Proceedings of Bioc 462a Honors

Abstract

Acid sensing ion channels (ASIC) are proton activated membrane proteins located in the nervous system. The structure of ASIC1, recently solved by Jasti et al. (2007), shows that ASIC are homotrimer proteins with a large extracellular domain coupled to a helical transmembrane domain. The individual monomers of the trimer protein show symmetry in the extracellular domain but none in the transmembrane domain. The transmembrane domain consists of helices arranged into a narrow pore opening that is specific for sodium ions and gated by residues located at the junction between the extracellular and transmembrane domains. The extracellular domain is shaped like a cup or a closed fist with many different regions. In the desensitized state the extracellular domain has many small cavities, but no direct opening pores that would allow ion passage. Acidic carboxyl-carboxylate residue pairs are locked at proton-binding sites in the acidic pocket located near the thumb and palm domain of the extracellular domain. Upon proton binding, the thumb domain undergoes conformational change. This conformation change is coupled with the transmembrane domain, which also undergoes conformation change, thus opening the ion channel and allowing passage of sodium ions into the cell (Jasti et al., 2007). The determination of the structure of ASIC1 provides great insight into the structure and function of other homologous membrane channel proteins.

Introduction
Acid sensing ion channels (ASIC) are part of a superfamily of sodium channels called epithelial sodium channels (ENaC) that are activated by an increase in extracellular acidity. This superfamily of proton sensing proteins are related to degenerin channels (DEG) and FMRF-amide peptide-gated channels (FaNaCh) (Alexendar et al., 2007). ENaCs, DEGs, and FaNaChs can all be found in the sensory neurons where they relate tissue acidosis and a physical perception of pain (Waldmann and Lazdunski, 1998). As such, they are important in the neurological understanding and treatment of pain.
Because EnaCs, DEGs, and FaNaChs are homologues, understanding of ASICs can lead to insight into hard to crystallize proteins such as other ENaCs, DEGs, and FaNaChs. ASICs are cation selective receptor/ion channel proteins found in the central nervous system of mammals, where they are involved in the detection and processing of sensory information (Lingueglia, 2007). ASICs are also able to sense proton gradients and aid in the regulation of sodium homeostasis (Kellenberger and Schlid, 2002). In mammals, there are four genes that code for six ASIC isoforms, ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4. ASIC1, the protein of interest in this review, is found in the central nervous system as well as the sensory neurons of the peripheral nervous system (Lingueglia et al., 1997). ASIC1a is found predominately in the brain, while ASIC1b is predominately found in the sensory neurons. In the central nervous system, ASIC1 facilitate spatial understanding and memory (Wemmie et al, 2002). Evidence indicates that stroke caused ASIC1 activation leads to ischaemic cell death. However, inhibition of ASIC1 can reduce cell damage and cell death (Xiong et al., 2004). Other ASIC, such as ASIC3 are involved in pain sensations (Sutherland et al., 2001). Better understanding of ASIC and its related sodium channel family proteins could lead to the formation of antagonists that have therapeutic value for both pain relief and stroke-induced cell death.
Because ASIC, ENaC, and DEG are all homologous proteins, the structure of one isoform of ASIC could provide great insight into the structures of other isoforms of ASIC, ENaC and DEG. Past research has lead to discrepancies in the stoichiometric ratio of subunits. A wide variety of models have been offered that include anywhere from tetramers to nine subunit complexes (Coscoy, 1998; Snyder, 1998). The crystal structure of ASIC1 described below is the first look at proteins in the superfamily of ENaC. As such, an understanding of ASIC1 is important to the understanding of sodium channel protein structure and function.
Desensitiazed form of ASIC1
The crystal structure of ASIC1 obtained by Jasti et al. (2007) is in the desensitized form (PDB:2QRTS). The desensitized form of the protein occurs at a low pH when the ion channel is closed and the protein is inactive. Under native conditions the desensitized form is the form in which the pore has closed up after opening of the ion channel. It does not conduct ions, although there are still protons bound the extracellular domain. These bound protons were used to initially activate the channel opening (Canessa, 2007).
Overall Structure
ASIC1 exists as a homotrimer related by a three-fold axis of molecular symmetry in the extracellular domain and no clear symmetry in the transmembrane domain (Fig 1). While the extracellular domain remains relatively similar among the subunits, the transmembrane domain of each subunit has considerably different conformations. Like all other transmembrane channel proteins, ASIC1 is composed of a short region that faces the cytoplasm and a large domain that resides outside the cell. The length of the entire structure as it exists imbedded in the cellular membrane is 130 Å, with 80 Å protruding from the membrane plane. The transmembrane domain is much smaller in width compared to the 85 Å extracellular domain (Jasti et al., 2007). In this sense, ASIC1 can be thought of as in the shape of a side view of a closed hand, with the narrow transmembrane domain being the wrist and arm and the bulky extracellular domain being the closed fist.

