The mechanosensitive BKα/β1 channel localizes to cilia of principal cells in rabbit cortical collecting duct (CCD)

Abstract

Within the CCD of the distal nephron of the rabbit, the BK (maxi K) channel mediates Ca²⁺- and/or stretch-dependent flow-induced K⁺ secretion (FIKS) and contributes to K⁺ adaptation in response to dietary K⁺ loading. An unresolved question is whether BK channels in intercalated cells (ICs) and/or principal cells (PCs) in the CCD mediate these K⁺ secretory processes. In support of a role for ICs in FIKS is the higher density of immunoreactive apical BKα (pore-forming subunit) and functional BK channel activity than detected in PCs, and an increase in IC BKα expression in response to a high-K⁺ diet. PCs possess a single apical cilium which has been proposed to serve as a mechanosensor; direct manipulation of cilia leads to increases in cell Ca²⁺ concentration, albeit of nonciliary origin. Immunoperfusion of isolated and fixed CCDs isolated from control K⁺-fed rabbits with channel subunit-specific antibodies revealed colocalization of immunodetectable BKα- and β1-subunits in cilia as well as on the apical membrane of cilia-expressing PCs. Ciliary BK channels were more easily detected in rabbits fed a low-K⁺ vs. high-K⁺ diet. Single-channel recordings of cilia revealed K⁺ channels with conductance and kinetics typical of the BK channel. The observations that 1) FIKS was preserved but 2) the high-amplitude Ca²⁺ peak elicited by flow was reduced in microperfused CCDs subject to pharmacological deciliation suggest that cilia BK channels do not contribute to K⁺ secretion in this segment, but that cilia serve as modulators of cell signaling.

Keywords: Ca2+ channel; Ca2+ signaling; dietary K+ adaptation; flow-induced K+ secretion; intercalated cell; maxi K channel.

Fig. 1.

Immunodetectable maxi K channel subunit (BKα) is expressed in cilia in fixed microperfused rabbit cortical collecting duct (CCD): 3D reconstructions of confocal images. Top: CCD immunolabeled with anti-BKα antibody (Ab) and appropriate 2° Ab (A) and stained with 4′-6-diamidino-2-phenylindole (DAPI; B), viewed en face, with merged image shown in C. Middle: same CCD as in A–C, immunolabeled for BKα (D) and stained with DAPI (E), viewed down the internal axis of the tubule, with merged image in F. Bottom, negative control: CCD immunolabeled with anti-BKα Ab preadsorbed with the control peptide (G) and stained with DAPI (H), viewed en face, with merged image shown in I. Cilia are identified by arrows in A, C, D, and F. Similar patterns of expression were observed in at least 10 rabbit CCDs.

Fig. 2.

Immunodetectable BKα colocalizes with polycystin 2 (PC2) in cilia in principal cells in microperfused rabbit CCD. Anti-BKα Ab (arrows; red, middle column) colocalizes with anti-PC2 Ab (arrows; artificial green in A) in organelles that label with acetylated (Ac) α-tubulin (arrows; green in B) in aquaporin-2 (AQP2)-positive (*; green in C) cells, as demonstrated by merged images in right column. Note apical membrane colocalization of anti-BKα Ab with immunoreactive PC2 and AQP2 (A and C, merged images). Bar = 10 µm. Similar patterns of expression were observed in at least 4 CCDs labeled with each Ab set.

Fig. 3.

Immunodetectable BKα colocalizes with β1-subunit in cilia in principal cells in microperfused rabbit CCD. Anti-BKα Ab (red, left) in cilia (arrows) colocalizes with the immunoreactive β1-subunit in cilia as well as in the apical membrane of principal cells (top, middle, and right) but not with the β4-subunit, which appears diffusely distributed throughout the cytoplasm of ciliated principal cells (bottom, middle, and right). Similar patterns of expression were observed in 4 CCDs labeled with each Ab set.

Fig. 4.

Effect of dietary K⁺ on BKα expression in immunoperfused rabbit CCDs. Representative 3D reconstructions of CCDs, isolated from low-K⁺ (LK; top)- and high-K⁺ (HK; bottom)-fed animals, fixed and immunoperfused with Abs directed against pendrin (green, left) and BKα (red, middle). In CCDs from LK-fed animals, immunoreactive BKα was observed in principal cell cilia (arrows) but barely detected in pendrin (+) and thus β-type intercalated cells (open arrowheads). In HK-fed rabbits, apical BKα expression was clearly apparent in both pendrin (+) intercalated and ciliated principal cells. Similar observations were made in 3 additional HK- and LK-fed animals.

Fig. 5.

