Recording the Electrical Current Flowing Through a Single Acetylcholineactivated Ion Channel

Throughout our discussion of the membrane properties of excitable cells, we have made extensive use of the notion of ions crossing the membrane through specific pores or channels. For example, we saw that the effect of ACh on the muscle membrane is mediated via ion channels in the postsynaptic membrane that open in the presence of ACh. As discussed in Chapters 5 and 7, the flow of ions across the cell membrane constitutes a transmembrane electrical current that can be measured with electrical techniques like the voltage clamp. Recently, a new technique was developed by Neher and Sakmann to record transmembrane ionic currents, and the technique has sufficient resolution to measure the minute electrical current flowing through a single open ion channel. The technique is called the patch clamp, and it is illustrated in Figure 8-13.

Figure 8-13 Schematic illustration of the procedure for recording the current through a single ACh-activated channel in a cell membrane. A micropipette with a tip diameter of 1-2 |im is placed on the external surface of the membrane. A tight electrical seal is made between the membrane and the glass of the micropipette, so that a resistance greater than 1010 ohms is imposed in the extracellular path for current flow through the channel. When a channel in the patch of membrane inside the micropipette opens, a current-sensing amplifier connected to the interior of the pipette detects the minute current flow.

The basic idea behind the patch clamp is to isolate electrically a small patch of cell membrane that contains only a few ion channels. The electrical isolation is achieved by placing a specially constructed miniature glass pipette in close contact with the membrane, so that a tight seal forms between the membrane and the glass. When one of the ion channels in the isolated patch opens, electrical current flows across the membrane; in the case of the ACh-activated channel that current would be a net inward (that is, depolarizing) current under normal conditions. We know from the basic properties of electricity that current must flow in a complete circuit. As shown in Figure 8-13, the return current path through the extracellular space is broken by the presence of the glass pipette; there is a high electrical resistance imposed by the seal between the cell membrane and the pipette. Under these conditions, the ionic current through the open channel is forced to complete its circuit through the current-sensing amplifier connected to the interior of the pipette. In order for the patch-clamp technique to achieve sufficient sensitivity to measure the current through a single channel, the electrical resistance between the interior of the patch pipette and the extracellular space must be greater than about 109 ohms, which is a very large resistance indeed. Fortunately for neurophysiologists, it is possible to achieve resistances greater than 1010 ohms.

Using the patch clamp, it is possible to record the current through ACh-activated channels of the postsynaptic membrane of muscle cells by placing a small amount of ACh (or structurally related compounds that are recognized by the receptors on the gate) inside the patch pipette. As shown in Figure 8-4, when the receptors are occupied, the gate opens and the channel allows ions to cross the membrane. Schematically, then, we might expect to record an electrical current like that shown in Figure 8-14a when the channel opens. There

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Figure 8-14 The current through single ACh-activated ion channels. (a) A schematic diagram of the current expected to flow through a single ACh-activated channel if the conducting state of the channel is controlled by a gate that is either completely open or completely closed. When ACh binds, the channel opens and a stepwise pulse of inward current flows through the channel. When ACh unbinds, the channel closes and the current abruptly disappears. (b) Actual recordings of currents flowing through single ACh-activated channels. (Data provided by D. Naranjo and P. Brehm of the State University of New York at Stony Brook.)

would be a rapid step of inward current that occurs as the gate opens, the current would be maintained at a constant level for as long as the channel is open, and the step would terminate when ACh unbinds from one of the receptor sites, causing the gate to close. If a second channel opens while the first is still open, the two currents simply add to produce a current twice as large as the single-channel current. This is also shown in Figure 8-l4a.

Actual patch-clamp recordings of currents through single ACh-activated channels of human muscle cells are shown in Figure 8-l4b. These records show that the currents through the channels are the rectangular events expected from the simple gating scheme of Figure 8-4. Experiments like that of Figure 8-l4b confirm directly the view of ion permeation and channel gating that we have used to explain the electrical behavior of the membranes of excitable cells: the gated ion channels carrying electrical current across the plasma membrane are not just figments of the neurophysiologist's imagination. The development of the patch-clamp technique has led to a flurry of new information about ion channels of all types; for example, the currents flowing through single voltage-sensitive sodium and potassium channels that underlie the action potential (see Chapters 6 and 7) have also been observed using this technique.

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