Figure 1. Structure of ASIC1. The structure is shown relative to the lipid bilayer (left) and from above (right). Each of the three subunits are colored differently. The extracellular domain is 80 Å, while the transmembrane is 50 Å in length. The width of the extracellular domain is 85 Å. PDB ascession code: 2QTS.

The transmembrane portion of the protein is composed of six long helices, two from each subunit. The N and C termini lie in the cytoplasmic side of the membrane. However, little of the protein exists in the cytoplasmic side of the membrane and there is no well ordered protein structure observable. This shows that the ion sensing and specificity domains are located outside the cell, which indicates that ASIC1 transports extracellular material into the cell as opposed to out of it. This is reemphasized by the extravagant, clearly ordered structures of the extracellular domain. Ordered loops are found at the “wrist” like connection between the helices of the transmembrane domain and the β-sheets in the extracellular domain. The extracellular domain is composed of primarily large β-sheets located around the central area and small β-sheets and α-helices throughout the rest of the extracellular domain. Although there are many cavities in the structure, at low pH there is no continuous pore that would allow the passage of ion into the cell. Thus, it is understandable that the structure must undergo a conformational change and open in response to pH change in order to function (Jasti et al., 2007).

The various subdomains of the protein can be observed in Figure 2. The extracellular portion of the protein can be divided into several parts following the closed fist analogy: palm, thumb, β-ball, knuckle, and finger (Jasti et al., 2007). The large β-sheets of the palm subdomain that dominates the extracellular domain are directly joined to the transmembrane α-helices via strands 1 and 12 and the thumb subdomain by strands 9 and 10. This proves to be crucial in coupling the conformational changes that are associated with channel opening. Adjacent to the palm subdomain is the thumb subdomain that consists mostly of α-helices and loops. Above the palm subdomain are α-helices and loops called the knuckle and finger subdomain, respectively. The thumb, finger, and palm subdomains surround a small five strand β-ball located relatively in the middle of the extracellular domain of the subunit. The extracellular domain of each ASIC1 subunit is held together by seven disulfide bonds. Five of these disulfide bonds are found in the thumb subdomain, starting from the thumb-finger junction straight down to the wrist junction. These disulfide bonds serve to provide structural integrity in order to facilitate the conformational changes that occur with pore opening (Jasti et al., 2007).

Figure 2. Structure of one subunit of ASIC1. One subunit of the protein is shown with the different subdomains colored differently. The red color labels the transmembrane region, cyan is the palm, green in the thumb, tan is the β-ball, pink is the knuckle, and orange is the finger. PDB ascession code: 2QTS.

Subunit Interactions
Figure 3 shows the key interactions of the various subunits of ASIC1 that contributes to the quantarny structure. The three subunits of ASIC1 interact through palm subdomains, palm-thumb subdomains, and knuckle and finger to palm and β-ball subdomains. The tight interaction of the three subunits serves to assemble and stabilize the resulting trimer protein. Three subunit-subunit interactions in the extracellular domain are of particular interest in structure stability. The first involves hydrogen bonds between Asp79 of one subunit and His74 and Gln421 of another subunit at the wrist junction. Previous studies show that these residue interactions are crucial to the rate of desensitization in ASICs (Jasti et al., 2007). Mutations of these residues into alanines results in anything from drastically lowered rates of desensitization to complete inactivity by protons (Coric et al., 2003). Another subunit interaction region of interest involves the loops in the palm subdomain in one subunit and the carboxyl terminus of the helix α5 thumb subdomain in another subunit. Arg176 forms hydrogen bonds to three consecutive carbonyl oxygens on α5. Lys212 of one subunit inserts into an adjacent subunit by binding to a buried chloride ion. This interaction with the chloride ion performs a role in the assembly and gating of ASICs (Jasti et al., 2007). The final interaction area of interest is the envelopment of the α6 helix of the knuckle subdomain by a concave surface on the finger subdomain of another subunit. The interactions of subunits are crucial to stabilization of the protein during conformational changes to open the ion channel (Jasti et al., 2007).

