Our previous studies using renal inner medullary collecting duct cells (IMCD-3 cell-line) have identified the presence of a large spontaneously active apical Cl^– conductance (Stewart et al. 2001) that is increased upon elevation of intracellular calcium ([Ca²⁺]_i) by kinins, and external ATP (Stewart et al. 2001). In order to more directly confirm the regulation and sensitivity of the IMCD anion conductance to [Ca²⁺]_i, fast whole cell patch clamp experiments were conducted with a pipette solution (pH 7.2), containing (mM) TEA Cl (110), MgCl₂ (2), ATP (1) and EGTA (5) in which CaCl₂ was added to give buffered free [Ca²⁺]_i values of 10, 300 and 1000 nM. Cells were initially bathed in a Na⁺-rich Krebs solution (2.8 mM Ca²⁺) and voltage clamped using a 10 s cycle with the holding potential (V_hold) at 0 mV with 500 ms excursions to ± 60 mV.

At 10 nM [Ca²⁺]_i, initial (1.5 min after establishing whole-cell) recordings demonstrated an outwardly rectifying current (current densities ± 60 mV, 59 ± 13 pA pF^-1 -32 ± 8 pA pF^-1 (means ± S.E.M.), n = 11 respectively) with an E_rev of -8.1 ± 0.6 mV, close to the estimated Cl^– equilibrium potential of -8.7 mV). At 5 min mean current densities had declined to 31 ± 8 and -21 ± 5 pA pF^-1 at ± 60 mV, n = 9), whilst E_rev was unchanged (-8.6 ± 0.3 mV). Replacement of 100 mM bath NaCl by 100 mM sodium aspartate at this time gave a positive shift in E_rev of 15.1 ± 1.9 mV (n = 4). With increased [Ca²⁺]_i, initial current densities in the Na⁺-rich bath solution were elevated compared to 10 nM (124 ± 30 and -73 ± 17 pA pF^-1 at ± 60 mV (300 nM) and 92 ± 26 and -60 ± 17 pA pF^-1 at ± 60 mV (1000 nM)) with E_rev values of -5.1 ± 0.6 and -4.5 ± 0.8 mV, respectively (n = 10 for both data sets). In contrast to the decline in current densities seen at 10 nM Ca²⁺, for elevated [Ca²⁺]_i mean current densities were maintained (at + 60 mV, 121 ± 66 and 93 ± 23 pA pF^-1 for 300 and 1000 nM, respectively, n = 10). Replacement of bath NaCl by N-methyl-D-glucamine-Cl did not significantly alter current densities. Upon replacement of bath 100 mM N-methyl D-glucamine-Cl isosmotically by mannitol positive shifts in E_rev of 16.7 ± 1.5 mV (n = 5) and 17.6 ± 1.3 mV (n = 7), respectively, were observed.

Finally we investigated the behaviour of EGTA buffer capacity upon whole cell currents at a fixed [Ca²⁺]_i of 10 nM. Initial (1.5 min) current densities in a Na⁺-rich bath were inversely correlated with EGTA concentration. At +60 mV these were, 91 ± 16, 76 ± 11 and 59 ± 3 pA pF^-1 for 0.1, 2 and 5 mM EGTA, respectively (n = 8-11). Furthermore current density for 0.1 mM EGTA was elevated compared to 5.0 mM EGTA at 5 min (at +60 mV, 72 ± 28 and 31 ± 8 pA pF^-1, respectively).

The maintenance of whole-cell Cl^– currents at low (10 nM) buffered [Ca²⁺]_i but clear dependence upon EGTA buffer capacity suggests a model in which the Cl^– conductance is responsive to a raised local [Ca²⁺] possibly via a Ca²⁺ influx pathway at the apical plasma membrane of IMCD cells.

This work was supported by an NKRF studentship award to J.L.