New Chapt 7-new-Molec struture of ion channels (1)

New Chapt 7-new-Molec struture of ion channels (1) -...

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Unformatted text preview: Chapter 7 The Molecular Structure of Ion Channels In the previous sections, we showed how voltage gated Na+ and K+ channels work and how the cooperative operation of those channels generates and shapes the action potential. We showed that depolarization opens Na+ and K+ channels, but that Na+ channels automatically shut off, i.e., inactivate, while K+ channels do not, and only close when the cell repolarizes. These are the features that characterize ion channels as shown by electrophysiological studies. Electrophysiological studies, however, cannot show the structural underpinnings of the channel. In other words, voltage clamp and other electrophysiological studies show what the channel does and its electrical behavior, but they cannot reveal its structure nor how the particular structure it possesses produces each aspect of its behavior. The questions that we explore in this chapter concern the molecular structures of channels and how those structures impart activation, inactivation, and selectivity for passing only one type of ion. Since each channel is a protein, we will examine the molecular structure of a channel, its complement of amino acids, and how the arrangement of amino acids in different parts of the channel enables the channel to operate in a way consistent with its behavior revealed in voltage clamp studies. Before turning to the structures of ion channels, a brief review of amino acids and proteins will be helpful. A brief review of amino acids and proteins Individual amino acids are linked together to from polypeptide chains by peptide bonds in which the carboxyl group (COOH) of one amino acid is attached to the amino group (NH2) of the next. Each polypeptide thus has a free NH2 group at one end (the amino or N terminus) and a free COOH group at the other (the carboxyl or C terminus). The side groups of the amino acids (R1 and R2 in the example below) protrude from the peptide backbone and vary with the amino acid. Twenty amino acids are commonly used in the construction of proteins. Each amino acid within a polypeptide or protein is called a residue because amino acids lose two hydrogens and one oxygen (a water molecule) during the formation of each peptide bond when they are polymerized into a larger molecule. Figure 1. Amino acids are linked by peptide bonds to form polypeptides. Amino acids are connected by a peptide bond formed between the COOH ­ (carboxyl) group of one residue and the NH2 + (amino) group of the next. R1 and R2 indicate the amino acid side chains. Some amino acids are acidic and positively charged, some are basic and negatively charged, some are polar but uncharged whereas others are non ­polar. Non ­polar residues are hydrophobic (water 66 hating) and stretches of hydrophobic residues therefore tend to be found in the portions of ion channels that are embedded in the lipid membrane. The portions of the protein that are embedded in the membrane are called the transmembrane segments of the protein. The protein in this case is an ion channel. In contrast, charged and polar residues, which are hydrophilic or water loving, are more likely to be located in regions of the protein that extend into the extracellular or intracellular fluids. Finally, some amino acids have additional features that are important for channel functioning. Serine, threonine and tyrosine residues, for example, can be modified by the attachment of a negatively charged phosphate group (these residues can be phosphorylated). This changes the electrostatic properties of the amino acid, alters the local structure and thus changes the function of the protein. As we shall see in later sections, phosphorylation modulates the properties of many ion channels. For example, phosphorylation of some channels increases its conductance, thereby allowing more ions to flow through the channel than when it is not phosphorylated. Proteins assume one of two main types of structures: the α ­helix and the β ­sheet. Both result from hydrogen bonding between the NH and C=O groups of the polypeptide backbone. The α ­helix is the most prevalent form and is particularly important for determining which portions of the protein comprise the transmembrane segments (which parts of the protein are embedded in the membrane). The polypeptide backbone is twisted into a helix such that about 20 amino acid residues comprise a length of about 30 angstroms along the helix. Since the cell membrane is also about 30 angstroms wide, a stretch of about 20 amino acids is just sufficient to span the membrane. Below we illustrate other factors which further show that the transmembrane segments of ion channels are formed from α ­helices comprised of ~20 amino acid residues . The molecular structure of the Na+ channel The first voltage gated ion channel whose structure was determined was the Na+ channel. Na+ channels are particularly rich in skeletal muscle and in the electric organ of some eels, the eels that generate hundreds of volts used to stun or even kill their prey. In all cases, a very large glycoprotein was purified and was called the α subunit. Messenger RNA was obtained and the full amino acid sequence of the α subunit of the Na+ channel was deduced from the sequence of nucleotides of the complementary DNA generated from the mRNA. The Na+ channel protein of the α subunit is comprised of about 2000 amino acids and is the product of a single gene. But so what? What can be learned about the structure of the Na+ channel by simply knowing its sequence of amino acids? Luckily there are a few rules that provide major insights into which regions of the protein lie within the lipid membrane and how the protein itself is structurally and functionally organized. Of importance in this regard is the hydrophobicity plot. Each amino acid is placed in the plot as it occurs in the protein and each is assigned a hydrophobicity value, which reflects its ability to interact with water. Recall from the previous discussion of amino acids that non ­polar amino acids are hydrophobic (water hating) and are assigned a high positive value, whereas polar amino acids are hydrophilic and are given a negative value. A running average of these values over several amino acids is then calculated around each amino acid in the protein sequence, and plotted as a function of position along the protein chain. Also recall from the previous description that a sequence of about 20 hydrophobic α ­helictial amino acids would span the cell membrane. Thus, sequences of ~20 hydrophobic amino acids in the hydrophobicity plot represent a transmembrane segment. Let’s examine the hydrophobicity plot of a Na+ channel shown in the top panel of Fig. 2 in greater detail. Look first at the pattern of the initial 400 amino acids. Beginning at amino acid #75 and ending at about #225, there is a stretch of about 150 amino acids that are all hydrophobic. Since 30 hydrophobic amino acids would span the membrane, this suggests that there should be five membrane 67 spanning segments from 75 ­225. If you look carefully, and use a little imagination, you can also see five little peaks in this region. Each “peak” is about 20 amino acids long, and each is indicated by a colored box above that span from amino acid 75 ­225. We refer to these peaks as a segment (S), and they are labeled S1 ­S5, as shown above each colored box and in the middle panel of Fig. 2. This is followed by a short span of 100 hydrophilic residues, from about #225 ­325, followed by about more 30 hydrophobic residues. The last set of hydrophobic residues thus forms a sixth transmembrane segment and is labeled S6 (magenta box) in Fig. 2. These six transmembrane segments are collectively called domain I, and it is apparent that three other domains, labeled domains II, III and IV, closely resemble domain I, in that domains II ­IV also contain six transmembrane segments. Figure 2. Top panel: Hydrophobicity plot of the amino acid sequence of a Na+ channel. The colored regions show the hydrophobic amino acids. The hydrophobic amino acids show a series of peaks, where each peak is assigned a colored box, where each colored box represents a transmembrane segment. There is a repeating pattern such that series of six peaks is repeated four times. Each series of six peaks is a domain of the channel. Middle panel: linear arrangement of transmembrane segments from amino acid sequence and hydrophobicity plot. Lower panel: top view of Na+ channel showing how four domains are actually arranged around a central pore. 68 The linear arrangement of the segments is shown in the middle panel of Fig. 2. Each segment in a domain is linked to its neighboring segment by an intra ­ or extracellular linker of hydrophilic amino acids. In addition each of the four domains is separated from its neighboring domain by a short intracellular hydrophilic segment. Notice that the hydrophilic stretches of amino acids are not embedded in the membrane but rather are in the extracellular or intracellular fluids and link the transmembrane segments. The arrangement of the segments and domains is not stretched out linearly in the membrane. Rather the four domains are folded, each next to its neighbor, to form a circular ring with a central pore. This arrangement would be seen if you could look down on the channel from the extraceullar space, as shown in the bottom panel of Fig. 2. In short, the Na+ channel is a single, large protein composed of four domains, where each domain is, in turn, composed of six transmembrane segments. The transmembrane segments, together with the intracellular and extracellular linking segments, the hydrophilic linking regions, play specific roles in the channel operation. The above section showed the molecular arrangement of the Na+ channel. Another important channel that conducts cations is the Ca++ channel, which is exceptionally important and will be considered later in the course. The only point I wish to make here is that the structure of the Ca++ channel is strikingly similar to that of the Na+ channel (Fig. 3). It too is constructed from a single protein, has four domains each with six transmembrane segments. Figure. 3. Molecular structures of Na+ and Ca++ channels are similar. In the sections below we consider three features of the various segments; 1) which segment “senses” voltage (charge on the inside of the cell) and how that segment responds to membrane depolarization to open activation gates; 2) which portions form the inactivation gate and how the inactivation gate is coupled to the voltage sensor; 3) the pore region through which the ions travel and how the structure of the pore region confers exquisite selectivity for one particular ion and excludes 69 other ions. As a preview, the S4 segment is the voltage sensor, the intracellular linkage region between domains III and IV form the inactivation gate, and segments S5 ­6 form the pore. We first consider the voltage sensor in S4 and then turn to the inactivation gate. In the final portion we consider the ion selectivity filter in the pore, which is one of the great stories in molecular neurobiology. The S4 segment