Ion channel recordings

Vasilyev DV, Merrill TL, Bowlby MR (2005) Development of a novel automated ion channel recording method using inside-out whole-cell membranes. J Biomol Screen 10(8) 806-813... 

A nanoporous membrane to be used for high throughput ion channel recordings has been described... 

Schibel, A. E. P. Edwards, T. Kawano, R. Lan, W. White, H. S. Quartz nanopore membranes for sns-pended bilayer ion channel recordings. Anal Chem 2010, 82,7259-7266. 

The patch-clamp technique is based on the formation of a high resistance seal (109-10lon) between the tip of a glass micropipette and the cell membrane it touches (gigaohm-seal). This technique allows recordings of ionic currents through single ion channels in the intact cell membrane and in isolated membrane patches at a... 

FIGURE 1.9 Records of the minute electrical currents (downward deflections) that flow through single ligandgated ion channels in the junctional region of frog skeletal muscle. The currents arise from brief transitions of individual nicotinic receptors to an active (channel open) state in response to the presence of various agonists (ACh = acetylcholine SubCh = suberyldicholine DecCh = the dicholine ester of decan-1,10-dicarboxylic acid CCh = carbamylcholine). (From Colquhoun, D. and Sakmann, B., J. Physiol., 369,501-557, 1985. With permission.)... 

This chapter deals mainly with information that can be obtained from equilibrium, or at least steady-state, recordings of ion-channel receptor activity. However, a great deal of information has also been obtained from kinetic studies of ion channels where the aim has been to determine values for the rate constants in a receptor mechanism. In general, only equilibrium constants can be determined from equilibrium studies. 

The ion-channel blocking mechanism has been widely tested and found to be important in both pharmacology and physiology. Examples are the block of nerve and cardiac sodium channels by local anesthetics, or block of NMDA receptor channels by Mg2+ and the anesthetic ketamine. The channel-block mechanism was first used quantitatively to describe block of the squid axon K+ current by tetraethylammonium (TEA) ions. The effects of channel blockers on synaptic potentials and synaptic currents were investigated, particularly at the neuromuscular junction, and the development of the single-channel recording technique allowed channel blockages to be observed directly for the first time. 

Changes in the occupancy of the open-channel state of the receptor as a function of time (pA2R (t)) in response to a perturbation of the receptor equilibrium can be used to obtain information about the rates of channel gating and the interaction of dmgs with ion-channel receptors. The system is said to relax towards a new equilibrium. The time course of the relaxation is used to measure rates from the average behavior of many ion channels in a recording, while noise analysis uses the frequency of the moment-to-moment fluctuations in occupancy of the open-channel state at equilibrium to provide information about the rates in the receptor mechanism. 

A G-protein-mediated effect has an absolute requirement for GTP. Reference has already been made to the requirement for GTP in reconstituting hormone-stimulated adenylate cyclase activity. A similar requirement can be demonstrated when the effector is an ion channel, such as the cardiac atrial inward-rectifier K+ channel which is activated following stimulation of the M2 muscarinic acetylcholine receptor. Thus, in the experiment illustrated in Figure 7.8, the channel recorded with a cell-... 

The opening of masses of ion channels in nematode muscle membranes may be detected using the two-microelectrode voltage-clamp technique. In contrast, the opening of single ion channels may be recorded using the vesicle preparation and patch-clamp technique. These techniques are both described below. 

Fig. 21.3. Two-micropipette current-clamp recording and effect of maintained application of 30 pM levamisole, which produces a 15 mV depolarization (upward movement of trace). The downward transients are the result of injected current pulses used to measure membrane conductance. The trace gets narrower as the input conductance increases from 2.35 pS to 4.35 pS as the levamisole ion channels open. The peak amplitude of the membrane potential response and change in input conductance are used as an assay of the number and activity of the levamisole ion channel receptors present in the muscle cell membrane. The response was fully reversible on washing (not shown).

Chronopotentiometry has also been used to determine chloride ions in seawater [31]. The chloride in the solution containing an inert electrolyte was deposited on a silver electrode (1.1 cm2) by the passage of an anodic current. The cell comprised a silver disc as working electrode, a symmetrical platinum-disc counter-electrode and a Ag-AgCl reference electrode to monitor the potential of the working electrode. This potential was displayed on one channel of a two-channel recorder, and its derivative was displayed on the other channel. The chronopotentiometric constant was determined over the chloride concentration range 0.5 to 10 mM, and the concentration of the unknown solution was determined by altering the value of the impressed current until the observed transition time was about equal to that used for the standard solution. 

The SR may contribute to excitation-contraction (EC) coupling in two ways firstly, by the release of Ca2+ for contraction as described above, but secondly by modulating membrane excitability. As will be described elsewhere in this book, the SR is an important mediator of surface membrane ion channel activity, and hence, excitability. Spontaneous Ca2+ release from the SR can activate Ca2+-sensitive ion channels. Both K+ (Kca) and Cl- (Clsmooth muscle cell membrane can be activated by SR Ca2+. If Kca channels are activated there will be a hyperpolarization, as K+ ions leave the cell and spontaneous transient outward currents (STOCs) can be recorded (Carl et al 1996, Nelson Quayle... 

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