jphysiol.2010.188318

8. Capasso M et al. (2010) HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen species Nat Immunol 11:265-272 doi:10.1038/ni.1843

9. Musset B et al. (2012) NOX5 in human spermatozoa: expression, function, and regulation J Biol Chem 287:9376-9388 doi:10.1074/jbc.M111.314955

10. Smith SM, Morgan D, Musset B, Cherny VV, Place AR, Hastings JW, DeCoursey TE (2011) Voltage-gated proton channel in a dinoflagellate Proc Natl Acad Sci U S A 108:18162-18167 doi:10.1073/pnas.1115405108

11. Chaves G, Derst C, Franzen A, Mashimo Y, Machida R, Musset B (2016) Identification of an Hv1 voltage-gated proton channel in insects FEBS J 283:1453-1464 doi:10.1111/febs.13680

12. Musset B, Cherny VV, DeCoursey TE (2012) Strong glucose dependence of electron current in human monocytes Am J Physiol Cell Physiol 302:C286-295 doi:10.1152/ajpcell.00335.2011

 

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