Supplementary Figure 2: Cartoon showing patch-clamp configurations.

a, voltage clamp experiment in whole-cell configuration (upper cartoon): In a whole cell patch clamp experiment the extracellular surface faces the bath solution which is connected to the reference electrode (ground). The potential of the bath solution and the extracellular surface of the cell is 0 mV. The patch clamp pipette solution is connected to the cytosolic side of the cell. To clamp a cell at a membrane potential of e.g. -90 mV, a negative potential of – 90 mV at the headstage input with respect to ground needs to be delivered; the potential inside the patch clamp pipette (= command voltage) needs to be set to -90 mV. Thus, the membrane potential is directly proportional to the command voltage. The upper cartoon shows cations (e.g. Na+) flowing from the outside into the cell (cytosol). A flow of cations (here Na+) into the cells represents a depolarizing current. In voltage clamp experiments, this current is clamped by the amplifier by a flow of cations into the headstage, which is defined as a negative current. Thus, inward membrane currents correspond to inward command currents into the headstage. In whole cell patch clamp command currents and membrane currents are proportionally related. b, voltage clamp experiment in whole endolysosomal configuration (lower cartoon): Once the endolysosome is released from the cell, the membrane surface that was originally facing the cytosol is exposed to the bath solution, which is connected to the reference electrode (ground). Now the potential on this membrane surface is 0 mV (bath potential). The pipette is still connected to the luminal surface of the endolysosomal membrane. For a membrane potential of e.g. Vm = -90 mV, the potential inside the pipette must be adjusted to the more depolarized value of +90 mV (top). Thus, the potential across the endolysosomal membrane is inversely proportional to the command potential. In order to match the conditions outlined for whole cell recordings, the command voltage needs to be inverted. The lower cartoon shows cations (e.g. Na+) flowing from the endolysosomal lumen into the bath solution (corresponds to the cytosol). In voltage clamp experiments, this current is compensated by a flow of cations out of the headstage into the pipette and subsequently out of the pipette tip, which is defined as a positive current. Therefore a positive command current corresponds to a membrane current carried by cations flowing from the lysosomal lumen into the cytosol. In whole endolysosomal patch clamp command currents and membrane currents are inversely related. In order to match the conventions that flow of cations into the cytosol represents an inward current the command current needs to be inverted.

Summary: Flow of Na+ into the cytosol corresponds to a negative command current in a and a positive command current in b. To match conventions that the flow of cations into the cytosol is defined as a negative current the command current in b is inverted. In order to clamp the membrane voltage to e.g. -90 mV cytosol negative with respect to the extracellular side, a negative command voltage is applied in a while a positive command voltage is applied in b. To match conventions that membrane voltage Vm is given as the difference (Δ) of the cytosolic voltage Vcytosol minus the Vnon-cytosol voltage in the compartment on the other side of the membrane under investigation (extracellular side in the whole cell recording) voltage is inverted in b.

Top rows in a and b (right part of the upper panel): command voltage (left) at headstage input and command current (right) into or out of the headstage input. Definition: The flow of positive current out of the headstage into the patch clamp pipette and out of the pipette tip is termed positive current. A positive potential means a positive voltage at the headstage input with respect to ground (for details see e.g. The Axon Guide for Electrophysiology & Biophysics Laboratory Techniques). Bottom rows in a and b (right): Current and voltages are given with respect to the cytosol. Currents flowing into the cytosol are negative currents, membrane voltages are given as difference of cytosolic voltage minus the voltage in the non-cytosolic compartment (for details see e.g. The Axon Guide for Electrophysiology & Biophysics Laboratory Techniques).