Proteopedia - User contributions [en]

User:Lori Wetmore/Sandbox 2

2010-10-05T03:12:42Z

Lori Wetmore:

{{STRUCTURE_1k4c| PDB=1k4c | SCENE= }}

==Background Information==
Potassium, a major cation in most cells, is responsible (in addition to other cations such as sodium) for the creation of the cell membrane potential, responsible for the generation of an action potential, which is necessary for a number of cellular functions such as neurotransmission, muscle contraction, and heart function. The proper balance of potassium in the cell is maintained by potassium ion pumps in the cellular membrane. To date, there are five potassium ion channels with a resolved structure (KcsA, KirBac1.1, KirBac3.1, KvAP, MthK), with KirBac3.1 being the most recently resolved, and they are all tetramers with several conserved secondary structural elements. <ref name="Hellgren">PMID:16253415</ref> A basic diagram of a potassium channel is illustrated by the monomeric and <scene name='User:Amy_Kerzmann/Sandbox_8/Tetramer/1'>the tetrameric form of KcsA 1k4c</scene>.

There are four basic classes of potassium channels:
* Calcium-activated potassium channels (KCa), which open in response to the presence of calcium ions or other signaling molecules.
* Inward-rectifier potassium ion channel/Inwardly rectifying potassium channels (Kir, IRK), which pass current (positive charge) more easily into the cell than out of the cell.
* Tandem pore domain potassium channels (KCNK), which are constitutively open or possess high basal activation, such as the "resting potassium channels" or "leak channels" that set the negative membrane potential of neurons. When open, they allow potassium ions to cross the membrane at a rate which is nearly as fast as their diffusion through water.
* Voltage-gated potassium channels (KcsA, KvAP), which open or close in response to changes in the membrane potential/transmembrane voltage.

==Channel Structure==

There are over 80 mammalian genes that encode potassium channel subunits. However, potassium channels found in bacteria are amongst the most studied of ion channels, in terms of their molecular structure. Using X-ray crystallography, <ref name="Doyle">PMID:9525859</ref> <ref name="MacKinnon">PMID:9525854</ref> profound insights have been gained into how potassium ions pass through these channels and why sodium ions, which are much smaller than potassium ions, do not. <ref name="Armstrong">PMID:9556453</ref>
As previously mentioned, potassium channels have a tetrameric structure in which four identical protein subunits associate to form a homotetramer, or a fourfold symmetric complex arranged around a central ion conducting pore. The polypeptide chain of bacterial potassium channels comprise 158 amino acid residues folded into two transmembrane helices, a pore helix and a cytoplasmic tail of 33 residues.<ref name="Doyle">PMID:9525859</ref> The subunits pack together in such a way that there is a hole in the center which forms the ion pore through the membrane. Alternatively four related but not identical protein subunits may associate to form heterotetrameric complexes with pseudo-symmetry. All potassium channel subunits have a distinctive pore-loop structure that lines the top of the pore and is responsible for potassium selective permeability (i.e., the selectivity filter). This pore-loop structure is then connected to specialized gating domains unique to each type of potassium channel.
{{STRUCTURE_1bl8| PDB=1bl8 | SCENE= }}
The C-terminal transmembrane helix (the inner helix) faces the central pore while the N-terminal helix (the outer helix) faces the lipid membrane. The four inner helices of the molecule are tilted and kinked so that the subunits open outwards. The inner helices contain the region of the polypeptide chain between the two transmembrane helices, which is a segment of about 30 amino acid residues that contains the pore helix and loop regions which form the outer portion of the channel. It is these loop regions that together form the narrow selectivity filter that is responsible for the highly specific ion selectivity of these potassium ion channels.

A good illustration of this highly conserved structure within potassium channels can be seen in the potassium channel 1b18 from Streptomyces lividans, an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis (data to 3.2 angstroms) reveals that the four identical subunits create an inverted cone that cradles the selectivity filter of the pore in its outer end. The narrow selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large, water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to overcome attractive forces between K+ ions and the selectivity filter. This basic structure allows us to visualize the physical principles underlying selective K+ conduction. <ref name="Doyle">PMID:9525859</ref>

==Selecvitity Filter/s==
[[Image:1K4C.png|thumb|left|250px|'''Crystallographic structure of the bacterial KcsA potassium channel.'''<ref name="Zhou">PMID:11689936</ref> Only two of the four subunits of the tetramer are currently displayed for the sake of clarity, and the protein is displayed as a green cartoon diagram. In addition, backbone carbonyl groups and threonine sidechain protein atoms (oxygen = red, carbon = green) are displayed. Finally potassium ions (occupying the S2 and S4 sites) and the oxygen atoms of water molecules (S1 and S3) are depicted as purple and red spheres respectively.]]

Potassium ion channels remove the hydration shell from the ion when it enters the selectivity filter, which is formed by five residues (TVGYG-in prokaryotic species) in the P loop from each subunit which have their electro-negative carbonyl oxygen atoms aligned towards the center of the filter pore and form an anti-prism similar to a water solvating shell around each potassium binding site. The distance between the carbonyl oxygens and potassium ions in the binding sites of the selectivity filter is the same as between water oxygens in the first hydration shell and a potassium ion in water solution. Passage of sodium ions would be energetically unfavorable since the strong interactions between the filter and pore helix would prevent the channel from collapsing to the smaller sodium ion size.<ref name="Miloshevsky">PMID:18621821</ref> The selectivity filter opens towards the extracellular solution, exposing four carbonyl oxygens in a glycine residue (Gly79 in KcsA). The next residue towards the extracellular side of the protein is the negatively charged Asp80 (KcsA). This residue together with the five filter residues form the pore that connects the water filled cavity in the centre of the protein with the extracellular solution.<ref name="Hellgren">PMID:16253415</ref>

The carbonyl oxygens are strongly electro-negative and cation attractive. The filter can accommodate potassium ions at 4 sites usually labelled S1 to S4 starting at the extracellular side. In addition one ion can bind in the cavity at a site called SC or one or more ions at the extracellular side at more or less well defined sites called S0 or Sext. Several different occupancies of these sites are possible. Since the X-ray structures are averages over many molecules, it is, however, not possible to deduce the actual occupancies directly from such a structure. In general, there is some disadvantage due to electrostatic repulsion to have two neighbouring sites occupied by ions. The mechanism for ion translocation in KcsA has been studied extensively by simulation techniques. A complete map of the free energies of the 24=16 states (characterised by the occupancy of the S1, S2, S3 and S4 sites) has been calculated with molecular dynamics simulations resulting in the prediction of an ion conduction mechanism in which the two doubly occupied states (S1, S3) and (S2, S4) play an essential role. The two extracellular states, Sext and S0, were found in a better resolved structure of KcsA at high potassium concentration. In free energy calculations the entire ionic pathway from the cavity, through the four filter sites out to S0 and Sext was covered in molecular dynamics(MD) simulations.<ref name="Allen">PMID:10545352</ref> The amino acids sequence of the selectivity filter of potassium ion channels is conserved with the exception that an isoleucine residue in eukaryotic potassium ion channels often is substituted with a valine residue in prokaryotic channels.<ref name="Hellgren">PMID:16253415</ref>