Figure 3. ASIC1 subunit interaction. The interactions between two adjacent subunits at three distinct points (indicated by the boxes) are shown. The top box shows the interaction of the α6 helix of one subunit’s knuckle subdomain with the concave surface on an adjacent subunit’s finger subdomain. The middle box shows the interaction of Arg716 with α5 helix of an adjacent subunit’s thumb subdomain and Lys212 with a buried chloride ion. The bottom box shows the interaction of Asp79 on one subunit and His74 and Gln421 on another subunit. PDB ascession code: 2QTS.

Transmembrane Domain
The six helix transmembrane domains of acid sensing ion channels are distinguished from other membrane receptors, like the tetramer potassium channel receptors, in that they are trimers (MacKinnon, 1991). Figure 4 shows the transmembrane domain of ASIC. The transmembrane domain that transcends the lipid bilayer of the cellular membrane is composed of mostly aliphatic hydrophobic residues. These residues interact with the lipid bilayer of the cell membrane to influence structure and gating of the channel. A V-shaped cavity that extends 5-10 Å into the bilayer is the connection between the openings of the transmembrane and extracellular domains. The pore opening to the extracellular domain is about 12 Å wide in the desensitized state (Jasti et al., 2007). There are no ions present in the pores during the desensitized state, which shows a very narrow pore that only allows passage of one ion at a time. The narrowness of the pores also facilitates the stripping of water molecules from sodium ions prior to entering the channel. Furthermore, the narrowness of the channel allows for selectivity of sodium ions over larger potassium ions. Detergents with both hydrophobic and hydrophilic ends reside in the cavity pores and interact with the residues of the protein to influence structural stability and channel opening. The interior of the transmembrane domain is lined with negatively charged residues and contributes to an overall negative electrostatic potential. This feature allows for cation selectivity and stability as it travels down the channel. Furthermore, the residues that line the interior of the pore are often conserved, which suggests crucial implications on structure and function (Jasti et al., 2007). Replacement of one of these conserved residues by a bulky amino acid, such as tyrosine or tryptophan, resulted in constitutive opening of the ion channel (Pfister et al., 2006). This demonstrates that mutations involving large amino acid residues along the pore results in an inability to control gating across the ion channel. There is a kink in the transmembrane domain helices caused by the Gly435 of two subunits in the protein. This kink allows for a smaller pore opening by bringing the helices of the transmembrane protein closer together. The effect is that Leu440 is able to close the pore (Jasti et al., 2007). Past experiments show that this region has an important role in the selectivity displayed by ENaC/DEG families of ion channels, in which ASIC is a member (Kellenberger et al., 2001).

Figure 4. Transmembrane domain of ASIC1. The transmembrane domain of ASIC1 (left) is shown with the top pointing to the extracellular region and the bottom to the cytoplasm. The blue shows hydrophilic regions, while the tan shows hydrophobic regions. A cut away view of the transmembrane domain (right) shows the ion channel. The Leu440 (red) and Gly435 (green) that provides the size specificity of the channel are labeled. PDB ascession code: 2QTS.

