Wide nanoscopic pore of maxi-anion channel suits its function as an ATP-conductive pathway

Abstract

The newly proposed function of the maxi-anion channel as a conductive pathway for ATP release requires that its pore is sufficiently large to permit passage of a bulky ATP(4-) anion. We found a linear relationship between relative permeability of organic anions of different size and their relative ionic mobility (measured as the ratio of ionic conductance) with a slope close to 1, suggesting that organic anions tested with radii up to 0.49 nm (lactobionate) move inside the channel by free diffusion. In the second approach, we, for the first time, succeeded in pore sizing by the nonelectrolyte exclusion method in single-channel patch-clamp experiments. The cutoff radii of PEG molecules that could access the channel from intracellular (1.16 nm) and extracellular (1.42 nm) sides indicated an asymmetry of the two entrances to the channel pore. Measurements by symmetrical two-sided application of PEG molecules yielded an average functional pore radius of approximately 1.3 nm. These three estimates are considerably larger than the radius of ATP(4-) (0.57-0.65 nm) and MgATP(2-) (approximately 0.60 nm). We therefore conclude that the nanoscopic maxi-anion channel pore provides sufficient room to accommodate ATP and is well suited to its function as a conductive pathway for ATP release in cell-to-cell communication.

FIGURE 1

Single maxi-anion channel currents and I–V relationships in bi-ionic conditions. (A) Representative current traces recorded at different voltages from a patch containing two active channels. The pipette was filled with a solution containing 150 mM NaCl and 5 mM HEPES-NaOH (pH 7.4), and the maxi-anion channels were activated by excising the patch into control Ringer solution before challenging it with a solution of the same composition as that in the pipette. Arrowheads indicate the zero current level for each trace. (B) Unitary I–V relationship for the maxi-anion channel in symmetrical chloride conditions. Each data point represents the mean ± SE of 5–11 measurements from five different patches. The solid line is a linear fit with a slope conductance of 403.9 ± 1.7 pS. (C–E) Unitary I–V relationships for the maxi-anion channel recorded in asymmetrical conditions with 150 mM Cl⁻ in the pipette and 150 mM formate (n = 5–12), propanoate (n = 5–12), or lactobionate (n = 4–13) in the bath. Each data set was obtained from five different patches. The mean ± SE values are shown by vertical bars where the values exceed the symbol size. Solid lines are polynomial fits (Eq. 1). Reversal potentials calculated from Eq. 3 are −10.6 ± 0.6 mV, −24.1 ± 0.8 mV, and −53.0 ± 1.3 mV for formate, propanoate, and lactobionate, respectively.

FIGURE 2

Ionic selectivity of the maxi-anion channel. (A) Solid circles represent the permeability ratios derived from bi-ionic potentials. Open circles represent ratios of ionic conductivities for the same anions in the bath solution, calculated from Eq. 5 (see Materials and Methods). The dashed line is a fit to Eq. 7 with k = 1.71 ± 0.25 and R_P = 0.55 ± 0.04 nm. The solid line is a fit to Eq. 8 with k = 0.28 ± 0.05 and R_P = 0.75 ± 0.11 nm. (B) Linear correlation between permeability ratios and ionic conductivity ratios for different organic anions. The slope is 1.03 ± 0.11, and the correlation coefficient R = 0.96 at p < 0.0001.

FIGURE 3

Effects of PEGs added only from the intracellular side on single maxi-anion channel currents. (A) Representative single-channel events recorded in the absence (left panel) and in the presence of PEG 200 (middle panel) or PEG 4000 (right panel) in the bath. Pipettes were filled with control Ringer solution, and the maxi-anion channels were activated by excising the patch into the control Ringer solution before challenging it with the test polymer-containing solution. (B) Current-voltage relationships for the maxi-anion channel in control symmetrical control Ringer solution (left panel) and with PEG 200 (middle panel) or PEG 4000 (right panel) present in the bath. Each symbol represents the mean value of 5–20 observations obtained from at least five different patches. Solid lines are linear fits with slopes corresponding to the unitary inward conductances of 402.8 ± 8 pS, 297 ± 13 pS, 395.3 ± 6.1 pS, and outward conductances of 400 ± 8.0 pS, 366.8 ± 5.0 pS, and 409.0 ± 6.0 pS for control, PEG 200 and PEG 4000, respectively.

