Molecular and Cellular Signaling - Martin Beckerman

References 461

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462 18. Regulation by Viruses

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Problems

18.1Engineered viruses are being devised for use in cancer therapy. The goal of researchers is to find natural viruses, or create genetically modified ones, that can kill cancer cells while leaving normal cells unharmed. This main idea behind creation of these oncolytic viruses is to take advantage of the altered signaling pathways in the tumors by devising viruses that selectively target those cells.

Problems 463

The first viruses to be used in this way are the adenoviruses. These viruses and other like them stimulate host cells to pass through the cell cycle. If the host cell does not pass into S phase the viruses cannot replicate their DNA. Adenoviruses encode 30 to 40 proteins that are expressed sequentially. First immediate early genes, then early genes and finally late genes are expressed. The first protein to be made is E1A. This control protein binds to the pRB protein of the host cell and inactivates it (Figure 14.11). The E1B protein is made soon thereafter. It binds to p53 and inactivates it, too. These binding and inactivation events ensure entry of the host cells into S phase.

Recall from Chapter 14 that p53 and pRb central cell cycle controller circuitry and are mutated in most cancers. The inactivation of these genes is an important step in transforming the host cell into one that supports replication of the adenoviruses. How would you engineer an adenovirus to kill cancerous cells possessing mutated p53 proteins while leaving normal cells unharmed?

18.2Some cells are permissive with respect to a virus while others are not. In a permissive cell, the virus can gain entry by binding a receptor and once inside the cell the viruses can replicate themselves. In nonpermissive cells, there aren’t any receptors that can be exploited for entry, and if they do the signaling pathways do not support completion of the viral replication cycle.

One of the signaling pathways often altered in cancer cells is the Ras signaling pathway. One of the downstream targets in this growthpromoting pathway is the antiviral agent PKR. As mentioned earlier, when activated PKR shuts down proteins synthesis thereby preventing viral replication. Phosphorylation by one or more kinases acting downstream from Ras represses this activity. How would you engineer an oncolytic virus to kill cancerous cells possessing mutated Ras proteins while leaving normal cells unharmed? Note: dsRNA viruses often encode an anti-PKR protein.

The membrane separating inside and outside in part (a) of the figure is impermeable to the passage of ions. Since there is no membrane permeability there is no membrane potential. Consider now what happens when ion channels are added as in part (b) of Figure 19.1. Channels for K+, Na+, and Cl- ions are now present, but not for A- ions, which are too large to pass through an ion channel. The sodium and chloride channels will be neglected for the moment, with the main attention on the K+ and the A- ion concentration.

Potassium channels allow potassium ions to passively diffuse out of the cell. They will be driven to do so by the potassium concentration gradient set up because the potassium concentrations outside and inside the cell differ. As potassium ions diffuse out of the cell a net negative charge will build up within the cell since the negative charge associated with the A- ions exceeds the positive charge of the remaining potassium ions. The buildup of negative charge will oppose the outward flow of positively charged potassium ions, eventually reaching the point where it is strong enough to halt the flow of ions. At this point an equilibrium situation is reached. There is a potential difference across the membrane and this potential just balances the concentration gradient. The potential difference required to halt the flow of potassium ions through the potassium channels is the potassium membrane potential, similar to the situation for other ion species. These potentials are given by the Nernst equation:

In the Nernst equation, R is the universal gas constant, T is the absolute temperature, F is Faraday’s constant, z is the valence of the ion (+1 for sodium and potassium; +2 for calcium, and -1 for chloride), and X stands for the ion species under consideration. At a temperature of 37° Celsius, RT/F = 26.73 mV, and switching from natural logarithms to base 10 logarithms:

The potentials given by the Nernst equation are commonly referred to as reversal potentials, or alternatively, as equilibrium potentials. They can be either positive or negative, depending on the sign of z and on which concentration is greater—inside or outside. As noted in Table 19.1, the reversal potentials for sodium and calcium are positive while those for potassium and chloride are negative.

The plasma membrane of cells is more permeable to some ions than to others. It is far more permeable to potassium ions than to sodium ions, and

468 19. Ion Channels

TABLE 19.1. Values for typical concentrations of four main kinds of ions inside and outside mammalian skeletal muscle cells are presented along with the values for the equilibrium potential calculated from the Nernst equation.

somewhat more permeable to potassium ions than to chloride ions. The resting potential of a membrane (Vm) is given by combining the currents from the various ion species, taking into account not only the concentrations outside and inside but also the relative permeabilities. A simple expression, known as the Goldman–Hodgkin–Katz equation, incorporates both of these factors:

As is customary, the calcium contribution, which is small, has been neglected. In Eq. 19.3, pK, pNa and pCl are permeability constants. The appearance of their ratios in Eq. 19.3 takes into account the relative permeabilities of the membrane to the different ion species. Typical resting potentials are in the range -60 to -80 mV. These values are far closer to the potassium equilibrium potential than to the sodium equilibrium potential. The closeness of the membrane resting potential to the potassium reversal potential is a reflection of a far greater permeability for potassium than for either sodium or chloride. As their permeability ratios in the GHK equation become negligible, the expression for the resting potential approaches that of the Nernst equation for potassium. The reversal potential may be thought of as supplying a driving force for changes in resting potential.

