Properties of Single Inwardly Rectifying K Channels in Skeletal and Cardiac Muscle

An important requirement of the patch clamp technique, by which the functional properties of a single ion channel in a cell membrane may be studied, is a tight seal between the membrane and the glass pipette electrode. Hitherto, the properties of ionic channels in mammalian skeletal muscle have been studied mostly in cultured cells which present a relatively clean surface membrane. Here a method is described by which the surface membrane (sarcolemma) of adult skeletal muscle, which is normally covered by a basement membrane, is made accessible for patch clamping. It is a simplified version of a procedure previously used for the production of membrane vesicles from frog muscle by treatment with a high KCl solution containing collagenase. The method has proved applicable to human muscle biopsy samples and so may be useful in clinical investigations. In exploring sarcolemmal vesicles from rat and human muscles the following ion channels could be readily demonstrated in high KCl solution: (i) ATP-sensitive K channels, (ii) delayed rectifier K channels, (iii) Ca-activated K channels. In addition, the following channels could be found though more rarely: (iv) inwardly rectifying K channels, (v) a channel of small conductance, (vi) a chloride channel. In subsequent work, attention was focused on the inwardly rectifying K channel as that channel had hitherto been little studied. The role of Mg2+ in producing inward rectification was demonstrated by comparing the properties of the channel in patches left attached to vesicles and detached into Mg2+-free solution. In the presence of internal Mg2+ no current passes when an outward driving force is applied and the inward current flowing in response to a small inside negative potential shows frequent interruptions (flickery block). In the absence of internal Mg2+, flickery block disappears and current flows instantaneously as readily in outward as in inward direction. When Mg2+ does not block the channel the existence of an intrinsic gating process which operates so as to close the channel on depolarization becomes evident. The time and voltage dependence of this gating process is described. The relationship between channel open probability (Po) and voltage is a steep one with a slope factor of 4.13mV. These results are compared with the already known properties of inwardly rectifying K channels in cardiac muscle. With the patch clamp technique, membrane patches may be detached from cells or vesicles so as to allow access to the inner surface of the membrane. The effect of changes in the composition of the "intracellular" solution on channel activity may therefore be studied. To change the composition of the internal solution, detached patches were exposed to a stream of flowing solution of variable composition. Under such conditions it was found that the kinetic properties of the inwardly rectifying K channel were influenced by flow itself. This entirely new phenomenon was found also in patches detached from dispersed muscle fibres from rat flexor digitorum brevis. Analysis of the new phenomenon showed that as a result of flow, the maximum open probability is decreased and the midpoint of the Po-voltage relation is shifted to the right by more than 20mV. No evidence could be found for the existence of a local concentration gradient sensitive to flow. Application of suction to the patch pipette showed the inwardly rectifying channels not be sensitive to membrane stretch. The possibility is contemplated that shear stress upon the inner face of the patch modulates the kinetic behaviour of the channel. From experiments on whole cells, it is known that the inwardly rectifying K conductance is sensitive to changes in intracellular pH (pHi). This effect was studied at the single channel level on patches excised from rat sarcolemmal vesicles and subjected to changes in pHi under conditions of constant flow. When pHi was lowered from 7.4 to 6.9, 6.5 or 6. 0, the channel continued to show transitions between open and short closed states, but this activity became interrupted by long lasting closures, the more so the lower pHi On exposure to pHi 5.0, the channel soon closed down until pHi was returned to 7.4. During the periods of channel activity at pHi below 7.4, Po decreased in graded fashion the lower pHi. This reduction in Po during the periods of channel activity was not obviously voltage dependent and cannot therefore be attributed to a shift in the Po-voltage relation