==Channel Function==
Calcium-Activated potassium channels, which include BK (big cunductance), IK (intermediate conductance), and SK (small conductance) channels, are responsible for a number of important physiological properties, including smooth muscle tone, neuronal excitability<ref name="Wu">PMID:20574420</ref>, electrical tuning of hair cells in the cochlea, and are also thought to be involved in synaptic plasticity, thus playing important roles in memory and learning.<ref name="Stackman">PMID:12451117</ref>

Inward-rectifier potassium ion channels(Inwardly rectifying potassium channels) are ubiquitously expressed and serve functions as diverse as regulation of resting membrane potential, maintenance of K(+) homeostasis, control of heart rate, and hormone secretion. <ref name="Abraham">PMID:10545352</ref>

Tandem pore domain potassium channels, which underlie leak K+ currents, are expressed throughout the central nervous system,<ref name="Talley">PMID:11567039</ref> and currents through these channels contribute to the resting membrane potential of neurons and regulate their excitability.<ref name="Aller">PMID:16339039</ref>

Voltage-gated potassium channels are sensitive to voltage changes in the cell's membrane potential and are responsoble for returning a depolarized cell to its resting state during an action potential. <ref name="Zhang">PMID:11375270</ref>

==Gating Mechanism==
A 10 Å wide central pore is located near the center of the transmembrane channel where the energy barrier is highest for the transversing ion due to the hydrophobity of the channel wall. The water-filled cavity and the polar C-terminus of the pore helices ease the energetic barrier for the ion. Repulsion by preceding multiple potassium ions is thought to aid the throughput of the ions. The presence of the cavity can be understood intuitively as one of the channel's mechanisms for overcoming the dielectric barrier, or repulsion by the low-dielectric membrane, by keeping the K+ ion in a watery, high-dielectric environment.

==Ongoing Research==
{{STRUCTURE_1qdv| PDB=1qdv | SCENE= }}One important aspect of ongoing research on potassium channels concerns determining the precise role that specific domains within the protein play in channel function. For example, all Kv voltage-gated potassium channels share a cytoplasmic assembly domain, T1. Research into whether or not this T1 domain plays a direct role in gating mechanisms has suggested that structural changes involving the buried polar T1 surfaces play a key role in the conformational changes leading to channel opening. <ref name="Minor">PMID:11007484</ref> In the 1QDV, a 4 chain structure of sequences from Rattus Norvegicus (the brown rat), an isosteric mutation causes surprisingly little structural alteration while stabilizing the closed channel and increasing the stability of T1 tetramers. Replacing T1 with a tetrameric coiled-coil destabilizes the closed channel, suggesting that in mammalian Kv1.2, gating depends critically on residues at complementary T1 surfaces in an unusually polar interface.

==References==
<references />

User:Lori Wetmore/Sandbox 3

2010-10-05T02:27:33Z

Lori Wetmore: /* '''Cl- ion Movement Through the Channel''' */

=='''ClC Channels and Transporters'''==
==='''Basic Function'''===
The ClC family of chloride channels and transporters are a group of proteins that transport Cl- ions across plasma or intracellular membranes. ClC channels are unrelated in sequence to all other ion-transporting channels, including other Cl- and anion-transporting channels. ClC channels serve many functions within the prokaryotic and eukaryotic cell. Within prokaryotes, ClC channels function to help maintain cell pH, such as in the extreme acid resistance response in ''E. coli'' <ref>PMID:12384697</ref>. Within eukaryotes, and mammals specifically, ClC channels are found in many different tissue types and provide a wide variety of services such as acidifying intracellular vesicles<ref>PMID:17110406</ref>, returning the resting membrane potentials of muscles to normal<ref>PMID:1659664</ref>, and allowing synaptic transmission in neurons<ref>PMID:17046694</ref>.

ClC channels are capable of moving ions in either direction across the membrane. However, in the majority of this article, for simplicity's sake, movement of ions will be described as if Cl- ions are moving from the extracellular→intracellular environment.

==='''Functions within Mammals'''===
Members of the ClC family of chloride channels are found in all of the kingdoms. Mammals contain 9 different types of ClC channels<ref>PMID:11917096</ref>. Many of the known functions of mammalian ClC channels have been determined based on the diseases caused in their absence. For example, a mutated form of the skeletal muscle ClC-1 channel in humans, mice, and goats leads to myotonia, a neuromuscular disease in which muscles have difficulty relaxing. This disease phenotype helped to implicate the ClC-1 channels in returning the resting membrane potential of skeletal muscles back to normal. 
Though the basic structure of these channels is the same (further explained in later sections), there are many differences in intracellular localization, tissue residence, Cl- transport vs. Cl-/H+ antiporter function, and gating properties<ref>PMID:16316975</ref> between the various members of the ClC family. The chart below shows the members of the ClC family that are present in humans and the various characteristics of these proteins.
 
 
<table border="1" cell padding="2">
<tr>
<TH COLSPAN=5 align="center">ClC Channels within Homo sapiens</TH>
</tr>
<tr>
<th>Channel Name</th>
<th>Tissue</th>
<th>Location Within Cell</th>
<th>Basic Function</th>
<th>Gated By</th>
</tr>

<tr>
<td>ClC-1</td>
<td>Skeletal Muscle</td>
<td>Plasma Membrane</td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage, pH</td>
</tr>
<tr>
<td>ClC-2</td>
<td>Many (Brain, Retina, Intestine, etc.)</td>
<td>Plasma Membrane</td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage, Cell Swelling</td>
</tr>
<tr>
<td>ClC-3</td>
<td>Many (Brain, Kidney, Liver, etc.)</td>
<td>Endosomes, Synaptic Vesicles</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage, Cell Swelling, Phosphorylation</td>
</tr>
<tr>
<td>ClC-4</td>
<td>Many (Brain, Skeletal Muscle, Heart, etc.)</td>
<td>Intracellular Membranes (tentative)</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage, pH</td>
</tr>
<tr>
<td>ClC-5</td>
<td>Kidney, Intestine, Liver</td>
<td>Endosomes</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage, pH</td>
</tr>
<tr>
<td>ClC-6</td>
<td>Many</td>
<td>Endosomes</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage</td>
</tr>
<tr>
<td>ClC-7</td>
<td>Many</td>
<td>Endosomes</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage</td>
</tr>
<tr>
<td>ClC-Ka</td>
<td>Kidney, Inner Ear</td>
<td>Plasma Membrane</td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage</td>
</tr>
<tr>
<td>ClC-Kb</td>
<td>Kidney, Inner Ear</td>
<td>Plasma Membrane</td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage</td>
</tr>
</table>
 

