sides, with their NPA regions overlapping in the middle of the membrane to form part of the specificity filter— the structure that allows only water to pass.

The residues that line the channel of each AQP-1 monomer are generally nonpolar, but carbonyl oxygens in the peptide backbone, projecting into the narrow part of the channel at intervals, can form hydrogen bonds with individual water molecules as they pass through; the two Asn residues (Asn76 and Asn192) in the NPA loops also hydrogen-bond with the water. The structure does not admit closely spaced water molecules that might form a chain to allow proton hopping (see Fig. 2-14), which would effectively move protons across the membrane. Critical Arg and His residues and electric dipoles formed by the short helices of the NPA loops provide positive charges in positions that repel any protons that might leak through the pore.

Ion-Selective Channels Allow Rapid Movement of Ions across Membranes

Ion-selective channels —first recognized in neurons and now known to be present in the plasma membranes of all cells, as well as in the intracellular membranes of eukaryotes—provide another mechanism for moving inorganic ions across membranes. Ion channels, together with ion pumps such as the Na+K+ ATPase, determine a plasma membrane's permeability to specific ions and regulate the cytosolic concentration of ions and the membrane potential. In neurons, very rapid changes in the activity of ion channels cause the changes in membrane potential (the action potentials) that carry signals from one end of a neuron to the other. In myocytes, rapid opening of Ca2+ channels in the sarcoplasmic reticulum releases the Ca2+ that triggers muscle contraction. We discuss the signaling functions of ion channels in Chapter 12. Here we describe the structural basis for ion-channel function, using as examples a bacterial K+ channel, the neuronal Na+ channel, and the acetylcholine receptor ion channel.

Ion channels are distinguished from ion transporters in at least three ways. First, the rate of flux through channels can be several orders of magnitude greater than the turnover number for a transporter— 107 to 108 ions/s for an ion channel, near the theoretical maximum for unrestricted diffusion. Second, ion channels are not saturable: rates do not approach a maximum at high substrate concentration. Third, they are "gated"—opened or closed in response to some cellular event. In ligand-gated channels (which are generally oligomeric), binding of an extracellular or intracellular small molecule forces an allosteric transition in the protein, which opens or closes the channel. In voltage-gated ion channels, a change in transmembrane electrical potential (Vm) causes a charged protein domain to move relative to the membrane, opening or closing the ion channel. Both types of gating can be very fast. A channel typically opens in a fraction of a millisecond and may remain open for only milliseconds, making these molecular devices effective for very fast signal transmission in the nervous system.

Ion-Channel Function Is Measured Electrically

Because a single ion channel typically remains open for only a few milliseconds, monitoring this process is be-

Erwin Neher

Bert Sakmann

Erwin Neher

Bert Sakmann yond the limit of most biochemical measurements. Ion fluxes must therefore be measured electrically, either as changes in Vm (in the millivolt range) or as electric currents I (in the microampere or picoampere range), using microelectrodes and appropriate amplifiers. In patch-clamping, a technique developed by Erwin Neher and Bert Sakmann in 1976, very small currents are measured through a tiny region of the membrane surface containing only one or a few ion-channel molecules (Fig. 11-47). The researcher can measure the size and duration of the current that flows during one opening of an ion channel and can determine how often a channel opens and how that frequency is affected by transmembrane potential, regulatory ligands, toxins, and other agents. Patch-clamp studies have revealed that as many as 104 ions can move through a single ion channel in 1 ms. Such an ion flux represents a huge ampli fication of the initial signal; for example, only two acetyl-choline molecules are needed to open an acetylcholine receptor channel (as described below).

The Structure of a K+ Channel Reveals the Basis for Its Specificity

The structure of a potassium channel from the bacterium Streptomyces lividans, determined crystallo-graphically by Roderick MacKinnon in 1998, provides much insight into the way ion channels work. This bacterial ion channel is related in sequence to all other known K+ channels and serves as the prototype for such channels, including the voltage-gated K+ channel of neurons. Among the members of this protein family, the similarities in sequence are greatest in the "pore region," which contains the ion selectivity filter that allows K+ (radius 1.33 A) to pass 10,000 times more readily than Na+ (radius 0.95 A)—at a rate (about 108 ions/s) approaching the theoretical limit for unrestricted diffusion.

The K+ channel consists of four identical subunits that span the membrane and form a cone within a cone surrounding the ion channel, with the wide end of the double cone facing the extracellular space (Fig. 11-48).


Micropipette applied tightly II Patch of membrane to plasma membrane / / pulled from cell

Micropipette applied tightly II Patch of membrane to plasma membrane / / pulled from cell

Patch of membrane placed in aqueous solution

Patch of membrane placed in aqueous solution

Electronics to hold transmembrane potential (Vm) constant and measure current flowing across membrane

FIGURE 11-47 Electrical measurements of ion-channel function. The

"activity" of an ion channel is estimated by measuring the flow of ions through it, using the patch-clamp technique. A finely drawn-out pipette (micropipette) is pressed against the cell surface, and negative pressure in the pipette forms a pressure seal between pipette and membrane. As the pipette is pulled away from the cell, it pulls off a tiny patch of membrane (which may contain one or a few ion channels). After placing the pipette and attached patch in an aqueous solution, the researcher can measure channel activity as the electric current that flows between the contents of the pipette and the aqueous solution. In practice, a circuit is set up that "clamps" the transmembrane potential at a given value and measures the current that must flow to maintain this voltage. With highly sensitive current detectors, researchers can measure the current flowing through a single ion channel, typically a few picoamperes. The trace showing the current as a function of time (in milliseconds) reveals how fast the channel opens and closes, how frequently it opens, and how long it stays open. Clamping the Vm at different values permits determination of the effect of membrane potential on these parameters of channel function.

Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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