The human H V 1

Proton currents in human granulocytes were first reported in 1993 (1.2) (Demaurex et al., 1993). Why is a proton current necessary at all in these specialized immune cells and how is this process controlled? Granulocytes become activated in response to microbial contact.

The bacteria are engulfed and killed by an increasing cytosolic acidification. In order to maintain the cell homeostasis the excess of cytosolic protons (low pHi) has to be regulated by a massive proton efflux. These outward currents were measured for cells overexpressing HV1 channels. These channels are opened due to membrane depolarization and pH changes.

Until now, many more functions of the human HV1 channel (hHV1) were described, which will be introduced in the next paragraphs.

The human genome encodes only one HV1 gene. Nevertheless, different isoforms exist due to alternative splicing events (DeCoursey, 2015). Channels are localized in the plasma membrane of human basophils (Musset et al., 2008), sperm cells (Lishko et al., 2010; Lishko

& Kirichok, 2010), B lymphocytes (Capasso et al., 2010), microglia (Eder & DeCoursey, 2001) and others. As mentioned, their common function is the restoring of the cytoplasmic pH.

This is of particular importance in cancer cells. Here, their extensive growth is enabled by a 10 times higher anaerobic glycolysis compared to normal tissue cells. The resulting acidification by increased concentrations of lactic acid due to the Warburg effect (Warburg, 1924) is most likely counteracted by the outward extrusion of protons by voltage-gated proton channels, thus, maintaining high proliferation rates of cancer cells (Wang et al., 2011;

Wang et al., 2012; Wang et al., 2013a; Wang et al., 2013b). Especially the synthesis of a shorter isoform, missing the first 20 N-terminal amino acids, was found to be upregulated in these cells (Capasso et al., 2010). An inhibition of hHV1 in malignant cells by polyvalent cations induced their apoptosis, which demonstrates its significant role in drug development

INTRODUCTION

(Wang et al., 2013b). As an example, the information that two Histidine residues are supposed to coordinate a zinc ion, His140 and His193, can be used for future developments of new blocking reagents (Figure 2B) (Ramsey et al., 2006). Furthermore, these channels are involved in the production of reactive oxygen species (ROS), which can cause severe tissue damage. As a result, hHV1 channels play a role in a variety of diseases like Alzheimer’s disease, ischemic stroke, Parkinson’s disease, Crohn’s disease, cystic fibrosis, breast cancer, colorectal cancer, and chronic lymphocytic leukemia underlining its importance for pharmaceutical research (Eder & DeCoursey, 2001; Haglund et al., 2013; Conese et al., 2014;

Wang et al., 2012; Morgan et al., 2015; Wang et al., 2013a). Further knowledge with regard to the protein structure and/or gating mechanism will accelerate future drug development to mitigate or even prevent the mentioned diseases.

Structurally, the hHV1 is composed of 273 amino acids (UniProtKB-Q96D96, [Consortium, 2017]). A model representing the voltage-sensing domain of hHV1 (hHV1-VSD) (amino acids 84-214) embedded in the membrane is shown in Figure 2.

The hHV1 is composed of a short N-terminal intracellular domain, four transmembrane helices connected by small loops and a large intracellular C-terminal domain. The latter is known to participate in dimer formation and is involved in the channeling process by influencing the S4 movement (Lee et al., 2008b; Li et al., 2010b; Fujiwara et al., 2012;

Fujiwara et al., 2014). The four transmembrane helices form the VSD. Based on the homology model S1 includes amino acids 99-120, S2 133-156, S3 172-186 and S4 194-210.