=='''The Structure of ClC Channels'''==
<applet load='1kpl' size='325' frame='true' align='left' caption='X-ray structure of ClC channel from ''S. typhimurium''' scene='User:Lori_Wetmore/Sandbox_3/Clc_channel_use/1' target='0' />
==='''Determining ClC Channel Structure'''===
Though the basic types and functions of many eukaryotic ClC channels have been elucidated, no exact structural information (i.e. crystallography structures) exists for eukaryotic ClC channels. Therefore, much of the current knowledge regarding ClC channel structure has come from the elucidation of the ClC structures of prokaryotes such as E. coli<ref>PMID:12649487</ref> and S. typhimurium<ref>PMID:11796999</ref>. How much information on the function of eukaryotic channels can be drawn from the prokaryotic structure? Overall, the sequence similarity between prokaryotic and eukaryotic ClC channels is low. In addition, prokaryotic ClC channels differ from eukaryotic ClC channels in the composition of their amino and carboxy terminal domains. Eukaryotes have a larger, intracellular carboxy terminal domain that is not present within prokaryotic ClC channels<ref>PMID:12885874</ref>. These differences, however, do not eliminate the ability to study eukaryotic ClC channel function with prokaryotic models. As will be elaborated on later, several selectivity filter and gating residues are conserved amongst prokaryotes and eukaryotes <ref>PMID:16554809</ref><ref>PMID:17710638</ref><ref>PMID:14985752</ref>, allowing Cl- transport within eukaryotic ClC channels to be studied within prokaryotic models. 

==='''Basic Structure'''===
On the left is the X-ray structure of the <scene name='User:Lori_Wetmore/Sandbox_3/Clc_channel_use/1'>ClC channel</scene> from the bacteria ''S. typhimurium''. The ClC channel is composed of two subunits, with each subunit consisting of 18 alpha helices. Each subunit is composed of two <scene name='User:Lori_Wetmore/Sandbox_3/Clc_channel_antiparallel/2'>antiparallel segments</scene>, that, at their interface, form the selectivity filter of the Cl- ions. The two subunits form a dimer, and there is an extensive interface between the two subunits. However, the interaction between the two dimers is not necessary for pore formation<ref>PMID:11796999</ref>.Instead, the basic structure of ClC channels is that of a <scene name='User:Lori_Wetmore/Sandbox_3/Clc_channel_double_barrel/2'>"double barrel"</scene>, in which each of the subunits contains its own pore, and two subunit monomers combine to form a double-pore channel <ref>PMID:11917096</ref>.
 

=='''Gating and Ion Selectivity'''==
==='''Fast-Gating vs. Slow-Gating'''===

<applet load='1ots' size='325' frame='true' align='right' caption='X-ray structure of ClC channel from ''E. coli''' scene='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex/1' target='0' />
All studied ClC channels have been shown to be gated by voltage <ref>PMID:11182894</ref>. However, different ClC channels exhibit a great variety of responses to specific voltage changes. For example, within humans, the ClC-1 channel closes during hyperpolarization, while the ClC-2 channel opens as a result of the same change. Other ClC channels have been shown to open or close due to other factors such as pH, cell-swelling, or phosphorylation<ref>PMID:11182894</ref>. 

In addition, ClC channels are voltage-gated by two different mechanisms known as fast-gating and slow-gating. Within slow-gating, which takes several seconds, both pores are opened upon hyperpolarization of the membrane. Within fast-gating, on the other hand, pores react independently of each other, and react in a matter of milliseconds<ref>PMID:11182894</ref>. This fast-gating is due to a combination of Cl- movement and protonation of glutamate residue gates, and will be explained in greater detail in the following sections.

==='''Selectivity Filter'''===
In order to study the selectivity filter of the ClC channels, a complex was created between the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_no_fab/1'>ClC channel</scene> from E. coli and a <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_no_clc/1'>Fab antibody</scene>. The Fab antibody was attached to the extracellular surface of the Cl- channel, and was added to stabilize the ClC channel. 

Each pore contains a selectivity filter that connects the intracellular and extracellular aqueous environments and through which ions travel. This selectivity filter consists of a number of side chains and main-chain amide nitrogen atoms that allow Cl-, and, in some cases, H+ ions through the channel. 

The selectivity filter consists of three possible binding sites for the chloride ion. These binding sites are known as Sint, Scen, and Sext. The Sint and Sext sites are in contact with the '''int'''racellular and '''ext'''racellular environments, respectively, while the Scen site is located in '''cen'''trally, between the other two sites. In the image on the right, chloride ions are bound in the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_zoom/1'>S(cen) (upper) and S(int) (lower) sites</scene>. Within the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_sint/1'>S(int)</scene> site, the chloride ion interacts with main-chain amide nitrogens of '''Gly106''' and '''Ser107'''<ref>Accardi, A. Structure and Function of CLC Chloride Channels and Transporters. Advances in Molecular and Cell Biology. 2006:56-82.</ref>. Within the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_scen/5'>S(cen)</scene> site, the chloride ion interacts with the side chains of the conserved '''Ser107''' and '''Tyr445''' residues as well as nitrogen atoms from the main-chain amide groups of '''Gly149''', '''Ile356''', and '''Phe357'''<ref>PMID:18977737</ref>. Within the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_sext/2'>S(ext)</scene> site, the chloride ion interacts with main-chain amide nitrogens from '''Gly315''', '''Gly316''', and '''Phe317'''<ref>PMID:14747318</ref>. When the pore is closed (as in this image), the Sext site is blocked by the '''Glu148 residue''', both preventing a Cl- ion from inhabiting the Sext site as well as keeping other Cl- ions from entering the channel. 
==='''Cl- ion Movement Through the Channel'''===
When the extracellular gate, or Glu148 residue, is deprotonated, it remains in a closed conformation and Cl- ions are incapable of entering the channel. When this residue is protonated, it swings outward, allowing a Cl- ion to bind in the Sext site <ref>PMID:11796999</ref>. The Cl- ion is then transferred to the Scen site, at which point the Glu148 residue moves back to occlude the pore entrance. The Cl- ion then moves to the the Sint site and out of the channel. By this mechanism, two Cl- ions can be within the channel when it is closed, while three Cl- ions are capable of being in the channel when it is open<ref>PMID:12649487</ref>. A mutation in which the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_e148q/1'>Glu148 residue was replaced with a glutamine</scene> resembles the theorized structure of the open conformation of the ClC channel. A change in Cl- ion localization during the open and closed states of the channel can be observed by comparing <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_e148q_q_view/3'>the E148Q mutation</scene> with the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_sext/3'>wild-type</scene>, respectively.

