LS1a 10-21 part1r-1

LS1a 10-21 part1r-1 - Some molecules can freely diffuse...

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Unformatted text preview: Some molecules can freely diffuse across the plasma membrane – these include small hydrophobic molecules such as oxygen, as well as small uncharged, polar molecules such as water. In contrast, large uncharged polar molecules such as glucose, and ions cannot freely diffuse across the membrane, or only do so very slowly. This kinetic barrier is the result of unfavorable interactions between charged or polar molecules and the hydrophobic interior of the bilayer. Molecules that are large and polar or charged must be moved across membranes using specialized transport proteins. 1 There are many different types of membrane transport proteins, each selective for a certain class of molecule - ions, amino acids, or sugars, for example. Many transport proteins are highly selective, transporting, for example, only potassium ions, but not sodium. Transport through membrane proteins can be: (1) passive, requiring no input of energy because the molecule being transported is moving down its concentration gradient (e.g. from higher concentration outside to lower concentration inside). Passive transport can be mediated by channel proteins or by carrier proteins. Channel proteins create protein-lined pores through which ions that are the right size and charge can move. Carrier proteins have binding sites for the molecule to be transported; binding of the molecule to be transported triggers a conformational change in the carrier that releases the molecule on the other side of the membrane. More than one molecule can move through a channel at one time, but carrier proteins typically move only one or a small number of molecules one at a time. (2) active - this means that the molecule being transported is moving against its concentration gradient and movement therefore has to be coupled to a process that provides energy. Only carrier proteins can mediate active transport. 2 Io n C o n c e n tr a tio n s In s id e a n d O u ts id e o f a C e ll Na + (145 mM) Na+ K+ (5-15 mM) (5 mM) Cl- K+ (140 mM) (5-15 mM) Cl (110 mM) o r g a n ic a n io n s inside cell Because of transport processes and the barrier imposed by the cell membrane, the concentrations of ions inside living cells is very different from that in the external environment. These differences are crucial for the normal functioning of all cells, and for the electrical activity generated in nerve cells. The things that are important for you to remember are: sodium is the most abundant ion outside the cell; and potassium is the most abundant inside. In order to avoid the buildup of too much electrical charge, the amount of positive and negative charge charge the cell must be equal; the same is true for the outside environment. Outside the cell, chloride anions are approximately equal in concentration to the sodium cation. Inside the cell, the potassium cations are balanced by many different organic anions (carbon-containing negatively charged molecules). However, this balance of positive and negative charge is not exact, and as a consequence, a small excess of charge does accumulate near the plasma membrane and, as we will see, it has important consequences. The difference in potassium and sodium concentrations inside and outside the cell are generated by the sodium-potassium ATPase, an active transporter that pumps sodium out of the cell and potassium into the cell. The pump powers the movement of ions against their concentration gradients using the energy from ATP. For every 3 sodium ions it pumps out of the cell, it pumps 2 potassium ions in. 3 The simplest case of passive transport is that of an uncharged molecule moving across the bilayer. For uncharged molecules, the driving force for movement comes only from the difference in concentration across the bilayer – referred to as the concentration gradient. Molecules move from regions of high concentration to those of lower concentration. If the molecule that is moving is charged, then two factors influence transport – the concentration gradient and any difference in charge across the membrane (called the membrane potential). These two forces combine to form the electrochemical gradient. In a few slides we will discuss where the membrane potential comes from. As we will see, cells have a membrane potential and this is crucial for all cellular functions, and particularly those of neurons. In this slide, the width of the purple arrow represents the magnitude of the electrochemical gradient for the same positively charged ion in three different situations. In the left panel there is a concentration gradient, but no membrane potential (see the zeros on either side of the membrane). The cation moves down its concentration gradient, from the outside of the cell to the inside (through a channel not shown; since it is charged it cannot diffuse). In the middle panel there is a concentration gradient AND a membrane potential (represented by the excess charge accumulated on either side of the membrane). The same concentration gradient exists as in the left panel, but now there is an extra driving force from the membrane potential, which is negative inside and therefore favors the movement of positively charged cations to the inside of the cell (movement of cations is favored in this case because the inside of the cell has a slight excess of negative charge). In the right panel the membrane potential reduces the driving force caused by the concentration gradient. In this case, the membrane potential is opposite to that in the middle panel, with a slight excess of positive charge inside the cell that will disfavor movement of cations to the inside. 