Membrane conductance, single-channel current records

Fig. 11. NMDA-activated channel properties of rat cerebellar granule cells at different days in culture. A Single-channel currents recorded from an outside-out patch. Currents were evoked by the application of 20 pm NMDA on the bath solution. The membrane potential was kept at —80 mV. Conductance values of 12 pS were observed at the third day in culture (DIC), 31 pS at the fifth DIC and 52 pS at the 11th DIC. B Maximum conductance values are plotted against the time in culture.

Fig. 6.23 Single-channel currents flowing across the membrane between the protoplast and vacuole of Chara corallina. Among several channels with different conductivity the recordings of the 130 pS channel are recorded here. The zero line is at the top of each curve. (By courtesy of F. Homble)...

Another versatile mode is the cell-excised configuration (Hamill 1993). It is obtained by suddenly removing the patch-pipette from the cell, so that the membrane patch is pulled off the cell. This mode easily allows to expose the channel proteins to drugs by changing the bath solution. The single channel currents are recorded on a videotape and are analyzed off-line by a computer system. Various parameters are evaluated, such as the single channel conductance, open-and closed-times of the channel, and the open-state probability, which is the percentage of time the channel stays in its open state. 

Figure 5. Single-channel currents from lipid bilayers containing the synthetic T4M28 and T4M28(S —> A) proteins. Currents were recorded at 100 mV in symmetric 0.5-M KCl from PC membranes containing T4M28 (A) and T4M28 (S —> A) (B). Calculated single-channel conductances are 26 and 20 pS, respectively. For other details, see reference 49.

NMDA-activated channels were recorded from 8-day-old rat cerebellar granule cells at different days in culture. Single-channel currents were recorded from outside-out membrane patches, and the agonist was applied directly to the bath solution. In Fig. IIA single-channel currents, in the presence of NMDA, recorded at different days in culture are shown. We observed an increase on the maximum conductance of the NMDA-activated channels with days in culture. The conductance increases during the first 5 days in culture, reaching a steady value after the sixth day in culture (Fig. IIB). 

A parallel development came from studies on artificial lipid bilayer membranes. Hladky and Hay don (1984) found that when very small amounts of the antibiotic gramicidin were introduced into such a membrane, its conductance to electrical current flow fluctuated in a stepwise fashion. It looked as though each gramicidin molecule contained an aqueous pore that would permit the flow of monovalent cations through it. Could the ion channels of natural cell membranes act in a similar way To answer this question, it was first necessary to solve the difficult technical problem of how to record the tiny currents that must pass through single channels. 

The vesicle assay previously used ves a good survey of transporter activity under a fixed set of conditions. However it is not suited to exploration of transport activity as a function of the sign and magmtude of the transmembrane potential. Bilayer conductance, or single channel recording techniques are required (21). In this techmque, a lipid bilayer is formed across a small hole in a Teflon barrier, by direct application of the lipid in decane to the hole. Under favorable conditions, Ae lipid tl s to a bilayer membrane which electrically isolates the two halves of the cell. T icdly KCl is used as an electrol, and electrical contact is made via Ag/AgQ wires directly inserted into the solutions. A high impedance operational amplifier circuit (bUayer clamp) can then be used to apply a fixed transmembrane potential and to monitor the current which flows as a function of time. The two sides of the membrane are independently accessible, so different sequences of transporter addition, control of pH, and other variables are in principle j ssible. 

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