=='''Cl-/H+ Transportation'''==
<applet load='2fee' size='325' frame='true' align='left' scene='User:Lori_Wetmore/Sandbox_3/Clc-ec1/1' target='0' />

There is some debate as to the function of various members of the ClC family. While the ClC channel from ''E. coli'' was originally assumed to be simply a Cl- channel, work by Accardi and Miller suggested that the ClC-ec1 channel was not a Cl- channel, but instead a transporter that coupled Cl- and H+ transport <ref> PMID:14985752</ref>.
As it turns out, several members of the family of ClC channels are Cl-/H+ transporters rather than simple Cl- ion channels. Given the conserved structure amongst members of the ClC family, how can these different two separate functions be justified? 

==='''Ion Channel vs. Antiporter'''===
In order to understand the mechanisms behind transport of Cl- alone as opposed to Cl- transport coupled to H+ transport, it is worth noting the differences in the gating mechanisms of ion channels versus their active transport counterparts. Ion channels generally take on a simple "open" or "closed" state, dependent on whether they are allowing or preventing ion movement through the channel, and therefore often require only one gate that may then be switched on or off. Antiporters, on the other hand, must be gated on at least one end at all times in order to properly coordinate the transport of two separate ions in different directions, and therefore often require multiple gates or gating mechanisms to regulate this transport. 
It seems counterintuitive that a similar structure within all ClC channels could yield two decidedly different functions. Within ClC channels that act solely to transport Cl- ions, the conserved Glu148 residue acts as the single gate that allows or prevents Cl- flow by the mechanism mentioned above. However, the glutamate gate is only a small part of the necessary components of Cl-/H+ antiporter gating.
===='''Coordinating Cl- Transport'''====
While the Glu148 residue, or <scene name='User:Lori_Wetmore/Sandbox_3/Clc-ec1_e148/1'>extracellular gate</scene>, ensures that extracellular ions are maintained by blocking the Sext entrance, an <scene name='User:Lori_Wetmore/Sandbox_3/Clc-ec1_intracellular_gate/2'>intracellular gate</scene> is created by the Ser107, Tyr445, and Phe348 residues<ref>PMID:20303857</ref>, preventing ions from exiting or entering via the intracellular pore. 

===='''Coordinating H+ Transport'''====
The Glu148 residue acts as the "end of the line" for H+ ion transport. In order for H+ ions to be transported, a residue on the intracellular side of the selectivity filter, Glu203 is protonated. The H+ ions are then transported through the channel to the Glu148 residue and into the extracellular environment. 

The distance between the Glu148 and the Glu203 residues is <scene name='User:Lori_Wetmore/Sandbox_3/Clc-ec1_e148_angstroms/1'>approximately 15 angstroms</scene><ref>PMID:17710638</ref>, and there must, therefore, be an intermediate location for protonation. The exact pathway of H+ transport has not yet been elucidated, but it has been suggested that the <scene name='User:Lori_Wetmore/Sandbox_3/Clc-ec1_e148_tyrosine/1'>Tyr445</scene> residue within the Cl- selectivity filter may be involved<ref>PMID:17710638</ref>.

==='''A Model for Cl-/H+ Transport'''===
Given what is known separately about H+ and Cl- transport within the channel, a model<ref>PMID:18977737</ref> (Figure 1) has been developed that may explain the coordination of Cl- and H+ transport within the ClC antiporter. The movement of the Cl- and H+ ions is believed to be coordinated and to occur simultaneously. As ClC channels are capable of moving ions in either direction depending on the ion gradient, this steps in this model may be reversed. However, the model will be described as if Cl- ions are being moved in an extracellular→intracellular direction, while H+ ions are being moved in an intracellular→extracellular direction. (1) Initially, both glutamate gates are deprotonated, the Glu148 residue is blocking the extracellular entrance, and a Cl- ion is present in the Scen site. (2) The intracellular gate, Glu203, is protonated by an intracellular H+ ion, which then proceeds to move to the Scen site (3) and protonate the Cl- ion. When a proton is present at the Scen site, the intracellular gate opens. (4) This opening allows Cl- to leave the channel via the intracellular exit, while the H+ ion protonates the extracellular Glu148 residue. (5) When this glutamate gate is protonated, it opens, allowing two Cl- ions to inhabit the Scen and Sext sites. The extracellular glutamate gate is then deprotonated as the H+ ion leaves, causing it to want to close. However, as a Cl- ion is blocking the exit, it is incapable of closing. (6) Therefore, the glutamate residue pushes the Cl- ions through the channel, causing one to exit the intracellular pore while the other remains in the Scent site (return to (1)). By this mechanism, the ClC transporter transports 2 Cl- ions for each proton transported<ref>PMID:18977737</ref>.

<center>[[Image:Clc_channel.jpg]]</center>

'''Figure 1:''' The coupled transport of Cl- ions and H+ ions. The blue rectangle represents one subunit of the ClC channel. The dotted circle in the middle represents the Scen site. The upper dip in the rectangle represents the extracellular pore (Sext site), while the lower dip represents the intracellular pore (Sint site). The extracellular Glu148 gate and the intracellular Glu203 residue are in blue, as is the intracellular gate composed of the Ser107, Tyr445, and Phe348 residues and represented by a blue line. The Cl- ion is represented by a purple dot, while the H+ ion is represented by a smaller red dot. The mechanism is described in detail above.

=='''References'''==

<references />

2010-10-05T01:16:21Z

Lori Wetmore:

=='''ClC Channels and Transporters'''==
==='''Basic Function'''===
The ClC family of chloride channels and transporters are a group of proteins that transport Cl- ions across plasma or intracellular membranes. ClC channels are unrelated in sequence to all other ion-transporting channels, including other Cl- and anion-transporting channels. ClC channels serve many functions within the prokaryotic and eukaryotic cell. Within prokaryotes, ClC channels function to help maintain cell pH, such as in the extreme acid resistance response in ''E. coli'' <ref>PMID:12384697</ref>. Within eukaryotes, and mammals specifically, ClC channels are found in many different tissue types and provide a wide variety of services such as acidifying intracellular vesicles<ref>PMID:17110406</ref>, return resting membrane potentials of muscles<ref>PMID:1659664</ref>, and synaptic transmission in neurons<ref>PMID:17046694</ref>.

ClC channels are capable of moving ions in either direction across the membrane. However, in the majority of this article, for simplicity's sake, movement of ions will be described as if Cl- ions are moving from the extracellular→intracellular environment.

==='''Functions within Mammals'''===
Members of the ClC family of chloride channels are found in all of the kingdoms. Mammals contain 9 different types of ClC channels<ref>PMID:11917096</ref>. Many of the known functions of mammalian ClC channels in particular have been determined based on the disease-states caused in their absence. For example, a mutated form of the skeletal muscle ClC-1 channel in humans, mice, and goats leads to myotonia, a neuromuscular disease in which muscles have difficulty relaxing. This disease phenotype helped to implicate the ClC-1 channels in returning the resting membrane potential of skeletal muscles back to normal. 
Though the basic structure of these channels is the same (further explained in later sections), there are many differences in intracellular localization, tissue residence, Cl- transport vs. Cl-/H+ antiporter function, and gating properties<ref>PMID:16316975</ref> between the various members of the ClC family. The chart below shows the members of the ClC family that are present in humans and the various characteristics of these proteins.
 
