An intracellular ATP-activated, calcium-permeable conductance on the basolateral membrane of single renal proximal tubule cells isolated from Rana temporaria

Abstract

1. The following study describes the properties of a non-selective cation channel, which has a unit conductance below the resolving power of the single channel technique, located on the basolateral membrane of single proximal tubule cells isolated from frog kidney. The conductance was examined using cell-attached, inside-out and outside-out patches. Due to the small single channel magnitude, macroscopic patch currents were measured. 2. Addition of 2 mM ATP to the intracellular surface of excised patches activated an outwardly rectifying conductance (MCANS): outward (Gout) and inward (Gin) conductances increased by 46.8 +/- 6.7 and 11.6 +/- 2.1 pS, respectively (n = 29). MCANS was more selective for cations than anions, with a cation:anion selectivity ratio of 10.1 +/- 1.7 (n = 7), but did not discriminate between Na+ and K+. It was more selective for Ca2+ over Na+, with a Ca2+:Na+ selectivity ratio of 4. 49 +/- 0.69 (n = 7). 3. In cell-attached patches addition of 100 microM strophanthidin to the bath increased both Gout and Gin. However this increase in conductance was absent in the presence of Gd3+, which inhibits MCANS. 4. These data suggest that single proximal tubule cells isolated from frog kidney contain an ATP-activated, non-selective cation conductance. The conductance does not discriminate between Na+ and K+, but is more selective for Ca2+ over Na+. Considering the prevailing electrochemical gradients for these ions, functional activation of the conductance would be expected to lead to a rise in intracellular Ca2+. MCANS is linked to the activity of the Na+, K+-ATPase and may therefore provide a link between the ATPase and K+ channel activity in the basolateral membrane and form an integral part of the pump-leak mechanism in transporting epithelia.

Figure 1. Effect of 2 mM ATP

A, excised inside-out patch currents taken from a typical recording in the absence (left) and presence (right) of 2 mm ATP. B, I–V curves (patch current plotted against minus pipette potential, -V_p) recorded from these traces (•, 0 ATP; ▪, 2 mm ATP).

Figure 2. Dose-response curve for ATP

The graph shows the mean current recorded in excised inside-out patches at a patch potential of +40 mV plotted against ATP concentration. Each data point is the mean of nine cells and the error bars show the s.e.m. The line though the data is the best fit to the Hill equation.

Figure 3. Estimation of single channel current

The graph shows the variance of the current recorded at +40 mV in excised inside-out patches against mean current. Data points were all taken from the same patch and the line through the data is the best fit to eqn (1).

Figure 4. Effect of Ba²⁺ and Gd³⁺ on the ATP-dependent activation of currents

All I–V curves show typical excised inside-out recordings obtained: top left, control circumstance; top right, presence of Gd³⁺; bottom left, presence of Ba²⁺ (▪, 0 ATP; •, ⋇ and ▴, 2 mM ATP). The bottom right plot shows the ATP-activated current taken from the three preceding I–V curves (^, control circumstance; ⋇, presence of Gd³⁺; ▵, presence of Ba²⁺).

Figure 5. Irreversibility of Gd³⁺ inhibition

Excised outside-out patch conductance under the control circumstance, in the presence of 100 μM Gd³⁺ and on washout of Gd³⁺ from the bath, in the absence of pipette ATP (A) and in the presence of pipette ATP (B). * Significant difference from the conductance recorded under the control circumstance (P < 0.05). There was no significant difference between the patch conductance recorded in the presence of Gd³⁺ and on washout of Gd³⁺ from the bath.

Figure 6. The cation:anion selectivity of patch current

I–V curves for recordings taken from typical excised inside-out patches. Currents were recorded in response to the dilution of bath NaCl in the absence of ATP (A) and in the presence of ATP (B). ▪, 100 mM NaCl; •, 20 mM NaCl.

Figure 7. The effect of strophanthidin on cell-attached patch current

I–V curves for recordings from two typical cell-attached patches in response to addition of 100 μM strophanthidin to the bath in the control circumstance (A) and with 100 μM Gd³⁺ in the pipette (B). ▪, control current; •, current recorded in the presence of strophanthidin.