Single K+ channel currents of anomalous rectification in cultured rat myotubes.

Abstract

The currents through single K+ channels of the anomalous (or inward) rectifier were recorded in tissue cultured rat myotubes by using the "gigohm seal" patch clamp technique developed by Sigworth and Neher. These unitary currents were detected as current fluctuations due to the blocking and unblocking of channels by Ba'+. The single-channel conductance was obtained from the slope of the linear relationship between unitary current amplitude and membrane potential. When the external solution contained 155 mM K+, the single-channel conductance was 10.4 ± 2.6 pS (±SD; n = 6). This value was independent of the concentration of blocking ions but increased with increasing external K+ concentration. The behavior of the unitary current agreed with that expected from the blocking kinetics of Ba2+ on the macroscopic K+ current of the anomalous rectifier. The density of the channel is likely to be small and may even be less than 1/Fm2.One of the most direct ways to demonstrate ionic channels in the cell membrane is to record single-channel currents. Neher and Sakmann (1) first made successful recordings ofthe current flowing through single acetylcholine-activated channels by using a "patch-clamp" technique. The low signal-to-noise ratio of this original technique, however, precluded its application to membrane channels of smaller unitary conductance, such as Na' channels. Recently, Sigworth and Neher (2) improved the signal-to-noise ratio of this technique by at least a factor of 10, when they increased the seal resistance of the tip of the patch electrode to the cell surface from 108 to 1010 fQ. With this improved technique they successfully recorded single Na' channel currents in tissue cultured rat muscle cells.Since its original discovery in frog skeletal muscle fibers by Katz (3), the anomalous (or inward) rectifier of the cell membrane K conductance has been found in various preparations and its properties have been analyzed extensively. In the present work our objective was to record the currents from single K+ channels of the anomalous rectifier by using this "gigohmseal" patch-clamp technique. We chose to use tissue cultured myotubes in this study for several reasons. Kidokoro (4) has shown that the resting myotube membrane behaves almost as a perfect K electrode. The membrane shows clear anomalous rectification with properties similar to those found in other preparations: (a) the rectification depends on V-VK (V, membrane potential; VK, K+ equilibrium potential) rather than on V alone and (b) the membrane conductance for the inward current increases when the external K+ concentration is increased. The C1 conductance, which is significant in adult muscle fibers, is negligible in the myotubes. Additional important reasons are: (a) the gigohm-seal patch-clamp technique has already been successfully applied to this preparation (5), and (b) the cell is large enough so that current flowing through the patch membrane does not cause a significant change in the potential of the rest of the cell membrane. MATERIALS...

Single inwardly rectifying potassium channels in cultured muscle cells from rat and mouse.

“…One of the most remarkable findings in this work is that the inwardly rectifying K+ channel of skeletal muscle showed transitions between open and closed states through intrinsic gating kinetics in the absence of blocking ions (but see Ohmori et al 1981). Gating properties of the cardiac inwardly rectifying K+ channel during hyperpolarization have been studied by a number of workers (Kameyama et al 1983;Sakmann & Trube, 1984b;Kurachi, 1985;Payet et al 1985).…”

“…2). This may correspond to that recorded by Ohmori et al (1981) in the presence of Ba2+ in the pipettes. In the following experiments, we analysed the voltage dependence of inactivation of the channel with the larger conductance, the channel which was more frequently observed.…”

“…However, relatively little is known at the single-channel level in skeletal muscle fibres (Ohmori, Yoshida & Hagiwara, 1981; Burton, Dbrstelmann & Hutter, 1987), although years have passed since the introduction of the patch-clamp technique (Neher & Sakmann, 1976). A major reason for the slow progress in this aspect of single-channel analysis is a very low density of inward rectifier channels in skeletal muscle fibres.…”

Studies of single calcium channel currents in rat clonal pituitary cells.

“…As a first step we obtained a probability density histogram ofthe patch current; this was an effective method for determining the amplitude of single, anomalous, rectifier channels under the condition 651 of multiple-operating channels (Ohmori, Yoshida & Hagiwara, 1981). The result obtained for the current traces, shown in Fig.…”

Single-channel recordings from cultured human retinal pigment epithelial cells.

“…The fraction of time spent at a given current level was determined from the sum of the time spent at that level divided by the total time in the record. Po was found by fitting these fractions with the binomial theorem (Ohmori et al, 1981;Patlak and Horn, 1982;Benham and Bolton, 1983).…”