 
<table border="1" cell padding="2">
<tr>
<TH COLSPAN=5 align="center">ClC Channels within Homo sapiens</TH>
</tr>
<tr>
<th>Channel Name</th>
<th>Tissue</th>
<th>Location Within Cell</th>
<th>Basic Function</th>
<th>Gated By</th>
</tr>

<tr>
<td>ClC-1</td>
<td>Skeletal Muscle</td>
<td>Plasma Membrane</td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage, pH</td>
</tr>
<tr>
<td>ClC-2</td>
<td>Many (Brain, Retina, Intestine, etc.)</td>
<td>Plasma Membrane</td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage, Cell Swelling</td>
</tr>
<tr>
<td>ClC-3</td>
<td>Many (Brain, Kidney, Liver, etc.)</td>
<td>Endosomes, Synaptic Vesicles</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage, Cell Swelling, Phosphorylation</td>
</tr>
<tr>
<td>ClC-4</td>
<td>Many (Brain, Skeletal Muscle, Heart, etc.)</td>
<td>Intracellular Membranes (tentative)</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage, pH</td>
</tr>
<tr>
<td>ClC-5</td>
<td>Kidney, Intestine, Liver</td>
<td>Endosomes</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage, pH</td>
</tr>
<tr>
<td>ClC-6</td>
<td>Many</td>
<td>Endosomes</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage</td>
</tr>
<tr>
<td>ClC-7</td>
<td>Many</td>
<td>Endosomes</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage</td>
</tr>
<tr>
<td>ClC-Ka</td>
<td>Kidney, Inner Ear</td>
<td>Plasma Membrane</td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage</td>
</tr>
<tr>
<td>ClC-Kb</td>
<td>Kidney, Inner Ear</td>
<td>Plasma Membrane</td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage</td>
</tr>
</table>
 

=='''The Structure of ClC Channels'''==
<applet load='1kpl' size='325' frame='true' align='left' caption='X-ray structure of ClC channel from ''S. typhimurium''' scene='User:Lori_Wetmore/Sandbox_3/Clc_channel_use/1' target='0' />
==='''Determining ClC Channel Structure'''===
Though the basic types and functions of many eukaryotic ClC channels have been elucidated, no exact structural information (i.e. crystallography structures) exists for eukaryotic ClC channels. Therefore, much of the current knowledge regarding ClC channel structure has come from the elucidation of the ClC structures of prokaryotes such as E. coli<ref>PMID:12649487</ref> and S. typhimurium<ref>PMID:11796999</ref>. How much information on the function of eukaryotic channels can be drawn from the prokaryotic structure? Overall, the sequence similarity between prokaryotic and eukaryotic ClC channels is low. In addition, prokaryotic ClC channels differ from eukaryotic ClC channels in the composition of their amino and carboxy terminal domains. Eukaryotes have a larger, intracellular carboxy terminal domain that is not present within prokaryotic ClC channels<ref>PMID:12885874</ref>. These differences, however, do not eliminate the ability to study eukaryotic ClC channel function with prokaryotic models. As will be elaborated on later, the selectivity filter and gating residues are conserved amongst prokaryotes and eukaryotes (CITE), allowing Cl- transport within eukaryotic ClC channels to be studied within prokaryotic models. 

==='''Basic Structure'''===
On the left is the X-ray structure of the ClC channel from the bacteria ''S. typhimurium''. The ClC channel is composed of two subunits, with each subunit consisting of 18 alpha helices. Each subunit is composed of two <scene name='User:Lori_Wetmore/Sandbox_3/Clc_channel_antiparallel/2'>antiparallel segments</scene>, that, at their interface, form the selectivity filter of the Cl- ions. The two subunits form a dimer, and there is an extensive interface between the two subunits. However, the interaction between the two dimers is not necessary for pore formation<ref>PMID:11796999</ref>.Instead, the basic structure of ClC channels is that of a <scene name='User:Lori_Wetmore/Sandbox_3/Clc_channel_double_barrel/2'>"double barrel"</scene>, in which each of the subunits contains its own pore, and two subunit monomers combine to form a double-pore channel <ref>PMID:11917096</ref>.
 

=='''Gating and Ion Selectivity'''==
==='''Fast-Gating vs. Slow-Gating'''===

<applet load='1ots' size='325' frame='true' align='right' caption='X-ray structure of ClC channel from ''E. coli''' scene='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex/1' target='0' />
All studied ClC channels have been shown to be gated by voltage <ref>PMID:11182894</ref>. However, different ClC channels exhibit a great variety of responses to specific voltage changes. For example, within humans, the ClC-1 channel closes during hyperpolarization, while the ClC-2 channel opens as a result of the same change. Other ClC channels have been shown to open or close due to other factors such as pH, cell-swelling, or phosphorylation<ref>PMID:11182894</ref>. 

In addition, ClC channels are voltage-gated by two different mechanisms known as fast-gating and slow-gating. Within slow-gating, which takes several seconds, both pores are opened upon hyperpolarization of the membrane. Within fast-gating, on the other hand, pores react independently of each other, and react in a matter of milliseconds<ref>PMID:11182894</ref>. This fast-gating is due to a combination of Cl- movement and protonation of glutamate residue gates, and will be explained in greater detail in the following sections.

==='''Selectivity Filter'''===
In order to study the selectivity filter of the ClC channels, a complex was created between the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_no_fab/1'>Clc channel</scene> from E. coli and a <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_no_clc/1'>Fab antibody</scene>. The Fab antibody was attached to the extracellular surface of the Cl- channel, and was added to stabilize the ClC channel. 

Each pore contains a selectivity filter that connects the intracellular and extracellular aqueous environments and through which ions travel. This selectivity filter consists of a number of side chains and main-chain amide nitrogen atoms that allow Cl-, and, in some cases, H+ ions through. 

The chloride channel consists of three possible binding sites for the chloride ion. These binding sites are known as Sint, Scen, and Sext. In the image on the right, chloride ions are bound in the Scen (upper) and Sint (lower) sites. The Sint and Sext sites are in contact with the '''int'''racellular and '''ext'''racellular environments, respectively, while the Scen site is located in '''cen'''trally, between the other two sites. Within the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_sint/1'>S(int)</scene> site, the chloride ion interacts with main-chain amide nitrogens of '''Gly106''' and '''Ser107'''(CITE structure and function of clc chloride channels and transporters by Accardi). Within the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_scen/4'>S(cen)</scene> site, the chloride ion interacts with the side chains of the '''Ser107''' and '''Tyr445''' residues as well as nitrogen atoms from the main-chain amide groups of '''Gly149''', '''Ile356''', and '''Phe357'''<ref>PMID:18977737</ref>. Within the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_sext/2'>S(ext)</scene> site, the chloride ion interacts with main-chain amide nitrogens from '''Gly315''', '''Gly316''', and '''Phe317'''<ref>PMID:14747318</ref>. When the pore is closed, the Sext site is blocked by the '''Glu148 residue''', both preventing a Cl- ion from being in the Sext site as well as keeping other Cl- ions from entering the channel. 

