Supplementary Figure 1

Transcription

1 Supplementary Figure 1 Activation of P2X2 receptor channels in symmetric Na + solutions only modestly alters the intracellular ion concentration. a,b) ATP (30 µm) activated P2X2 receptor channel currents were recorded in symmetric Na + solutions using a protocol similar to Fig. 1b. The 15 s ATP activation at -20 mv (a) caused the reversal potential to shift from 0.2±0.2 mv to -2.9±0.9 mv (n=3 cells), while activation at +20 mv (a) shifted the reversal potential to the right 3.2±0.6 mv (n=3 cells), indicating that the intracellular Na + concentration increased or decreased by about 15 mm.

2 Supplementary Figure 2 Changes in ion concentrations resulting from ionic fluxes under different recording conditions. a) Schematic illustration for how ion concentrations will change more dramatically in bi-ionic solutions compared to symmetric solutions. b) Net current (black), outward Na + current (yellow), and inward NMDG + current (blue) as a function of voltage for a membrane with a selectivity of P Na+ : P NMDG+ = 20:1 and conductance of 100 ns described by the GHK flux equation. For voltages near the reversal potential (V rev = - 75mV ), the net current is considerably smaller than the flux of either ionic species.

3 Supplementary Figure 3 Schematics of the whole-cell reservoir model. a) Illustration of the whole cell reservoir model. The concentration of each ionic species in the bath, ρ j,bath (t), the concentration of each ionic species near the pipette electrode, ρ j,pip, and the voltage near the pipette, V pip (t) can be controlled experimentally. The cell voltage, V cell (t) and concentration of each ionic species in the cell, ρ j,cell (t) change in response to currents flowing across the cell membrane and between the pipette and cytoplasm. The movement of the j-th ionic species between the pipette and the cell is described by the current, I j,pip (t), while the flow from the cell to the bath across the membrane is described by I j,mem (t). In the example shown, the net current flow between the pipette and cell, and the net current flow between the cell and bath are both zero so the charge and voltage of the cell will not change. However, even though the cell is at the reversal potential, the cellular concentration of the yellow ions will decrease because the outward flux of yellow ions moving from the cell into the bath is greater than the flux of yellow ions entering the cell from the pipette. Similarly, the cellular concentration of blue ions will increase because the inward flux of blue ions from the bath is greater than the flow of blue ions from the cell into the pipette. b) Illustration of the quasi one dimensional trajectories of ions moving towards the pipette tip with diameter, d tip, and tip angle, θ. At a distance x from the tip, the flux of each species flowing through the cross-sectional area, A(x), can be described by the current, I j (x,t).

4 Supplementary Figure 4 Effects of water permeation on steady-state ion accumulation. Influence of membrane water permeability (P f ) and initial conductance at the reversal potential (di/dv) on the steady-state depletion of intracellular Na + ([Na + ] pip -[Na + ] cell ; a), accumulation of intracellular NMDG + ([NMDG + ] cell ; b), intracellular Cl - ([Cl - ] cell ; c), and reversal potential shift (ΔV rev ; d). For the calculation, R access was held constant at 5 MΩ, the membrane conductance was modelled using the GHK flux equation with a permeability ratio of P Na+ /P NMDG+ = 20:1, and the cell area was held constant at A cell = 10 3 μm 2. The steadystate ion concentrations and reversal potential shifts were then determined by holding the cell at -60mV (~ 15mV above the initial reversal potential) until the current reached steady-state. As expected, increasing the ion channel density (di/dv) increases the depletion of intra-cellular Na + and accumulation of intracellular NMDG +. When the membrane is relatively impermeable to water (P f ~ 0 μm.s -1 ), the negative holding voltage can cause NMDG + to accumulate within the cell to higher concentrations than in the bath ([NMDG + ] bath = 150mM) which limits the reversal potential shift at even the highest channel densities. In contrast, when the membrane is more permeable to water, any increase in cellular osmotic pressure causes water to flow from the bath through the cell into the pipette. This water flow reduces the intra-cellular concentration of all species so that the intracellular osmotic pressure is closer to that of the bath. The resulting increased depletion of [Na + ] cell and reduced accumulation of [NMDG + ] cell both cause the reversal potential shift (d) to increase with membrane water permeability.

5 Supplementary Figure 5 Shifts in equilibrium potentials after prolonged activation of P2X2 receptors in bi-ionic NMDG + out/k + in solutions. a) Macroscopic currents recorded from a HEK cell transfected with P2X2 in pie vector. The voltage protocol (green trace) and the periods of extracellular ATP application (grey bars) are presented above the current trace. Voltage ramps from -90 mv to -20 mv (500 ms duration) were applied in the presence of 30 µm ATP to estimate the reversal potential before (1) and after (2) a prolonged (15-s) activation of the channel by ATP at -60 mv in NMDG out /K in solutions. R access for this recording was 6 MΩ, and the cell capacitance was 12 pf. b), I-V relations measured before (black, 1) and after (red, 2) prolonged activation of the channel by ATP from the current trace shown in a. c) Summary of the reversal potentials before (black, 1), and after (red, 2) the 15-s ATP activation in NMDG out /K in solutions from 4 cells (error bars represent S.E.M.).

6 Supplementary Figure 6 Effects of internal NMDG + on the Shaker Kv channel. a, b) Ionic currents obtained from an inside-out patch pulled from a HEK cell expressing the Shaker Kv channels in K out /K in or K out /NMDG in solutions. Currents were elicited by voltage steps from -100 mv in 10 mv increments and tail voltage was -100 mv. c) Steady-state I-V relations from the same patch as in a and b. Current amplitude at the end of each test pulse is plotted as a function of test voltage. d) Normalized G-V relations obtained from tail currents for Shaker Kv channels in K out /K in or K out /NMDG in solutions (n=3 cells). Fitting of a single Boltzmann function to these G-V relations yield midpoints of mv with K + in solutions and mv with NMDG + in solutions. e) I-V relations recorded from the same patch shown in A and B using voltage ramps from -100 mv to +50 mv in 500 ms. The I-V relations obtained from voltage steps and ramps are similar. f) I-V relations obtained with inside-out patch recordings in different internal solutions.