Proton-Binding Site
Acid sensing proton channels, as the name would suggest, change confirmation and open their channel upon binding to protons. The crystal structure and electrostatic potential mapping showed the acidic pocket that binds protons is formed by intrasubunit interactions between the thumb, β-ball, and finger subdomains of one subunit and the palm subdomain of an adjacent subunit. The acidic pocket is lined with highly negative residues, as seen in Figure 5. There are three unusually close carboxy-carboxylate interactions between residue side chains in the pocket. Asp238-Asp350 and Glu239-Asp346 are interactions between the finger and thumb subdomain of one subunit. Glu220-Asp408 interaction is located on the palm of an adjacent subunit. The interactions between these acidic amino acids are between 2.8-3.0 Å, which suggests that one of the carboxyl groups in each pair is protonated (Jasti et al., 2007). Studies show that placing two carboxyl groups in close proximity shifts the pK_a of one group 2-3 units to the basic side and shifts the pK_a of the other group 1-2 units to the acidic side (Sawyer and James, 1982). Previous studies on extracellular cation permeability showed that the permeability of calcium ion to ASIC appears to be a voltage-independent pathway of entry into the cell (Waldmann et al., 1997). This is because at high pH, calcium serves to stabilize the resting closed state of the channel, the conformation of ASIC that is unbound to protons (Todorvic et al., 2005). Calcium regulates the channel by competitively binding against protons for ASIC. Calcium ions block the protons and thus modulate the affinity of ASIC to protons. When protons bind the extracellular domain there is a conformational change in the extracellular domain subunits that releases a calcium ion, induces a conformational change in the transmembrane domain, and opens the ion channel (Paukert et al., 2004). This suggests that some, or all, of the pairs of carboxyl-carboxylate interactions coordinate calcium. Mutation of one member of the carboxyl-carboxylate pairs showed dramatic changes in pH_50, or the apparent Hill coefficient, or both (Jasti et al., 2007). This shows that both partners of the pair are important in proton sensing. Furthermore, the observation that there are three pairs of amino acids that sense protons indicates that proton sensing is distributed over multiple residues, perhaps to accommodate mutations (Jasti et al., 2007).

Figure 5. Proton binding site of ASIC1. Three close carboxy- carboxylate interactions, Asp238-Asp350 (red), Glu239-Asp346 (blue), and Glu220-Asp408 (yellow), form the acidic pocket that recognizes and binds protons. The interactions between these amino acid pairs are 2.8-3.0 Å. PDB ascession code: 2QTS.

Mechanism
It has long been known that an extracellular current voltage caused by a proton gradient activates acid-sensing ion channels. An acidic level of pH 5.9-6.4, lower than the physiological level of 7.4, activates ASIC1. In the presence of protons, the channel opens and allows the passage of sodium into the cell. It is believed that the thumb subdomain, stabilized in both open and closed conformations by disulfide bonds, undergoes movement depending on whether protons are bound or unbound. These movements of the thumb are transmitted to the transmembrane domain at the wrist junction. Tyr288, located at the base of the thumb subdomain, acts like a ball sitting in the ball-and-socket joint of the transmembrane domain. When the thumb subdomain moves, its movements are coupled with that of the transmembrane domain via movement of Tyr 288 in its socket joint (Jasti et al., 2007).
Discussion
The crystal structure of ASIC1 provides a view into and better understanding of proteins homologous to ASIC1, such as ENaC, DEG, and FaNaCh. The structure of ASIC shows that other members in its family are most likely trimers, either heterotrimers or homotrimers. Furthermore, the general motif of the extracellular domain being riddled with protrusions and cavities, but no central opening pore, probably also holds true for ENaC, DEG, and FaNaCh channels. The proton binding site distributed among different acidic amino acid residue pairs in the binding cleft is another facet of ASIC that probably holds true for homologous proteins (Jasti et al., 2007). Furthermore, the transmembrane pore for this class of channel proteins is very specific to sodium. The conformational change of the thumb region coupled with that of the transmembrane domain accomplishes the task of opening the ion channel and allowing sodium to enter the cell. Regulation of the ASIC ion channel comes from competitive binding of calcium ions to the proton binding site (Paukert et al., 2004). All this information can be applied to an understanding of the general structure and function of protein in the ENaC/DEG family.
The family of six ASIC proteins, coded by four genes, resides in the central and peripheral nervous system. ASICs are involved in pain perception correlated with acidosis. Ischemia, shortage of blood supply to a particular body part, and hyperglycemia, high blood sugar, can cause tissue pH to fall to 6.0-6.5. It is also known that drastically lowered pH can amplify ischemia (Xiong et al., 2004). ASIC1 activation, between pH 5.9-6.4, might have some connection to physiological damage caused by ischemia and hyperglycemia. However, the mechanism between acidosis and ischemia and the relationship between ischemia and ASIC1 are unclear. Although experiments have shown that eliminating ASIC1 and blocking ASIC expression in neuron cells reduces acidosis stimulated cell damage (Yermolaieva et al., 2004). A better understanding of the structure and function of ASIC allows for development of therapeutic remedies to prevent extreme pain perception and extensive cell death due to ischemia.
The next step in the understanding of ASIC and its related family of sodium channels is to obtain a crystal structure of the protein in its active form. This form involves an open sodium channel and bound protons. A crystal structure would provide insight and proof for the specific mechanism of ion channel opening and conformational change of the protein.

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