When the extracellular gate, or Glu148 residue, is deprotonated, it remains in a closed conformation and Cl- ions are incapable of entering the channel. When this residue is protonated, it swings outward, allowing a Cl- ion to bind in the Sext site <ref>PMID:11796999</ref>. The Cl- ion is then transferred to the Scen site, at which point the Glu148 residue moves back to occlude the pore entrance. The Cl- ion then moves to the the Sint site and out of the channel. By this mechanism, two Cl- ions can be within the channel when it is closed, while three Cl- ions are capable of being in the channel when it is open<ref>PMID:12649487</ref>. A mutation in which the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_e148q/1'>Glu148 residue was replaced with a glutamine</scene> resembles the theorized structure of the open conformation of the ClC channel. A change in Cl- ion localization during the open and closed states of the channel can be observed by comparing <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_e148q_q_view/3'>the E148Q mutation</scene> with the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_sext/3'>wild-type</scene>, respectively.

 

=='''Cl-/H+ Transportation'''==
<applet load='2fee' size='325' frame='true' align='left' scene='User:Lori_Wetmore/Sandbox_3/Clc-ec1/1' target='0' />

There is some debate as to the function of various members of the ClC family. While the ClC channel from ''E. coli'' was originally assumed to be simply a Cl- channel, work by Accardi and Miller suggested that the ClC-ec1 channel was not a Cl- channel, but instead a transporter that coupled Cl- and H+ transport <ref> PMID:14985752</ref>.
As it turns out, several members of the family of ClC channels are Cl-/H+ transporters rather than simple Cl- ion channels. However, given the conserved structure amongst members of the ClC family, how can these different two separate functions be justified? 

==='''Ion Channel vs. Antiporter'''===
In order to understand the mechanisms behind transport of Cl- alone as opposed to Cl- transport coupled to H+ transport, it is worth noting the differences in the gating mechanisms of ion channels versus their active transport counterparts. Ion channels generally take on a simple "open" or "closed" state, dependent on whether they are allowing or preventing ion movement through the channel, and therefore often require only one gate that may then be switched on or off. Antiporters, on the other hand, must be gated on at least one end at all times in order to properly coordinate the transport of two separate ions in different directions, and therefore often require multiple gates or gating mechanisms to regulate this transport. 
It seems counterintuitive that a similar structure within all ClC channels could yield two decidedly different functions. Within ClC channels that act solely to transport Cl- ions, the extracellular Glu148 residue acts as the single gate that allows or prevents Cl- flow by the mechanism mentioned above. However, the glutamate gate is only one half of the necessary components of Cl-/H+ antiporter gating.
===='''Coordinating Cl- Transport'''====
While the Glu148 residue, or <scene name='User:Lori_Wetmore/Sandbox_3/Clc-ec1_e148/1'>extracellular gate</scene>, ensures that extracellular ions are maintained by blocking the Sext entrance, an <scene name='User:Lori_Wetmore/Sandbox_3/Clc-ec1_intracellular_gate/1'>intracellular gate</scene> is created by the Ser107, Tyr445, and Phe348 residues<ref>PMID:20303857</ref>, preventing ions from exiting or entering via the intracellular pore. 

===='''Coordinating H+ Transport'''====
The Glu148 residue acts as the "end of the line" for H+ ion transport. In order for H+ ions to be transported, a residue on the intracellular side of the selectivity filter, Glu203 is protonated. The H+ ions are then transported through the channel to the Glu148 residue and into the extracellular environment. 

The distance between the Glu148 and the Glu203 residues is <scene name='User:Lori_Wetmore/Sandbox_3/Clc-ec1_e148_angstroms/1'>approximately 15 angstroms</scene><ref>PMID:17710638</ref>, and there must, therefore, be an intermediate location for protonation. The exact pathway of H+ transport has not yet been elucidated, but it has been suggested that the Tyr445 residue within the Cl- selectivity filter may be involved<ref>PMID:17710638</ref>.

==='''A Model for Cl-/H+ Transport'''===
Given what is known separately about H+ and Cl- transport within the channel, a model<ref>PMID:18977737</ref> has been developed that may explain the coordination of Cl- and H+ transport within the ClC antiporter. The movement of the Cl- and H+ ions is believed to be coordinated and to occur simultaneously. As ClC channels are capable of moving ions in either direction depending on the ion gradient, this steps in this model may be reversed. However, the model will be described as if Cl- ions are being moved in an extracellular→intracellular direction, while H+ ions are being moved in an intracellular→extracellular direction. Initially, both glutamate gates are deprotonated, the Glu148 residue is blocking the extracellular entrance, and a Cl- ion is present in the Scen site. The intracellular gate, Glu203, is protonated by an intracellular H+ ion, which then proceeds to move to the Scen site and protonate the Cl- ion. When a proton is present at the Scen site, the intracellular gate opens. This opening allows Cl- to leave the channel via the intracellular exit, while the H+ ion protonates the extracellular Glu148 residue. When this glutamate gate is protonated, it opens, allowing two Cl- ions to inhabit the Scen and Sext sites. The extracellular glutamate gate is then deprotonated as the H+ ion leaves, causing it to want to close. However, as a Cl- ion is blocking the exit, it is incapable of closing. Therefore, the glutamate residue pushes the Cl- ions through the channel, causing one to exit the intracellular pore while the other remains in the Scent site. By this mechanism, the ClC transporter transports 2 Cl- ions for each proton transported<ref>PMID:18977737</ref>.

 
[[Image:Clc_channel.jpg]]

=='''References'''==

<references />

2010-10-04T21:37:56Z

Lori Wetmore:

=='''ClC Channels'''==
==='''Basic Function'''===
ClC channels are unrelated in sequence to all other ion-transporting channels, including other Cl- and anion-transporting channels. ClC channels serve many functions within the prokaryotic and eukaryotic cell. Within prokaryotes, ClC channels function to help maintain cell pH, such as in the extreme acid resistance response in ''E. coli'' <ref>PMID:12384697</ref>. Within eukaryotes, and mammals specifically, ClC channels are found in many different tissue types and provide a wide variety of services such as acidifying intracellular vesicles<ref>PMID:17110406</ref>, return resting membrane potentials of muscles<ref>PMID:1659664</ref>, and synaptic transmission in neurons<ref>PMID:17046694</ref>. In order to perform these functions properly, ClC channels transport Cl- (and, in some cases, H+ ions), across the plasma membrane or intracellular membranes.

ClC channels are capable of moving ions in either direction across the membrane. However, in the majority of this article, for simplicity's sake, movement of ions will be described as if Cl- ions are moving from the extracellular→intracellular environment.

