Amiloride-sensitive sodium channels in confluent M-1 mouse cortical collecting duct cells

Abstract

Confluent M-1 cells show electrogenic Na⁺ absorption and possess an amiloride-sensitive Na⁺-conductance (Korbmacher et al., J. Gen. Physiol. 102:761–793, 1993). In the present study, we further characterized this conductance and identified the underlying single channels using conventional patch clamp technique. Moreover, we isolated poly(A)⁺ RNA from M-1 cells to express the channels in Xenopus laevis oocytes, and to check for the presence of transcripts related to the epithelial Na⁺ channel recently cloned from rat colon (Canessa et al., Nature 361:467–470, 1993).

Patch clamp experiments were performed in 6–13-day-old confluent M-1 cells at 37°C. In whole-cell experiments application of 10−5 m amiloride caused a hyperpolarization of 24.9, sem±2.2 mV (n = 35) and a reduction of the inward current by 107±10 pA (n = 51) at a holding potential of -60 mV. Complete removal of bath Na⁺ had similar effects, indicating that the amiloride-sensitive component of the inward current is a Na⁺ current. The effect of amiloride was concentration-dependent with half-inhibition at 0.22 μm. The Na⁺ current saturated with increasing extracellular Na⁺ concentrations with an apparent K _m of 24 mm. Na⁺ replacement for Li⁺ demonstrated a higher apical membrane conductance for Li⁺ than for Na⁺. In excised inside-out (i/o) or outside-out (o/o) patches from the apical membrane, we observed single-channels which showed slow kinetics and were reversibly inhibited by amiloride. Their average conductance for Na⁺ was 6.8±0.5 pS (n = 15) and for Li⁺ 11.2±1.0 pS (n = 14). They had no measurable conductance for K⁺. In o/o patches, channel activity was slightly voltage dependent with an open probability (NP _o ) of 0.46±0.14 and 0.16±0.05 at a holding potential of -100 and 0 mV, respectively (n = 8, P<0.05).

Using the two-microelectrode voltage-clamp technique, we assayed defolliculated stage V–VI Xenopus oocytes for an amiloride-sensitive inward current 1–6 days after injection with H₂O or with 20–50 ng of M-1 poly(A)⁺ RNA. In poly(A)⁺ RNA-injected oocytes held at -60 or -100 mV application of amiloride (2 μm) reduced the Na-inward current by 25.5±4.6 nA (n = 25) while it had no effect in H₂O-injected oocytes (n = 19). Northern blot analysis of M-1 poly(A⁺) RNA revealed the presence of transcripts related to the three known subunits of the rat colon Na⁺ channel (Canessa et al., Nature 367:463–467, 1994).

We conclude that the channel in M-1 cells is closely related to the amiloride-sensitive epithelial Na⁺ channel in the rat colon and that the M-1 cell line provides a useful tool to investigate the biophysical and molecular properties of the corresponding channel in the cortical collecting duct.