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Department of Molecular Physiology

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patch clamp
Introduction

Ion channels mediate a controlled passage of ions over the plasma membrane of cells. Due to their electric charge, ions cannot enter the plasma membrane itself. The much lower dielectrical constant of the membrane (about 2) compared to water (about 80) strongly opposes entry of ions into the membrane. One way for ions into or out of the cell are the gated pores of ion channel proteins. These pores are short tubular structures in the middle of the protein that serve to strip the hydration shell off the ion and allow its passage to the opposite side of the membrane. Ion channels are passive transporters: they do not supply energy for ion transport, they simply allow electrodiffusion which is solely driven by the electrochemical potential gradient across the membrane. Most ion channels conduct only one species of ion. The most prominent channels are sodium channels, potassium channels, calcium channels and chloride channels.

Ion channel pores are not always open. They can be opened and closed according to the cell┤s requirements. Specialized structures within the protein serve as gates that control the passage of ions. These gates have a fundamental significance for all living organisms: Opening and closing of channel gates ("gating") is the basis of most forms of communication between cells, as virtually every biological signal acts directly or indirectly on a channel gate. Gates can be controlled chemically (ligand-gated channel), as in transmitter-gated or calcium-gated channels, or electrically, as in the voltage-gated channels in excitable cells. In both cases: when the gate opens, current flows over the membrane, the current stops when the gate is closed.

The current that flows through single channel molecules can be recorded using the patch-clamp method. The opening and closing of the gate produces two distinct current levels: a background-current when the gate is closed and a larger current when the gate is open. The probability that the gate opens depends on the control mechanism, for example in a chemically controlled channel on the concentration of the ligand. The example here shows 5-s-recordings of a cAMP-gated channels at six different concentrations of cAMP (top trace: 5 ÁM cAMP, bottom trace: 300 ÁM cAMP). Opening of the gate causes a sudden current increase by 1.5 pA (1.5 x 10-12 A). The higher the cAMP concentration, the higher is the open probability of the channel. The patch-clamp method thus makes it possible to watch a single molecule doing its work.



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