Apical nonspecific cation channels in everted collecting tubules of potassium-adapted ambystoma.

We observed intermediate conductance channels in approximately 20% of successful patch-clamp seals made on collecting tubules dissected from Ambystoma adapted to 50 mm potassium. These channels were rarely observed in collecting tubules taken from animals which were maintained in tap water. Potassium-adaptation either leads to an increase in the number of channels present or activates quiescent channels. In cell-attached patches the conductance averaged 30.3 +/- 2.4 (9) pS. Since replacement of the chloride in the patch pipette with gluconate did not change the conductance, the channel carries cations, not anions. Notably, channel activity was observed at both positive and negative pipette voltages. When the pipette was voltage clamped at 0 mV or positive voltages, the current was directed inward, consistent with the movement of sodium into the cell. The pipette voltage at which the polarity of the current reversed (movement of potassium into the pipette) was -29.6 +/- 6.5(9) mV. Open probability at 0 mV pipette voltage was 0.08 +/- 0.03 and was unaffected when the apical membrane was exposed to either 2 x 10(-6) or 2 x 10(-5) m of amiloride. Exposure of the basolateral surface of the tubule to a saline containing 15 mm potassium caused a significant increase (P less than 0.001) in the open probability of these channels to 0.139 +/- 0.002 without affecting the conductance of the apical channel. These data illustrate the presence of an intermediate conductance, poorly selective, amiloride-insensitive cation channel in native vertebrate collecting tubule. We postulate that, at least in amphibia, this channel may be used to secrete potassium.

Elevation of basolateral K+ induces K+ secretion by apical maxi K+ channels in Ambystoma collecting tubule.

We previously reported that exposure of aquatic-phase Ambystoma tigrinum to a solution containing 50 mM K+ (K+ adaptation) caused a nearly 10-fold increase in the number of detectable maxi K+ channels on the apical membrane of their initial collecting tubules. In apparent contradiction to the notion that maxi K+ channels contribute to K+ secretion, these channels were not routinely active at the resting membrane potential (0 mV voltage clamp). To test the possibility that hyperkalemia yields maxi K+ channels that are secreting K+ (i.e., active at 0 mV), we patch-clamped the apical membranes of initial collecting tubules under conditions of elevated basolateral K+ (15 mM). Seven patches containing maxi K+ channels were studied. Six of the seven patches showed maxi K+ channel activity when voltage was clamped at 0 mV. Open probability and unitary current averaged 0.059 +/- 0.016 and 1.65 +/- 0.50 pA, respectively. This activity, together with the high density of channels observed (1.06 channels/micrometer2), indicates that after K+ adaptation, maxi K+ channels contribute to the ability of the late distal nephron of amphibians to secrete K+.

Environmental KCl causes an upregulation of apical membrane maxi K and ENaC channels in everted Ambystoma collecting tubule.

Patch clamp methods were used to characterize the channels on the apical membrane of initial collecting ducts from Ambystoma tigrinum. Apical membranes were exposed by everting and perfusing fragments of the renal tubule in vitro. Tubules were dissected from two groups of animals; one maintained in tap water, and the other kept in a solution of 50 mM KCl from seven to nineteen days. Patches of apical membranes on tubules taken from animals exposed to tap water expressed low-conductance amiloride sensitive sodium channels (ENaC) in 22 of 49 patches. Only three maxi K channels were observed in this group. In animals exposed to KCl, low-conductance amiloride sensitive sodium channels, 3.7 +/- 0.2 pS (36 of 45 patches) and high-conductance 98.3 +/- 5.0 pS (19 of 45 patches) potassium channels were observed. The estimated density of apical maxi K channels increased dramatically from 0.08 to 0.76 channels/mu 2 in tubules taken from animals exposed to KCl. All but four of nineteen patches which contained maxi K channels also expressed the low conductance sodium channels. Therefore, at least 85% of the maxi K channels studied were in principal cells. We speculate that the increase in maxi K channel activity may represent a mechanism for enhancing the potassium secretory capacity of the initial collecting duct. As expected, exposure of the animals to 50 mM KCl prior to dissection of the initial collecting ducts also increased the estimated density of ENaC from 0.99 to 3.89 channels/mu 2. This upregulation of sodium channel activity is presumably related to the widely recognized effect of potassium loading to increase the plasma aldosterone level.

Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells.

We have previously demonstrated that apical Na+ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971-6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na+ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882-C888); however, it is unknown if the Na+ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na+ channels to the membrane cytoskeleton is a site for modulation of Na+ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na+ channels to the membrane cytoskeleton is a site for regulation of Na+ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na+ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na+ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na+ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 +/- 5.42%) when compared to control (unstimulated) (f = 14.2 +/- 0.77%) and vasopressin-treated (20 min) (f = 12.7 +/- 2.4%) monolayers. The twofold increase in mobile fraction of Na+ channels corresponds to the average increase in Na+ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na+ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na+ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na+ channels.

Amiloride-sensitive apical membrane sodium channels of everted Ambystoma collecting tubule.

Patch clamp methods were used to characterize sodium channels on the apical membrane of Ambystoma distal nephron. The apical membranes were exposed by everting and perfusing initial collecting tubules in vitro. In cell-attached patches, we observed channels whose mean inward unitary current averaged 0.39 +/- 0.05 pA (9 patches). The conductance of these channels was 4.3 +/- 0.2 pS. The unitary current approached zero at a pipette voltage of -92 mV. When clamped at the membrane potential the channel expressed a relatively high open probability (0.46). These characteristics, together with observation that doses of 0.5 to 2 microM amiloride reversibly inhibited the channel activity, are consistent with the presence of the high amiloride affinity, high sodium selectivity channel reported for rat cortical collecting tubule and cultured epithelial cell lines. We used antisodium channel antibodies to identify biochemically the epithelial sodium channels in the distal nephron of Ambystoma. Polyclonal antisodium channel antibodies generated against purified bovine renal, high amiloride affinity epithelial sodium channel specifically recognized 110, 57, and 55 kDa polypeptides in Ambystoma and localized the channels to the apical membrane of the distal nephron. A polyclonal antibody generated against a synthetic peptide corresponding to the C-terminus of Apx, a protein associated with the high amiloride affinity epithelial sodium channel expressed in A6 cells, specifically recognized a 170 kDa polypeptide. These data corroborate that the apically restricted sodium channels in Ambystoma are similar to the high amiloride affinity, sodium selective channels expressed in both A6 cells and the mammalian kidney.