Fig 2: Functional validation of KCNN4 mRNA copy number changes in detected human pyramidal cells. Waveforms of action potentials evoked by depolarizing current injections (a) and of extracellularly evoked EPSPs (b) responded differently to the serial application of the small- and intermediate-conductance calcium activated potassium channel activator NS309 (500 nM) and TRAM34 (1 µM), an inhibitor of intermediate-conductance calcium activated potassium channels. The descending phase of action potentials and EPSPs was shortened in pyramidal cells recorded in brain slices prepared from the Edema group, but remained unchanged in pyramidal neurons of the Control group and in fast spiking interneurons of the Edema group. Traces shown are population averages. Confocal images of immunoreactions with antibodies against Kcnn4 performed simultaneously on samples of the Control and Edema groups showing a cross section of the gray matter (c) and part of layer 3 similar to areas where electrophysiological experiments were performed (d). Pyramidal cells were not labeled in the Control group and moderate Kcnn4 positivity was detected in pyramidal cells (p) of the Edema group. In addition, intense immunolabeling for Kcnn4 was detected in glial cells resembling astrocytes and interlaminar glia in both groups of patients

Fig 3: 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 4: The effect of TRPV4 activation and KCa channel blockers on transepithelial electrical resistance (TEER) of mCCDcl1 cells grown to confluency on permeable supports (see Materials and Methods).Addition of GSK101 or the blockers is indicated by the length of the “bar” below each symbol in the individual tracings. A. Representative time course of TEER measurements under basal conditions showing stable TEER values over the time course of the experiments. B. Representative example of the effect of adding GSK101 to the outside basolateral compartment (Basolateral GSK) or to the luminal (apical) compartment (Luminal GSK). GSK101 had no apparent affect when added to the basolateral media, but showed a consistent decrease in TEER (reflecting activation of TRPV4 and other channels) when added to the luminal compartment. C. Example showing the effect of addition of the cocktail of KCa blockers (as in Fig 8) on the GSK101-induced reduction in TEER. Addition of the blockers lead to an increase in TEER levels, results consistent with inhibition of [Ca2+]i activated KCa channels. D. Representative example showing the effect of SK inhibition (Apamin) on the GSK101-activated TEER values. E. Representative example showing the effect of BK inhibition (IbTX) on the GSK101-activated TEER values. F. Representative example showing the effect of IK1 inhibition (TRAM-34) on the GSK101-activated TEER values. G. Representative example showing the effect of ROMK inhibition (TQ) on the GSK101-activated TEER values. H. Summary results showing the effect of GSK101 activation of TRPV4 on TEER values (GSK, luminal compartment) and the subsequent individual KCa channel blockers (Luminal Blockers) on the TRPV4-activated TEER measurements. Each blocker (TRAM-34, Apamin, IbTX, TQ) or a cocktail (4X Blockers) of KCa channel blockers showed an increase in TEER. The decrease in TEER upon addition of GSK101 averaged 0.74 ± 0.027 (n = 3). The average increase in the relative TEER for the blockers in the presence of GSK101 was 0.17 ± 0.03 for the 4 Blockers, 0.08 ± 0.02 (n = 3) for TRAM-34, 0.09 ± 0.02 (n = 3) for Apamin, 0.09 ± 0.02 (n = 3) for IbTX, and 0.09 ± 0.02 (n = 3) TQ. Although the response to the 4 Blockers appears much larger, statistically there was no significant difference in the responses among the blocker sets.

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).