Effect of dietary K⁺ on subcellular localization of BKα in rabbit CCD. Representative confocal images of principal (PC) and intercalated (IC) cells from CCDs isolated from LK (n = 4)- and HK (n = 4)-fed rabbits and immunoperfused with Abs directed against BKα (red in A and B) and pendrin (green in A) or acetylated α-tubulin (Ac α-tubulin; green in B) are shown. A: fluorescence intensity of BKα in the apical+subapical region (delineated by yellow dots, left) relative to that in the same whole cell (white dots) for individually identified cells in the tubular wall was calculated and averaged for cell types (ciliated PC, pendrin (+) IC) and diet; averages were normalized to cell-specific values obtained in LK-fed animals. This analysis (right) revealed a relative increase in apical+subapical BKα distribution in response to dietary K⁺ loading for both PC and IC, but the increase [(Δ(HK − LK)] in IC was greater than that detected in PC. B: cilia labeled with Abs directed against BKα and Ac α-tubulin were outlined for analysis. The ratios of BKα/Ac α-tubulin fluorescence intensity signals were calculated for each cilium, and ratios were averaged for LK and HK diets. There was no significant difference in the relative cilia BKα expression between the 2 diets (right). Values are means ± SE; n = number of cells or cilia. *P = 0.017. #P ≤ 0.002.

Fig. 6.

Effect of dietary K⁺ on cilia length in rabbit CCDs. Cilia length, measured as described in methods, was ~2.5 times longer in LK-fed rabbits (4.2 ± 1.4 µm in 57 cilia in 4 CCDs) than in HK-fed animals (1.8 ± 0.5 µm in 116 cilia in 4 CCDs). Values are means ± SE. *P ≤ 0.001.

Fig. 7.

Cilia possess functional high-conductance K⁺ channels. Single-channel recordings were performed in cilia, identified by their green fluorescence (A, arrows), in Ssr3::GFP mpkCCD cells (gift from B. Yoder, U. of Alabama Hepato/Renal Fibrocystic Disease Core Center). Channel activity was recorded with 140 mM KCl in the pipette and 140 mM NaCl/5 mM KCl in bath. The channel closed state (C) is indicated by a dashed line in B, a representative recording of a cilia-attached patch at 0 mV. Channel openings are shown as downward deflections. The conductance of this channel was >200 pS. C: prevalence of conducting channels (number of patches with high-conductance K⁺ channels/total number of patches × 100%) was greater in cilia (9/62; 14.5%) than in the associated apical membrane (3/59; 5.1%) of these cells.

Fig. 8.

Effect of deciliation on CCD BK channel expression and structural integrity in microperfused rabbit CCDs. Confocal images of perfused CCDs fixed and labeled with anti-BKα Ab (red) and DAPI (blue) after treatment with luminal vehicle (A and C) or 1 mM dibucaine × 30 min (B and D) are shown. Dibucaine led to complete loss of cilia, although apical immunoreactivity for BKα was retained (B). Dibucaine did not disrupt F-actin cytoskeletal integrity, as evidenced by similar phalloidin (E and F) and FITC-DBA (G and H) staining in control and experimental CCDs.

Fig. 9.

Effect of deciliation on CCD BK channel function in microperfused rabbit CCDs. A 5-fold increase in luminal flow rate in microperfused rabbit CCDs from ~1 (slow; black bars) to 5 (fast; grey bars) nl·min^–1·mm^–1 led to a significant increase in J_Na (left) and J_K (right) in control (n = 4) CCDs. Flow-stimulated J_Na and J_K (FIKS) were similar in control and deciliated (n = 5) CCDs (P > 0.1). Values are means ± SE. *P < 0.05 compared with J_x at 1 nl·min^–1·mm^–1 in same tubules.

Fig. 10.

Effect of deciliation on the flow-induced increase in fura 2 fluorescence intensity ratio (FIR), a measure of intracellular Ca²⁺ concentration ([Ca²⁺]_i), in microperfused CCDs. Summary tracings of the effect of an acute increase in flow rate on the normalized fura 2 FIR in bright (presumably intercalated) and dull (principal) cells in control (vehicle-treated) and deciliated (dibucaine-treated) CCDs loaded with the Ca²⁺ indicator are shown. In control CCDs, an acute increase in luminal flow rate led to a typical biphasic response including an immediate rapid increase in FIR to a peak value within ~10 s, presumably secondary to release of Ca²⁺ from internal stores, followed by a gradual decay to a plateau [Ca²⁺]_i value that significantly exceeds baseline for at least 10 min of sustained high flow. The latter plateau elevation in FIR is believed to represent mechano-induced Ca²⁺ influx into cells. Treatment of CCDs with dibucaine eliminated the immediate peak response but not the plateau elevation in FIR. Values are means ± SE; n = 3 CCDs/group. *P ≤ 0.001 compared with baseline FIR in that subset of cells.