4 Sometimes the cell needs to move ions against the electrochemical gradient, in a process that is energetically unfavorable – ΔG > 0. So how can the cell do this? As you heard from Dan, a common theme in biology is to drive energetically unfavorable processes by coupling them to favorable ones. This energetically unfavorable movement is called active transport and can be carried out by three different types of transporters: (1) Coupled transporters move molecules against their electrochemical gradient by coupling the transport to the favorable transport of another molecule. In the case shown on the left portion of the slide the orange molecule is being moved against its electrochemical gradient. The cost of this movement is paid for by coupling the transport to the energetically favorable movement of the blue molecule in the direction of its electrochemical gradient (the gradient for the blue molecule is not shown, but it is favorable to move the blue molecule into the cell). (2) ATP-driven pumps like those diagrammed in the middle portion of this slide pay the energetic cost of moving the orange molecule into the cell, against its electrochemical gradient, by coupling this movement to the energetically favorable hydrolysis of ATP. In this case the free energy liberated in ATP hydrolysis “pays” the cost of the energetically unfavorable movement of the orange molecule into the cell. (3) Light driven pumps like those shown on the right hand portion of the slide couple unfavorable movement to the favorable absorption of photons from light. Several important light driven pumps are found in bacteria. 5 Ion Channels - K+ Channel δ+ δ− As we mentioned earlier, channel proteins create protein-lined pores in the bilayer through which ions can move. Ion channels can have amazing properties – they can transport ions rapidly and they can be selective in that they only allow ions of a certain size and charge to pass through. We know a tremendous amount about the function and structure of a potassium channel from bacteria – its structure was determined by x-ray crystallography (Rod MacKinnon won the Nobel Prize in 2003 for this work). This potassium channel consist of 4 identical subunits, each consisting of three alpha helices. In the diagram shown on this slide only 2 of the subunits are shown for simplicity (one is green and the other yellow). Two long helices in each subunit are transmembrane helices, and the 3rd shorter pore helix plays is inside the bliayer, but does not span the entire membrane (circled in black on the yellow subunit) During its movement from the cytoplasm to the extracellular space, a potassium ion encounters the following important features of the ion channel: (1) The cytoplasmic entrance to the pore is surrounded by an abundance of acidic amino acids. These amino acids are negatively charged at neutral pH and help attract cations (like potassium) into the channel and repel anions. (2) Once ions enter the pore they reach the vestibule, a protein-lined space in the membrane which is large enough that potassium ions can remain associated with water molecules (hydrated). (3) Ions leave the vestibule and enter the selectivity filter on their way to the outside of the cell. The selectivity filter is composed of a series of carbonyl groups from the peptide backbone of the channel subunits; as we will see, the selectivity filter plays an important role in ensuring that only potassium ions are transported. The potassium ions are attracted into the selectivity filter by interactions with the negatively charged helix dipole at the end of the pore helix, and they move through the selectivity filter in single file to the outside of the cell. 6 Helix Macrodipole O C N δ+ H δ− δ− C-terminus δ+ N-terminus As Dan already told you, alpha helices have a macrodipole, with the amino terminus of the helix having a partial positive charge and the carboxy terminus having a partial negative charge. Note that these are different from the formal charges that the amino and carboxyl groups at the N- and C-terminus have. The helix macrodipole arises because the peptide bond itself is a dipole, and in a helical structure the peptide bonds are all oriented in the same direction. In this diagram, a peptide bond is drawn on the left, and the dipole is indicated with the arrow (pointing towards -). On the right, the peptide bond dipoles are labeled in the helical structure. They align in the same direction to form the macrodipole of the helix. 7 δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ The potassium channel has remarkable selectivity - it conducts potassium 10,000-fold more efficiently than sodium. How can the channel do this, given that sodium and potassium are both the same shape (spheres) and almost the same size (diameter 0.133 nm for potassium vs. 0.095 nm for sodium)? A single amino acid substitution in the selectivity filter of the channel can destroy this selectivity. So what is the basis for the high selectivity of this channel? As we pointed out previously, the ions are hydrated in the vestibule – they make favorable interactions with multiple water molecules (top right). To enter the selectivity filter the ions must give up their interactions with water molecules (they cannot fit otherwise). In the selectivity filter carbonyl oxygens from the protein are positioned precisely to interact with a dehydrated potassium ion (a potassium ion that has lost its water molecules). The dehydration of the