FIGURE 4

Effects of PEGs added from the extracellular side or both sides of the patch on single maxi-anion channel currents. (A) Representative single-channel events recorded when PEG 200 was added only to the pipette solution (left panel) and to both the pipette and bath solutions (middle panel), and when PEG 4000 was added to both the pipette and bath solutions (right panel). (B) Current-voltage relationships for the maxi-anion channel with PEG 200 present only in the pipette (left panel) and in both the pipette and bath (middle panel), and PEG 4000 present in both the pipette and the bath solution (right panel). Each symbol represents the mean of 5–19 observations obtained from at least five different patches. Solid lines are linear fits with slopes corresponding to the unitary inward conductances of 327.0 ± 9.7 pS, 227.5 ± 4.9 pS, and 404.5 ± 3.2 pS, and the outward conductances of 257.7 ± 5.8 pS, 216.8 ± 7.1 pS, and 395 ± 11 pS in the left, middle, and right panels, respectively.

FIGURE 5

Effects of PEG 200 and PEG 4000 on unitary maxi-anion channel conductances and bulk solution conductivities. (A) Pore conductance changes by addition of PEG 200 and PEG 4000 from the inside, outside, and both sides of membrane patches. Open bars represent the inward conductance, and hatched bars the outward conductance. Asterisk symbol means significantly different from the control at p < 0.05. (B) Changes in bulk conductivity by addition of PEG 200 and PEG 4000. The electrical conductivity of control Ringer solution (control) was 15.24 ± 0.03 mS/cm (n = 5). Asterisk symbol means significantly different from the control at p < 0.05.

FIGURE 6

Relative changes in unitary maxi-anion single-channel conductances as a function of hydrodynamic radii (R) of nonelectrolyte polymer molecules upon one-sided application of polymer. Open circles represent the inward conductance of channel when polymers were added only to the bath solution. The ascending straight line on the left is a linear fit to the data points for PEG 200–PEG 1540. The point of the intersection with the plateau level is at R = 1.16 nm. Solid circles represent the outward conductance of channel when polymers were added only to the pipette solution; the ascending straight line on the right is a linear fit to the data points for PEG 1000–PEG 2000 and crosses the plateau level at R = 1.42 nm. EG represents ethylene glycol, and Gl is glycerol. Mean single-channel amplitudes at different voltages (n = 5–20 from at least five different patches) were used to construct I–V relationships and calculate slope conductances. Error bars represent standard errors of the linear fits.

FIGURE 7

Relative changes in unitary maxi-anion single-channel conductances as a function of the hydrodynamic radii (R) of nonelectrolyte polymer molecules applied from both sides of membrane patches. The ascending straight line for channel inward conductances (open circles) is a linear fit to the data points for PEG 300–PEG 1540 and crosses the plateau level at R = 1.28 nm. The ascending straight line for outward conductances (solid circles) is a linear fit to the data points for PEG 600–PEG 2000 and crosses the plateau level at R = 1.34 nm. Relative decreases in the bulk conductivity of the polymer-containing control Ringer solutions used in these experiments (open triangles) are also plotted. Mean single-channel amplitudes at different voltages (n = 5–20 from at least five different patches) were used to construct I–V relationships and calculate slope conductances. Error bars represent standard errors of the linear fits.

FIGURE 8

Partitioning of PEGs into the maxi-anion channel pore. Partition coefficients for inward (A) and outward (B) unitary conductances were calculated according to Eq. 9 and plotted as a function of the polymer hydrodynamic radius (R). The solid descending line for the inward conductance is a linear fit to the data points for PEG 300–PEG 1540. The point of intersection with the lower plateau is at R = 1.29 nm. The solid straight descending line for the outward conductance is a linear fit to the data points for PEG 600–PEG 2000. The crossing point with the lower plateau is at R = 1.38 nm. The dashed lines are fits to Eq. 10 with R_p = 0.97 ± 0.02 nm and α = 3.07 ± 0.17 for the inward conductance and R_p = 1.06 ± 0.03 nm and α = 3.27 ± 0.33 for the outward conductance (see text for details).