15:  15.4 Intracellular Ion Environment and Membrane Electric Potential

Ionic Gradients and an Electric Potential Are Maintained across the Plasma Membrane

The specific ionic composition of the cytosol usually differs greatly from that of the surrounding fluid. In virtually all cells — including microbial, plant, and animal cells — the cytosolic pH is kept near 7.2 and the cytosolic concentration of K⁺ is much higher than that of Na⁺. In addition, in both invertebrates and vertebrates, the concentration of K⁺ is 20 – 40 times higher in cells than in the blood, while the concentration of Na⁺ is 8 – 12 times lower in cells than in the blood (Table 15-1). The concentration of Ca²⁺ free in the cytosol is generally less than 0.2 micromolar (2 × 10⁻⁷ M), a thousand or more times lower than that in the blood. Plant cells and many microorganisms maintain similarly high cytosolic concentrations of K⁺ and low concentrations of Ca²⁺ and Na⁺ even if the cells are cultured in very dilute salt solutions. The ATP-driven ion pumps that generate and maintain these ionic gradients are discussed later.

In addition to ion pumps, which transport ions against their concentration gradients, the plasma membrane contains channel proteins that allow the principal cellular ions (Na⁺, K⁺, Ca²⁺, and Cl⁻) to move through it at different rates down their concentration gradients. Ion concentration gradients and selective movements of ions through channels create a difference in voltage across the plasma membrane. The magnitude of this electric potential is ≈70 millivolts (mV) with the inside of the cell always negative with respect to the outside. This value does not seem like much until we realize that the plasma membrane is only about 3.5 nm thick. Thus the voltage gradient across the plasma membrane is 0.07 V per 3.5 × 10⁻⁷ cm, or 200,000 volts per centimeter! (To appreciate what this means, consider that high-voltage transmission lines for electricity utilize gradients of about 200,000 volts per kilometer!) As explained below, the plasma membrane, like all biological membranes, acts like a capacitor — a device consisting of a thin sheet of nonconducting material (the hydrophobic interior) surrounded on both sides by electrically conducting material (the polar head groups and the ions in the surrounding aqueous medium) — that can store positive charges on one side and negative charges on the other.

The ionic gradients and electric potential across the plasma membrane drive many biological processes. Opening and closing of Na⁺, K⁺, and Ca²⁺ channels are essential to the conduction of an electric impulse down the axon of a nerve cell (Chapter 21). In many animal cells, the Na⁺ concentration gradient and the membrane electric potential power the uptake of amino acids and other molecules against their concentration gradient; this transport is catalyzed by ion-linked symport and antiport proteins. In most cells, a rise in the cytosolic Ca²⁺ concentration is an important regulatory signal, initiating contraction in muscle cells and triggering secretion of digestive enzymes in the exocrine pancreatic cells.

Here we discuss the role of ion channels in generating the membrane electric potential. Later we examine the ATP-powered ion pumps that generate ion concentration gradients, and ion-linked cotransport proteins.

The Membrane Potential in Animal Cells Depends Largely on Resting K⁺ Channels

In the experimental system outlined in Figure 15-8a, the distribution of K⁺, Na⁺, and Cl⁻ ions is similar to that between an animal cell and its aqueous environment. A membrane separates a 15 mM KCl/150 mM NaCl solution on the right side (representing the “outside” of the cell) from a 150 mM KCl/15 mM NaCl solution on the left side (the “inside”). A potentiometer (voltmeter) is connected to the solution on each side to measure any difference in electric potential across the membrane. If the membrane is impermeable to all ions, no ions will flow across it; there will be no electric potential across it.

Now suppose that the membrane contains Na⁺-channel proteins that accommodate Na⁺ ions but exclude K⁺ and Cl⁻ ions. Na⁺ ions then tend to move down their concentration gradient from the right side to the left, leaving an excess of negative Cl⁻ ions compared with Na⁺ ions on the right side and generating an excess of positive Na⁺ ions compared with Cl⁻ ions on the left side. The excess Na⁺ on the left and Cl⁻ on the right remain near the respective surfaces of the membrane, since, as in a capacitor, the excess positive charges on one side of the membrane are attracted to the excess negative charges on the other side. The resulting separation of charge across the membrane can be measured by a potentiometer as an electric potential, or voltage, with the right side of the membrane negative (having excess negative charge) with respect to the left (Figure 15-8b).

As more and more Na⁺ ions move through channels across the membrane, the magnitude of this charge difference (i.e., voltage) increases. However, continued right-to-left movement of the Na⁺ ions eventually is inhibited by the mutual repulsion between the excess positive (Na⁺) charges accumulated on the left side of the membrane and by the attraction of Na⁺ ions to the excess negative charges built up on the right side. The system soon reaches an equilibrium point at which the two opposing factors that determine the movement of Na⁺ ions — the membrane electric potential and the ion concentration gradient — balance each other out. At equilibrium, no net movement of Na⁺ ions occurs across the membrane. Thus the excess negative (Cl⁻) charges bound to the right surface of the membrane are separated from and attracted to the excess positive (Na⁺) ones on the left. In this way, the phospholipid membrane, with its nonconducting hydrophobic interior bounded by the conducting polar head groups and adjacent aqueous medium, stores the charge across it exactly as does a capacitor in an electric circuit.

