Vm-related extracellular potentials observed in red blood cells

Abstract

Even in nonexcitable cells, the membrane potential V_m is fundamental to cell function, with roles from ion channel regulation, development, to cancer metastasis. V_m arises from transmembrane ion concentration gradients; standard models assume homogeneous extracellular and intracellular ion concentrations, and that V_m only exists across the cell membrane and has no significance beyond it. Using red blood cells, we show that this is incorrect, or at least incomplete; V_m is detectable beyond the cell surface, and modulating V_m produces quantifiable and consistent changes in extracellular potential. Evidence strongly suggests this is due to capacitive coupling between V_m and the electrical double layer, rather than molecular transporters. We show that modulating V_m changes the extracellular ion composition, mimicking the behaviour if voltage-gated ion channels in non-excitable channels. We also observed V_m-synchronised circadian rhythms in extracellular potential, with significant implications for cell-cell interactions and cardiovascular disease.

Figure 1

(A) A schematic of the cell, indicating the location of the electrical double layers, the shear plane (broken purple line) of ζ, the specific membrane conductance G_eff and capacitance C_eff, cytoplasm conductivity σ_cyto and membrane potential V_m. (B) The variation in potential from the cell surface, showing the location of the Stern layer and the shear plane at which ζ is located.

Figure 2

(A) The mean variation (± s.d.) in V_m and ζ for control RBCs in media containing 100% (square), 10% (circle) and 1% (triangle) physiological saline as described in the text. (B) The same values of ζ plotted against the calculated reciprocal Debye length κ for each medium, together with values adjusted by 0.37 V_m. The relationship between these variables theoretically follows a negative exponential to which the adjusted values offer a perfect (r² = 1) fit. All points represent the average of four donors.

Figure 3

The mean (n = 3) V_m (A) and ζ-potential (B) of RMCE immediately after resuspension into 10% solution (see text). As can be seen, in both instances the value immediately changes from a rest value (taken in 100% solution, denoted here as time 0), peaks and then increases to a rest value. The transition period was consistent for both V_m and ζ, but the former was observed to respond consistently across all three samples, whereas for ζ a delay of 5–15 min was observed before transition began.

Figure 4

(A) The mean variation (± s.d.) in V_m and ζ for RBCs suspended in media containing 100% (square), 10% (circle) and 1% (triangle) physiological saline as described in the text. RBCs were treated with valinomycin, neuraminidase, and a combination of both, as well as a DMSO was control. All points represent the average of four donors. (B–E) The values of ζ from Fig. 4A plotted against the reciprocal Debye length κ, together with values adjusted by ΞV_m as described in the text. The relationship between these variables theoretically follows a negative exponential, to which the adjusted values offer a perfect (R² = 1) fit. All points represent the average of four donors.

Figure 5

(A) The DEP spectra (points) of RBCs in media of different ionic strength (1% (grey), 10% (orange) and 100% (blue)) together with rolling average trendlines; (B) the values of G_eff vs. σ_cyto for 1% and 10% media, together with best-fit trendline; (C) Specific membrane conductance G_eff and capacitance C_eff as a function of medium ionic strength; (D) the relationship between cytoplasm conductivity σ_cyto and membrane potential V_m.

Figure 6

Circadian behaviour of ζ-potential in RBCs. (A) mean (n = 4) ζ for RBCs entrained and suspended in KHB and DEP medium, together with best-fit rhythm with period of 24.5 h and 24.3 h respectively. (B) ζ of RBCs taken directly from a participant over a 24 h period, measured within 60 s of donation, together with a best-fit rhythm with 22.2 h period (time 0 h corresponds to 10am).

Figure 7

(A) A schematic showing ion concentrations in proximity to the cell surface in high ionic strength (HIS) and low ionic strength (LIS) solutions. Where the surface potential of a cell is negative, it will electrostatically attract cations from solution, raising the cation concentration at the surface whilst depleting anions. Where the surface potential is positive, this situation is reversed, with the cation level in HIS solutions reaching similar values to those observed in LIS solutions. (B) A comparison of the voltage-gated cation channel behaviour reported by Kaestner et al., and the cation concentration at the surface relative to the bulk (green line) determined using the Poisson-Boltzmann equation, using ζ determined from Eq. (5).