==='''Functions within Mammals'''===
Members of the ClC family of chloride channels are found in all of the kingdoms. Mammals contain 9 different types of ClC channels<ref>PMID:11917096</ref>. Many of the known functions of mammalian ClC channels in particular have been determined based on the disease-states caused in their absence. For example, a mutated form of the skeletal muscle ClC-1 channel in humans, mice, and goats leads to myotonia, a neuromuscular disease in which muscles have difficulty relaxing. This disease phenotype helped to implicate the ClC-1 channels in returning the resting membrane potential of skeletal muscles back to normal. 
Though the basic structure of these channels is the same (further explained in later sections), there are many differences in intracellular localization, tissue residence, Cl- transport vs. Cl-/H+ antiporter function, and gating properties<ref>PMID:16316975</ref> between the various members of the ClC family. The chart below shows the members of the ClC family that are present in humans and the various characteristics of these proteins.
 
 
<table border="1" cell padding="2">
<tr>
<TH COLSPAN=5 align="center">ClC Channels within Homo sapiens</TH>
</tr>
<tr>
<th>Channel Name</th>
<th>Tissue</th>
<th>Location Within Cell</th>
<th>Basic Function</th>
<th>Gated By</th>
</tr>

<tr>
<td>ClC-1</td>
<td>Skeletal Muscle</td>
<td>Plasma Membrane</td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage, pH</td>
</tr>
<tr>
<td>ClC-2</td>
<td>Many (Brain, Retina, Intestine, etc.)</td>
<td>Plasma Membrane</td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage, Cell Swelling</td>
</tr>
<tr>
<td>ClC-3</td>
<td>Many (Brain, Kidney, Liver, etc.)</td>
<td>Endosomes, Synaptic Vesicles</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage, Cell Swelling, Phosphorylation</td>
</tr>
<tr>
<td>ClC-4</td>
<td>Many (Brain, Skeletal Muscle, Heart, etc.)</td>
<td>Intracellular Membranes (tentative)</td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage, pH</td>
</tr>
<tr>
<td>ClC-5</td>
<td>Kidney, Intestine, Liver</td>
<td></td>
<td>H+/Cl- Exchange Transporter</td>
<td>Voltage, pH</td>
</tr>
<tr>
<td>ClC-6</td>
<td>Many</td>
<td></td>
<td></td>
<td>Voltage, ???</td>
</tr>
<tr>
<td>ClC-7</td>
<td>Many</td>
<td></td>
<td></td>
<td>Voltage, ???</td>
</tr>
<tr>
<td>ClC-Ka</td>
<td>Kidney, Inner Ear</td>
<td></td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage, ???</td>
</tr>
<tr>
<td>ClC-Kb</td>
<td>Kidney, Inner Ear</td>
<td></td>
<td>Plasma Membrane Ion Channel</td>
<td>Voltage, ???</td>
</tr>
</table>
 

=='''The Structure of ClC Channels'''==
<applet load='1kpl' size='325' frame='true' align='left' caption='X-ray structure of ClC channel from ''S. typhimurium''' scene='User:Lori_Wetmore/Sandbox_3/Clc_channel_use/1' target='0' />
==='''Determining ClC Channel Structure'''===
Though the basic types and functions of many eukaryotic ClC channels have been elucidated, no exact structural information (i.e. crystallography structures) exists for eukaryotic ClC channels. Therefore, much of the current knowledge regarding ClC channel structure has come from the elucidation of the ClC structures of prokaryotes such as E. coli<ref>PMID:12649487</ref> and S. typhimurium<ref>PMID:11796999</ref>. How much information on the function of eukaryotic channels can be drawn from the prokaryotic structure? Overall, the sequence similarity between prokaryotic and eukaryotic ClC channels is low. In addition, prokaryotic ClC channels differ from eukaryotic ClC channels in the composition of their amino and carboxy terminal domains. Eukaryotes have a larger, intracellular carboxy terminal domain that is not present within prokaryotic ClC channels<ref>PMID:12885874</ref>. These differences, however, do not eliminate the ability to study eukaryotic ClC channel function with prokaryotic models. As will be elaborated on later, the selectivity filter and gating residues are conserved amongst prokaryotes and eukaryotes (CITE), allowing Cl- transport within eukaryotic ClC channels to be studied within prokaryotic models. 

==='''Basic Structure'''===
On the left is the X-ray structure of the ClC channel from the bacteria ''S. typhimurium''. The ClC channel is composed of two subunits, with each subunit consisting of 18 alpha helices. Each subunit is composed of two <scene name='User:Lori_Wetmore/Sandbox_3/Clc_channel_antiparallel/2'>antiparallel segments</scene>, that, at their interface, form the selectivity filter of the Cl- ions. The two subunits form a dimer, and there is an extensive interface between the two subunits. However, the interaction between the two dimers is not necessary for pore formation<ref>PMID:11796999</ref>.Instead, the basic structure of ClC channels is that of a <scene name='User:Lori_Wetmore/Sandbox_3/Clc_channel_double_barrel/2'>"double barrel"</scene>, in which each of the subunits contains its own pore, and two subunit monomers combine to form a double-pore channel <ref>PMID:11917096</ref>.
 

=='''Gating and Ion Selectivity'''==
==='''Fast-Gating vs. Slow-Gating'''===

<applet load='1ots' size='325' frame='true' align='right' caption='X-ray structure of ClC channel from ''E. coli''' scene='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex/1' target='0' />
All studied ClC channels have been shown to be gated by voltage <ref>PMID:11182894</ref>. However, different ClC channels exhibit a great variety of responses to specific voltage changes. For example, within humans, the ClC-1 channel closes during hyperpolarization, while the ClC-2 channel opens as a result of the same change. Other ClC channels have been shown to open or close due to other factors such as pH, cell-swelling, or phosphorylation<ref>PMID:11182894</ref>. 

In addition, ClC channels are voltage-gated by two different mechanisms known as fast-gating and slow-gating. Within slow-gating, which takes several seconds, both pores are opened upon hyperpolarization of the membrane. Within fast-gating, on the other hand, pores react independently of each other, and react in a matter of milliseconds<ref>PMID:11182894</ref>. This fast-gating is due to a combination of Cl- movement and protonation of glutamate residue gates, and will be explained in greater detail in the following sections.

==='''Selectivity Filter'''===
In order to study the selectivity filter of the ClC channels, a complex was created between the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_no_fab/1'>Clc channel</scene> from E. coli and a <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_no_clc/1'>Fab antibody</scene>. The Fab antibody was attached to the extracellular surface of the Cl- channel, and was added to stabilize the ClC channel. 

Each pore contains a selectivity filter that connects the intracellular and extracellular aqueous environments and through which ions travel. This selectivity filter consists of a number of side chains and main-chain amide nitrogen atoms that allow Cl-, and, in some cases, H+ ions through. 

