Fig 2: Differential mRNA expression levels of ion channels in mouse kidney.A. qPCR anlysis of mRNA expression levels of marker channels relative to the AQP2 water channel. As in mCCDcl1 cells, the mechanosensitive TRPV4 channel mRNA levels and ROMK mRNA levels are relatively high while mRNA levels for a second mechanosensitve TRP channel, TRPV2, are very low. For comparison mRNA expression levels for all channels are also given, relative to AQP2 mRNA levels, in the accompanying Table 1. B. qPCR analysis of mRNA expression levels of KCa channels in mouse kidney relative to the ROMK channel. It shows that SK1, SK2, and SK3 are expressed at relative high mRNA levels while IK1 and BKa are expressed at moderate mRNA levels. C. mRNA expression of BK channel subunits relative to BKa mRNA levels. The results show high relative mRNA levels for BKß4, moderate mRNA levels for BKß2, and low mRNA levels for BKß4.

Fig 3: Immunohistochemical staining of SK3 for thick ascending limb tubules.Sagital section (5 µm) of WT mouse kidney showing staining for Tamm-Horsefall protein (THP, red), a marker of TAL cells, and SK3 (green). Panels A, C, E, and G are low magnification images showing THP staining of TAL structrues (A), SK3 labeling of the same structures (C), and a merged image (E). As shown at higher resolution for one of the tubules (inset from A), THP strongly stains the luminal border of the TAL (B and F) with SK3 also showing strong labeling of the luminal border and, to a variable degree, the abluminal border (D and F). The merged image (F) clearly identifies SK3 staining in the TAL cells. Panel H is a magnified view of a proximal tubule (PT), located left of the TAL in A. The PT showed minimal staining for SK3, although light staining was apparent along the luminal brush border. Scale bar is 10 µm.

Fig 4: Effect of TRPV4-mediated activation of SK3 channels on membrane potential, Vm. A. Fluorescence image of a split-open CCD loaded with the voltage-sensitive fluorescence dye, DiSBAC2(3), showing loading of all cells. The fluorescence intensity is an index of Vm and is presented as relative fluorescence units (RFU). B. Effect of 50 mM K+ (High K+) application on Vm of CCD cells showing the expected membrane depolarization (increased RFU). C. Effect of 300 nM apamin or 50 nM IbTX application on Vm in basal conditions showing little or no effect of either apamin (Apa) or IbTX in the basal state (TRPV4 not activated). D. Effect of TRPV4 activation with GSK101 (50 nM) leading to membrane hyperpolarization of Vm (decreased RFU), as expected for SK3 and BK activation. Subsequent application of either 300 nM apamin or 50 nM IbTX now induce a marked depolarization of Vm (increased RFU) demonstrating inhibition of SK3 and BK, respectively. E. Summary graph showing mean changes in Vm in basal conditions upon addition of High K+ (High K+, n = 44 cells), 300 nM apamin, or 50 nM IbTX (Left panel, Basal). Right panel (GSK101: TRPV4 Activation) shows the results after activation of TRPV4 (Ca2+ influx). Both apamin and IbTX now bring about a significant depolarization of Vm (*P<0.01 compared to Basal). The combine addition of both apamin and IbTX (Apa + IbTX) displays an enhanced depolarization compared to addition of apamin or IbTX alone (**P<0.01). The number in parentheses is the number of cells for each group (n).

Fig 5: Differential expression of ion channels between principal cells (PC) and intercalated cells (IC) in the mouse kidney CCD.AQP2 staining was used to identify PC, the AQP2-positive cells, and IC, the APQ2-negative cells. Bar graphs give the mean ± SEM for the normalized intensities for each KCa channel for both PC and IC. The number of PC and IC cells analyzed, n, is indicated on the bar graphs. The images of CCD show representative immunofluorescence examples for each KCa channel and AQP2 (40X) where one or two PC (AQP2-positive) and IC (AQP2-negative) cells are labelled as “PC” or “IC.” The outer border of the tubule in each image is indicated by the dashed white line (indicating the basal side or anti-luminal side of tubular cells). “L” identifies the tubular lumen. The immunofluorescence intensity was determined for each KCa channel using ImageJ and normalized to the intensity levels of AQP2 expression in PC (see Methods). Each example gives staining for AQP2 (red), the KCa channel (green), and the merged image (AQP2, KCa channel) that includes DAPI staining to identify nuclei. A. Expression of SK1 in PC and IC showing dominant relative expression in IC over PC (P<0.001). SK1 expression was apparent within the cytosol and along the luminal border of both PC and IC as apparent in the representative images. Light staining along the basolateral border is also apparent in some cells. B. Expression of SK3 in PC and IC showing relatively more dominant expression in PC over IC (P<0.001). SK3 expression was apparent within the cytosol with light expression along the basal aspect of some cells and more dominant expression along the luminal border of PC. C. Expression of IK1 in PC and IC showing relatively more dominant expression in PC over IC (P<0.001). IK1 expression was apparent within the cytosol, especially of IC, but also apparent along the luminal border of PC with light staining along the basal aspect of the cells. D. Expression of BKa in PC and IC showing strong staining along the luminal border of both PC and IC. However, the intensity of staining between PC and IC was found to vary depending on the primary antibody used for the immunostaining. In our hands, the Alomone APC-151 BKa antibody staining was most apparent in PC over IC (P<0.001, compare bar graph labeled APC-151 with APC-107). In contrast, the Alomone APC-107 BKa antibody staining was most apparent in IC over PC. Since the antibodies were made to different epitopes of the BKa channel, such variations can be anticipated (see text).