potassium ion requires energy, which is mostly balanced by the energy regained by the interaction of that ion with the carbonyl oxygens (ΔH ~ 0). Since multiple water molecules are liberated in the process of dehydration (even more than the four that are shown), ΔS > 0. With ΔH ~ 0 and ΔS > 0, ΔG < 0 and the dehydration occurs spontaneously, it is energetically favorable. The sodium ion is too small to interact with the oxygens; therefore, it does not enter the selectivity filter because the energetic expense of losing its interactions with water molecules is too large because these interactions are not replaced by interactions with carbonyl oxygens. The diagram on the left depicts the structure of the potassium channel, consisting of four subunits (blue, green, purple, yellow; each representing one polypeptide chain, all four are identical). A view looking down onto the plasma membrane is shown. One potassium ion is shown in the process of moving through the selectivity filter. 8 Ion channels are regulated – they cannot be open all the time or the difference between the inside and outside of the cell would disappear. Ion channels can exist in a closed state where no ions can be transported, or in an open state where ions can freely pass through. These two states represent different conformations of the channel; the channel can spontaneously transition between the two states. As we will see on the next slide, the cell regulates the transition between the open and closed states of ion channels, effectively controlling the probability the channel will be in one state or the other. 9 The regulation of the transition between open and closed states of ion channels is referred to as gating. Cells use several different mechanisms to gate ion channels, controlling probability they will be in one state or the other: (A) changes in voltage across the membrane (e.g. in neurons) (B) binding of ligands to the outside of the channel (e.g. neurotransmitter) (C) binding of ligands to the inside of the channel (e.g. nucleotide or ions) (D) gating by mechanical stimulation (e.g. hair cells of ear). 10 The membrane potential is a slight imbalance of charge across the membrane. This charge builds up in a thin layer adjacent to the membrane, held in place by interaction with the oppositely charged ions on the other side of the membrane. In animal cells there is slightly more negative charge inside the cell and slightly more positive charge outside (left). Thus, the membrane potential in animal cells favors the entry of cations and disfavors the entry of anions. How does this imbalance of charge arise? 11 The membrane potential comes from the movement of potassium ions across the membrane Potassium is maintained at a high concentration, and sodium at a low concentration (both relative to the extracellular environment), by the sodium-potassium pump. The sodium potassium pump (not shown on this diagram) uses the energy of ATP hydrolysis to pump sodium out of the cell and pump potassium into the cell. If we think about the cell starting with no membrane potential (left), high potassium concentration inside (from the sodiumpotassium pump), and no potassium channels open – in this state there is no membrane potential. If the potassium channel now opens (right; these are special potassium channels called potassium leak channels and they are not gated so they spend some time in the open state), potassium will leave the cell because it will move down its concentration gradient. As potassium leaves the cell, it creates an imbalance in charge because its organic anions that were associated with it are left behind – therefore the inside of the cell develops a slight excess of negative charge and the outside of the cell develops a slight positive charge. The movement of potassium out the potassium channel continues until the force exerted on the potassium ion from the concentration gradient exactly balances the force exerted on the potassium ion from the voltage gradient (the charge difference). The imbalance of charge at that balancing point (a charge separation is a voltage or potential) is called the resting membrane potential. The resting potential is related to the difference in concentration of the ion inside and outside the cell (larger concentration differences lead to larger membrane potentials). If you go on to study neuroscience you will see how the interplay between membrane potential and ion channels is used for electrical signaling in nerve cells. Extra information on the sodium-potassium pump: This pump is an active transporter that uses energy from ATP hydrolysis to move potassium and sodium against their electrochemical gradients. For every two potassium ions it pumps in, it transports 3 sodium ions out. This creates a charge imbalance, but it only contributes ~10% of the membrane potential. Most of the membrane potential comes from potassium ions moving out through leak channels. 12 S u m m a r y o f M a in P o in ts • The membrane imposes a barrier to diffusion of large, polar and all charged molecules; these molecules must be moved across membranes through transport proteins • The inside and outside of the cell have very different ion compositions that are maintained by transporters and channels • Electrochemical gradient influences how ions will move across the membrane • Ion channels transport ions with tremendous selectivity and efficiency; structural features of the channel enable them to do so • Membrane potential originates from a charge imbalance due to the movement of potassium ions 13 ...
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