is the voltage sensor and opens the activation gate For an ion channel to respond to changes in the electric charge across the membrane, some charged part of it must act as a voltage sensor and must undergo some movement that triggers the opening of the activation gate. This idea was originally postulated by Hodgkin and Huxley in 1952. They proposed that the gating of a channel was accompanied by movement of a charge within the membrane. Such a movement of charge within the protein constitutes a small current, which is now called the gating current (Fig. 5). It should be noted that the gating current is very small because it is only due to the small movement of a charged element of the protein and it occurs before the channel actually opens, which then allows a much larger current, carried by millions of Na+ ions, to flow through the pore of the channel. The S4 segment has the exact properties required for a voltage sensor. In the S4 segment, basic (positively charged) amino acids occur at every third residue and form a spiral of positive charge on the outside of an α helical segment (an α helix makes a complete turn for each 3.4 amino acids). In the S4 transmembrane segment, each of these positive charges is paired to negative charges in the helices of neighboring segments, thereby helping to stabilize the segment or helix (Fig.4). The positive charges on the outside of the cell also aid in stabilizing the configuration of the helix at the normal resting potential. Figure 4. The positively charged amino acids in the S4 segment are attracted to negatively charged amino acids (E&D) in S2 and S3. This holds them in place at rest. Depolarization of the cell overcomes this electrostatic attraction, thereby repelling the positively charged S4 segment, which then initiates channel opening. When the cell is depolarized, and the inside of the cell gains positive charges, the positive charges act to repel the positively charged helix and displace it upward into the extracellular space. Each charge on the outside of the helix is shifted upward to the position of the next charged neighbor where it is stabilized. The net effect is to cause the helix to rotate upwards into the extracellular fluid and thereby add positive charges to the outside of the membrane. This movement of positive charges in the membrane repels positive ions in the extracellular fluid and generates a small current, called the gating current. The gating current is evoked before the channel opens and thus it occurs before the ionic current that flows through the open channel. The upward displacement of the S4 segment is coupled to and thus opens the activation gate, as shown in Fig. 5. Recall that there the Na+ channel has four domains, and each domain has an S4 segment. The S4 segments in domains I, II and III have to move 70 upward to open the activation gate, which allows ionic current to flow through the pore of the channel (Fig. 7, top panel). Figure 5. Schematic arrangement of the voltage sensor in S4 segment. In the resting state, the S4 segment is positioned in the membrane, attracted by the negative charges inside. When + charges enter the cell causing depolarization the + changes in the sensor are repelled by the + charges inside the depolarized cell. The repulsive + charges “push” the S4 segment upward into the extracellular space, which in turn repels + charges in the extracellular fluids, thereby generating the “gating current”. The gating current is transient, and lasts only as long as long as the S4 segment moves. The upward movement of the S4 segment is coupled to other segments and thereby opens the activation gate, allowing ions to flow through the pore. The flow of ions through the pore is called the ionic current. The effects of repolarization are exactly the opposite: the increase in negative charges on the inside (and/or the increase in positive charges on the outside) then attract the positive charges on the S4 helix, causing it to move downward back to its normal position. That movement, in turn, is also coupled to and closes the activation gate. Inactivation Activation of Na+ channels, the opening of the channel so that ions flow through the pore, is always shortly followed by inactivation, where a second gate automatically closes and prevents ions from flowing through the pore, even though the cell is depolarized. One might then ask whether the voltage sensor also comprises a portion of the inactivation gate? The answer is no; the voltage sensor and activation gates are not part of the inactivation gate and are independent of it. This was shown in the 1970s when investigators injected the enzyme, pronase into the axoplasm of the squid giant axon. Pronase is a protease that would cleave and therefore disable pieces of proteins that are exposed in the cytoplasm. When an experiment was conducted in voltage clamp, applying positive current to the inside of the cell caused an opening of the activation gate, and thus an inward current carried by Na, and when the applied current was stopped, the activation gate closed. Thus the operation of the activation gate was not affected by pronase. What was affected was inactivation; with pronase, there was no inactivation and inward current, carried by Na+, flowed so long as the cell was clamped at a positive membrane potential. In other words, the activation gate behaved normally but the inactivation gate was eliminated by intracellular pronase. This experiment showed something more; since the pronase was injected into the axon, the inactivation gate must be on the cytoplasmic side of the...
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