A four-electrode method to study dynamics of ion activity and transport in skeletal muscle fibers

Abstract

Ion movements across biological membranes, driven by electrochemical gradients or active transport mechanisms, control essential cell functions. Membrane ion movements can manifest as electrogenic currents or electroneutral fluxes, and either process can alter the extracellular and/or intracellular concentration of the transported ions. Classic electrophysiological methods allow accurate measurement of membrane ion movements when the transport mechanism produces a net ionic current; however, they cannot directly measure electroneutral fluxes and do not detect any accompanying change in intracellular ion concentrations. Here, we developed a method for simultaneously measuring ion movements and the accompanying dynamic changes in intracellular ion concentrations in intact skeletal muscle fibers under voltage or current clamp in real time. The method combines a two-microelectrode voltage clamp with ion-selective and reference microelectrodes (four-electrode system). We validate the electrical stability of the system and the viability of the preparation for periods of ∼1 h. We demonstrate the power of this method with measurements of intracellular Cl−, H+, and Na+ to show (a) voltage-dependent redistribution of Cl− ions; (b) intracellular pH changes induced by changes in extracellular pCO2; and (c) electroneutral and electrogenic Na+ movements controlled by the Na,K-ATPase. The method is useful for studying a range of transport mechanisms in many cell types, particularly when the transmembrane ion movements are electrically silent and/or when the transport activity measurably changes the intracellular activity of a transported ion.

Measurement of intracellular ion activity in skeletal muscle fibers: Four microelectrodes or no deal

“…The high Cl − conductance of the membrane implies that a change in membrane voltage leads to rapid transmembrane Cl − movements until the Cl − equilibrium potential meets the new value of membrane potential. This is exactly what Heiny et al (2019) show by using a Cl − -selective ISM both in voltage-and current-clamp conditions. Interestingly, they validate their methods by showing that the Cl − current produced by the voltage jump led to an amount of conducted Cl − charges and a resulting change in concentration that exactly matches the change in concentration measured with the ISM.…”

“…It is remarkable that in close to 90% of preparations for which all four microelectrodes were successfully inserted, the background current, the membrane potential read using the ISM, and the calibration values of the ISM did not deviate significantly at the end of the experiment from their initial values. Additionally, Heiny et al (2019) show that, although two of the four microelectrodes measure membrane potential at different locations, both electrodes measure very similar potentials. Therefore, for most purposes, the technique can be assuredly simplified to three electrodes: the singlebarreled ISM and the two in charge of voltage clamp.…”

“…The two main advantages of ISM over fluorescence approaches are that absolute intracellular ion activity can be accurately recorded over a very large range of concentrations and calibration can be easily achieved before impalement of the cell then checked at the end of the experiment by simply dipping the electrode into saline containing different concentrations of the ion of interest. Heiny et al (2019) have brought the ISM technique to a high level of reliability and accuracy in enzymatically isolated mouse flexor digitorum brevis (fdb) muscle fibers using homemade ISM. The large size of the muscle cell allows four microelectrodes to be impaled, ensuring physiologically relevant measurements of intracellular Na + , Cl − , or H + .…”

“…Moreover, one two-barreled ISM or one single-barreled ISM plus one voltage electrode often causes membrane damage, generating a short that changes the membrane potential, and inducing currents that alter the ion activity being measured. In the technique described by Heiny et al (2019), the measurement of ion activity with the ISM is not only separated from the measurement of membrane potential, but, by using two other intracellular microelectrodes, the membrane potential can be voltage clamped or adjusted to a desired value under current-clamp conditions. The function of these two additional microelectrodes is instrumental to a reliable measurement of intracellular activity by avoiding heavy impacts of membrane potential changes on intracellular ion activity and, reciprocally, by preventing the effect of large changes in ion concentrations on membrane potential and hence ion activity measurement.…”

“…Recording the background current or cell membrane resistance by these two microelectrodes also gives the benefit of checking cell integrity all along the course of the experiment. The impalement of four microelectrodes can be viewed as additional injuries caused to the cell, but, by monitoring membrane potential and background current in the voltage clamp configuration, Heiny et al (2019) provide evidence of very stable recording conditions without any signs of cell deterioration. It is remarkable that in close to 90% of preparations for which all four microelectrodes were successfully inserted, the background current, the membrane potential read using the ISM, and the calibration values of the ISM did not deviate significantly at the end of the experiment from their initial values.…”