The VSD responds to membrane depolarization by the movement of the S4 helix enabling the channeling of protons across the membrane barrier. Here, the countercharge positions of three arginine residues localized in S4 (Figure 1, Figure 2) (R205, R208 and R211) are supposed to be exchanged. In detail, countercharges D112, E119, D123 and D185 are described to be involved in the opening process whereas E153 and D174 stabilize the closed-state of hHV1 (Ramsey et al., 2010; Li et al., 2015; DeCoursey et al., 2016). A special role is assumed for the amino acids F150, V109 and V178 function as a plug, closing the pore as described for other voltage-gated cation channels (DeCoursey et al., 2016; Li et al., 2014;

Lacroix et al., 2014; Li et al., 2015). Potential phosphorylation sites are located in the N-terminal region of the channel, T29 and S97. Phosphorylation of T29 by protein kinase C enhances the channeling process in leukocytes (Morgan et al., 2007; Musset et al., 2010a).

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Figure 2: Homology model and sequence information of the hHV1-VSD embedded in the membrane. A A homology model of the hHV1-VSD (amino acids 75-223 applied for modeling, 84-214 shown), under investigation in this thesis, was created by SWISS-MODEL (Arnold et al., 2006; Benkert et al., 2011; Biasini et al., 2014). The X-ray structure of the voltage-sensor containing phosphatase from ciona intestinalis (4G7V, rcsb.org [Berman et al., 2000]) was used as the template with a sequence similarity of 33 % and sequence identity of 24.4 %. Subsequently, the resulting hH_V1-VSD structure was embedded in a model membrane of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid molecules (Lomize et al., 2012). Helices are shown in green and loop regions in orange. N- and C-terminus of the VSD are labeled. The membrane is represented by dots whereby the cytoplasmic barrier is shown in blue and the extracellular membrane part is shown in red. B The hHV1 sequence with highlighted residues is shown. The yellow box indicates the complete sequence of the modeled VSD structure (84-214), extended by the orange boxes which show the hHV1-VSD sequence used in this thesis (75-223) (Figure 2A). The green boxes represent possible phosphorylation sites, the light purple boxes a potential coordination site for polyvalent cations and the dark purple box residue D112, known to be involved in gating processes. The brown boxes show residues, which determine proton accessibility, and the red boxes additional potential countercharges for the three arginine residues, known as the voltage-sensors, here highlighted in blue.

Despite numerous attempts involving crystallization and computational modeling, the structure of the hHV1 could not be solved so far (Li et al., 2010b; Musset et al., 2010c; Wood et al., 2012; Kulleperuma et al., 2013; Takeshita et al., 2014; Pupo et al., 2014; DeCoursey et al., 2016; Randolph et al., 2016). Referring to the channel presence in a variety of different diseases, the knowledge of the structure and/or of the channeling mechanism is of tremendous importance for pharmaceutical research in drug development. If we understand how protons pass through the channel and also how conformational changes lead to its closing or opening, inhibitors could be designed that block proton extrusion e.g. from cancer cells.

INTRODUCTION

Consequently, the cells would die due to massive cytosolic acidification. These considerations were the basis of the present work.

In this thesis, I worked with a truncated version of hHV1 including amino acids 75-223, referred as hHV1-VSD, which was cut off shortly before the first intracellular helix and four amino acids after the calculated fourth transmembrane helix (Figure 3) (7.1). As the VSD was the preferred target for my studies, the C-terminal domain was left out to generate a construct of decent size for solution-state NMR studies.

Figure 3: Schematic representation of examined voltage-gated proton channels. The figure illustrates the different constructs under investigation in this thesis (hH_V1-VSD and DrVSD) in comparison to the respective full-length proteins (hHV1 and DrVSP). Helices are displayed as cylinders and named according their order S0-S4. The C-terminal helix domain of hHV1 is shown as a grey cylinder. The phosphatase domain of the zebrafish voltage-sensing phosphatase (DrVSP) is shown as an orange ellipse. Three plus symbols represent the three positive charged arginine residues, which are described to be the voltage-sensor unit. L90P and L164V indicate substitutions in the DrVSD construct compared to the wild-type protein.

A similar procedure was used for the second analyzed VSD from the zebrafish phosphatase construct, which will be introduced in the next section.