If a membrane is permeable only to Na⁺ ions, then the measured electric potential across the membrane equals the sodium equilibrium potential in volts, E_Na. The magnitude of E_Na is given by the Nernst equation, which is derived from basic principles of physical chemistry:

where R (the gas constant) = 1.987 cal/(degree · mol), or 8.28 joules/(degree · mol); T (the absolute temperature) = 293 K at 20 °C, Z (the valency) = +1, F (the Faraday constant) = 23,062 cal/(mol · V), or 96,000 coulombs/(mol · V), and [Na_l] and [Na_r] are the Na⁺ concentrations on the left and right sides, respectively, at equilibrium. The Nernst equation is similar to the equations used to calculate the voltage change associated with oxidation or reduction reactions (Chapter 2), which also involve movement of electric charges. At 20 °C, Equation 15-5 reduces to If [Na_l]/[Na_r] = 0.1, as in Figure 15-8b, then E_Na = −0.059 V (−59 mV), with the right side negative with respect to the left.

If the membrane is permeable only to K⁺ ions and not to Na⁺ or Cl⁻ ions, then a similar equation describes the potassium equilibrium potential E_K:

The magnitude of the membrane electric potential is the same (59 mV), except that the right side is now positive with respect to the left (Figure 15-8c), opposite to the polarity obtained with selective Na⁺ permeability.

As noted earlier, the membrane potential across the plasma membrane of animal cells is about −70 mV; that is, the cytosolic face is negative with respect to the exoplasmic (outside) face. These membranes contain many open K⁺ channels but few open Na⁺ or Ca²⁺ channels. As a result, the major ionic movement across the plasma membrane is that of K⁺ from the inside outward, leaving an excess of negative charge on the inside and creating an excess of positive charge on the outside. Thus the flow of K⁺ ions through these open channels, called K⁺ leak channels or resting K⁺ channels, is the major determinant of the inside-negative membrane potential. Quantitatively, the usual resting membrane potential of −70 mV is close to but less than that of the potassium equilibrium potential calculated from the Nernst equation. The K⁺ concentration gradient that drives the flow of ions through resting K⁺ channels is generated by an ion pump that transports K⁺ ions into the cytosol from the extracellular medium and Na⁺ ions out. In the absence of this pump, which is discussed later, the K⁺ concentration gradient could not be maintained and eventually the membrane potential would fall.

Recent cloning and molecular characterization of resting K⁺ channels show that the channel protein is built of four identical subunits. Each subunit contains two membrane-spanning α helices, which partially line the ion-conducting pore in the middle of the protein, and a shorter looped P segment, which acts as a filter to allow K⁺ but not other ions to enter the pore and cross the membrane. As we discuss in Chapter 21, the structure of resting K⁺ channels is generally similar to the structures of other ion channels that are critical to the function of nerve cells.

Although resting K⁺ channels play the dominant role in generating the electric potential across the plasma membrane of animal cells, this is not the case in plant and fungal cells. The inside-negative membrane potential in these cells is generated by transport of H⁺ ions out of the cell by an ATP-powered proton pump.

Na⁺ Entry into Mammalian Cells Has a Negative ΔG

As we’ve seen, two forces govern the movement of such ions as K⁺, Cl⁻, and Na⁺ across selectively permeable membranes: the voltage and the ion concentration gradient across the membrane. These forces may act in the same direction or in opposite directions. To calculate the free-energy change ΔG corresponding to the transport of any ion across a membrane, we need to consider the contribution from each of these forces independent of the other.

For example, in a reaction where Na⁺ moves from outside to inside the cell, the free-energy change generated from the Na⁺ concentration gradient is given by

At the concentrations of Na_in and Na_out shown in Figure 15-9, which are typical for many mammalian cells, ΔG_c would be −1.45 kcal/mol, the change in free energy for the thermodynamically favored transport of 1 mol of Na⁺ ions from outside to inside the cell if there were no membrane electric potential. The free-energy change generated from the membrane electric potential is given by where F is the Faraday constant and E is the membrane electric potential. If E = −70 mV, then ΔG_m would be −1.6 kcal/mol, the change in free energy for the thermodynamically favored transport of 1 mol of Na⁺ ions from outside to inside the cell if there were no Na⁺ concentration gradient. Given both forces acting on Na⁺ ions, the total ΔG will be the sum of the two partial values: In this typical example, the Na⁺ concentration gradient and the membrane electric potential contribute almost equally to the total ΔG for transport of Na⁺ ions. Since ΔG is <0, the inward movement of Na⁺ ions is thermodynamically favored. As discussed later, certain cotransport proteins use the inward movement of Na⁺ to power the uphill movement of several ions and small molecules into or out of animal cells.

SUMMARY

• ATP-driven ion pumps generate and maintain ionic gradients across the plasma membrane. As a result, the ionic composition of the cytosol usually differs greatly from that of the surrounding fluid (see Table 15-1).

• In both invertebrates and vertebrates, the K⁺ concentration is higher and the Na⁺ concentration is lower in cells than in the blood. The cytosolic Ca²⁺ concentration is maintained at less than 0.2 μM.

• An inside-negative electric potential (voltage) of 50 – 70 mV exists across the plasma membrane of all cells; this is equivalent to a voltage gradient of 200,000 volts per centimeter.

• In animal cells, the electric potential across the plasma membrane is generated primarily by movement of cytosolic K⁺ ions through resting K⁺ channels to the external medium. Unlike most other ion channels, which open only in response to various signals, these K⁺ channels are usually open.

• In plants and fungi, the membrane potential is maintained by the ATP-driven pumping of protons from the cytosol across the membrane.

• Two forces govern the movement of ions across selectively permeable membranes: the membrane electric potential and the ion concentration gradient, which may act in the same or opposite directions. For the thermodynamically favored inward movement of Na⁺ into animal cells, these forces act in the same direction (see Figure 15-9).