19.2 Membrane and Action Potentials Have

Regenerative Properties

Action potentials are generated by the joint action of fast-acting sodium channels and delayed-acting potassium channels. The generation of an action potential begins with a threshold-exceeding depolarization that opens sodium channels. When this happens the permeability of the membrane to sodium increases; according to the GHK equation the membrane depolarizes as its potential moves towards the equilibrium value for sodium. The open sodium channels permit the entry of sodium cations into the cell, which depolarize the membrane even further allowing for an

470 19. Ion Channels

even if the membrane potential has not changed from its depolarized value. That is, the sodium channels inactivate after being activated for a short time. Potassium channels activate more slowly than sodium channels, reaching their peak conductivity values after the sodium channels shut down. However, once they reach their peak conductivity values, the potassium channels remain open for some time. That is, potassium channels are noninactivating.

To summarize, sodium channels launch and generate the rising part of the action potential; positive feedback sustains it, and potassium channels generate the slowly declining following portion of the pulse. The positive feedback loop of the sodium channels is the key to generation of action potentials that propagate down axons without diminishing. Once a small depolarization is started in a limited region of space, the spread of ions laterally to neighboring spaces triggers a growing depolarization in that space and from there to the next neighboring space and so on. The pulse does not die out since a small depolarization triggers a large one, that is, the signal is amplified, as a result of the positive feedback. Thus, once started, a pulse propagates along without losing strength, that is, the pulses are regenerative. The existence of a threshold all-or-nothing response is a consequence of the positive feedback from the sodium channels and negative feedback from the potassium channels. Negative feedback is present because the depolarization-induced exit of potassium hyperpolarizes the membrane, thereby acting negatively to shut down the potassium ion flow. As a result of both forms of feedback there is an all-or-nothing response. It takes a certain amount of depolarization to trigger the positive feedback dominated sodium influx, and once started the process runs to completion.

19.3 Hodgkin–Huxley Equations Describe How Action

Potentials Arise

In 1952, Alan Hodgkin and Andrew Huxley constructed a mathematical model of the time course of sodium and potassium permeability changes in the giant axon of the squid. There are two ways to preserve and speed up the conduction of electrical pulses over long distances in an axon: either by increasing the diameter or by adding an insulating fatty sheath (myelination). The squid giant axon is unmyelinated and makes use of the first of the two solutions to the conduction problem. It is enormous, with a diameter that can reach 1 mm. It can be easily manipulated in the laboratory, and is thus is an ideal system for laboratory studies of how pulses are transmitted down nerves. The Hodgkin–Huxley model represents the culmination of decades of study by Hodgkin, Huxley, Katz, and many others on how electrical pulses are produced, and how they propagate, in nerve tissue. The development of the model was preceded by their detailed experimental studies of the flow of electrical current through the surface membrane of

the squid giant axon. In these studies, a method, the “voltage clamp,” was developed that allowed researchers to directly measure current flow across the membrane of the axon.

The basic elements of the Hodgkin–Huxley model are a set of ionic currents separated into distinct contributions from sodium and potassium ions, and from the leakage current, representing the fairly constant and voltageindependent, background leakage of ions through the plasma membrane. These three currents are related to membrane permeabilities, which are expressed in terms of ionic conductances gNa, gK and gL, and the displacements of the membrane potential from the reversal potentials for the ion species. The relationship between current, conductance, membrane potential, and reversal potential is from Ohm’s law,

In the above, X denotes the ion species under consideration, Vm the membrane potential, Ix is its current, Ex is its reversal potential, and gx is its conductance, the inverse of its resistance Rx, that is,

In elementary circuits, R is a constant, and Ohm’s law says that current and voltage are linearly related. This is not the case for a biological membrane. Although the linear form of Ohm’s law is retained in the Hodgkin–Huxley equations, the sodium and potassium conductance are no longer constants, but instead are treated as functions of the membrane voltage.

Because of the nonconducting nature of the lipid bilayer the plasma membrane can be thought of as a capacitor that permits a buildup of charge on its surfaces. If a current is applied part will contribute to charging up the plasma membrane and part will flow through the membrane into the cell. The net ionic current is the sum of the sodium, potassium, and leakage currents plus any external applied or synaptic currents. The capacitive current

IC is, from Faraday’s law,

In this expression, C is the capacitance, and dVm/dt is the time rate of change in the voltage. The membrane voltage does not instantaneously adapt to the flow of charge in and out of the cell. Instead, it takes a certain amount of time for charge to build up on the membrane and the voltage to adjust to the current flow. The complete circuit equation, expressing the conservation of charge, is

C dVm = -gK (Vm - EK ) - gNa (Vm - ENa ) - gL (Vm - EL ) + I , (19.7)

dt

where I is any applied or synaptic current and EL is leakage current.

An equivalent circuit for the membrane and its ion channels is presented in Figure 19.3. The Hodgkin–Huxley equation describes an electrical circuit