The chloride channel consists of three possible binding sites for the chloride ion. These binding sites are known as Sint, Scen, and Sext. In the image on the right, chloride ions are bound in the Scen (upper) and Sint (lower) sites. The Sint and Sext sites are in contact with the '''int'''racellular and '''ext'''racellular environments, respectively, while the Scen site is located in '''cen'''trally, between the other two sites. Within the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_sint/1'>S(int)</scene> site, the chloride ion interacts with main-chain amide nitrogens of '''Gly106''' and '''Ser107'''(CITE structure and function of clc chloride channels and transporters by Accardi). Within the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_scen/4'>S(cen)</scene> site, the chloride ion interacts with the side chains of the '''Ser107''' and '''Tyr445''' residues as well as nitrogen atoms from the main-chain amide groups of '''Gly149''', '''Ile356''', and '''Phe357'''<ref>PMID:18977737</ref>. Within the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_sext/2'>S(ext)</scene> site, the chloride ion interacts with main-chain amide nitrogens from '''Gly315''', '''Gly316''', and '''Phe317'''<ref>PMID:14747318</ref>. When the pore is closed, the Sext site is blocked by the '''Glu148 residue''', both preventing a Cl- ion from being in the Sext site as well as keeping other Cl- ions from entering the channel. 

When the extracellular gate, or Glu148 residue, is deprotonated, it remains in a closed conformation and Cl- ions are incapable of entering the channel. When this residue is protonated, it swings outward, allowing a Cl- ion to bind in the Sext site <ref>PMID:11796999</ref>. The Cl- ion is then transferred to the Scen site, at which point the Glu148 residue moves back to occlude the pore entrance. The Cl- ion then moves to the the Sint site and out of the channel. By this mechanism, two Cl- ions can be within the channel when it is closed, while three Cl- ions are capable of being in the channel when it is open<ref>PMID:12649487</ref>. A mutation in which the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_e148q/1'>Glu148 residue was replaced with a glutamine</scene> resembles the theorized structure of the open conformation of the ClC channel. A change in Cl- ion localization during the open and closed states of the channel can be observed by comparing <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_e148q_q_view/2'>the E148Q mutation</scene> with the <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_fab_complex_sext/1'>wild-type</scene>, respectively.

If the Glu148 residue is mutated to an alanine residue, <scene name='User:Lori_Wetmore/Sandbox_3/Ecclc_e148a/1'>you see something like this</scene>. 

=='''Cl-/H+ Transportation'''==
<applet load='2fee' size='325' frame='true' align='left' scene='User:Lori_Wetmore/Sandbox_3/Clc-ec1/1' target='0' />

There is some debate as to the function of various members of the ClC family. While the ClC channel from ''E. coli'' was originally assumed to be simply a Cl- channel, work by Accardi and Miller suggested that the ClC-ec1 channel was not a Cl- channel, but instead a transporter that coupled Cl- and H+ transport <ref> PMID:14985752</ref>.
As it turns out, several members of the family of ClC channels are Cl-/H+ transporters rather than simple Cl- ion channels. However, given the conserved structure amongst members of the ClC family, how can these different two separate functions be justified? 

==='''Ion Channel vs. Antiporter'''===
In order to understand the mechanisms behind transport of Cl- alone as opposed to Cl- transport coupled to H+ transport, it is worth noting the differences in the gating mechanisms of ion channels versus their active transport counterparts. Ion channels generally take on a simple "open" or "closed" state, dependent on whether they are allowing or preventing ion movement through the channel, and therefore often require only one gate that may then be switched on or off. Antiporters, on the other hand, must be gated on at least one end at all times in order to properly coordinate the transport of two separate ions in different directions, and therefore often require multiple gates or gating mechanisms to regulate this transport. 
It seems counterintuitive that a similar structure within all ClC channels could yield two decidedly different functions. Within ClC channels that act solely to transport Cl- ions, the extracellular Glu148 residue acts as the single gate that allows or prevents Cl- flow by the mechanism mentioned above. However, the glutamate gate is only one half of the necessary components of Cl-/H+ antiporter gating.
===='''Coordinating Cl- Transport'''====
While the Glu148 residue, or <scene name='User:Lori_Wetmore/Sandbox_3/Clc-ec1_e148/1'>extracellular gate</scene>, ensures that extracellular ions are maintained by blocking the Sext entrance, an <scene name='User:Lori_Wetmore/Sandbox_3/Clc-ec1_intracellular_gate/1'>intracellular gate</scene> is created by the Ser107, Tyr445, and Phe348 residues<ref>PMID:20303857</ref>, preventing ions from exiting or entering via the intracellular pore. 

===='''Coordinating H+ Transport'''====
The Glu148 residue acts as the "end of the line" for H+ ion transport. In order for H+ ions to be transported, a residue on the intracellular side of the selectivity filter, Glu203 is protonated. The H+ ions are then transported through the channel to the Glu148 residue and into the extracellular environment. 

The distance between the Glu148 and the Glu203 residues is <scene name='User:Lori_Wetmore/Sandbox_3/Clc-ec1_e148_angstroms/1'>approximately 15 angstroms</scene><ref>PMID:17710638</ref>, and there must, therefore, be an intermediate location for protonation. The exact pathway of H+ transport has not yet been elucidated, but it has been suggested that the Tyr445 residue within the Cl- selectivity filter may be involved<ref>PMID:17710638</ref>.

==='''A Model for Cl-/H+ Transport'''===
Given what is known separately about H+ and Cl- transport within the channel, a model<ref>PMID:18977737</ref> has been developed that may explain the coordination of Cl- and H+ transport within the ClC antiporter. The movement of the Cl- and H+ ions is believed to be coordinated and to occur simultaneously. As ClC channels are capable of moving ions in either direction depending on the ion gradient, this steps in this model may be reversed. However, the model will be described as if Cl- ions are being moved in an extracellular→intracellular direction, while H+ ions are being moved in an intracellular→extracellular direction. Initially, both glutamate gates are deprotonated, the Glu148 residue is blocking the extracellular entrance, and a Cl- ion is present in the Scen site. The intracellular gate, Glu203, is protonated by an intracellular H+ ion, which then proceeds to move to the Scen site and protonate the Cl- ion. When a proton is present at the Scen site, the intracellular gate opens. This opening allows Cl- to leave the channel via the intracellular exit, while the H+ ion protonates the extracellular Glu148 residue. When this glutamate gate is protonated, it opens, allowing two Cl- ions to inhabit the Scen and Sext sites. The extracellular glutamate gate is then deprotonated as the H+ ion leaves, causing it to want to close. However, as a Cl- ion is blocking the exit, it is incapable of closing. Therefore, the glutamate residue pushes the Cl- ions through the channel, causing one to exit the intracellular pore while the other remains in the Scent site. By this mechanism, the ClC transporter transports 2 Cl- ions for each proton transported<ref>PMID:18977737</ref>.

 

=='''References'''==

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