The epithelial potassium channel Kir7.1 is stimulated by progesterone

Abstract

The choroid plexus (CP) epithelium secretes cerebrospinal fluid and plays an important role in healthy homeostasis of the brain. CP function can be influenced by sex steroid hormones; however, the precise molecular mechanism of such regulation is not well understood. Here, using whole-cell patch-clamp recordings from male and female murine CP cells, we show that application of progesterone resulted in specific and strong potentiation of the inwardly rectifying potassium channel Kir7.1, an essential protein that is expressed in CP and is required for survival. The potentiation was progesterone specific and independent of other known progesterone receptors expressed in CP. This effect was recapitulated with recombinant Kir7.1, as well as with endogenous Kir7.1 expressed in the retinal pigment epithelium. Current-clamp studies further showed a progesterone-induced hyperpolarization of CP cells. Our results provide evidence of a progesterone-driven control of tissues in which Kir7.1 is present.

Figure 1.

Potentiation of an inwardly rectifying ion conductance by progesterone in murine CPECs. Whole-cell recordings from both male and female CPECs show a significant potentiation of an inwardly rectifying ion current upon exposure to 10 µM P4 applied via bath solution. (A) Representative traces from isolated lateral CPECs elicited in response to a voltage ramp, as indicated. P4 was administered three times with subsequent washout between readministrations. (B) Average fold increase of the inward current recorded at −80 mV from 6 female and 14 male CPECs after subsequent P4 applications, recorded from ramps shown in A. Statistical significance was calculated using the paired t test, and the significance of changes is indicated as follows: *, P ≤ 0.05. (C) Representative inward current amplitudes obtained at −80 mV from the experiments shown in A and plotted against time show a prolonged response to P4 in both male and female CPECs. The time of applications of 10 µM P4 to the bath solution is indicated by bars above. Bottom right: A differential interference contrast image of a typical CPEC with brush border indicated by the arrowhead. Top right: CPECs attached to a recording pipette. Cartoon depicts ion composition of the pipette and bath solutions. Data are means ± SEM.

Figure S1.

The relative gene expression of the lateral CPs isolated from Abhd2^+/+ and Abhd2^−/− male and female mice. Number of mRNA-sequencing reads from corresponding CPs mapped to the mouse genome. Abhd2 and Kcnj13 expression levels are shown for wild-type (A) and knockout (B). Red dotted lines represent an expected number of sequencing reads for genes with similar expression levels between two samples. Signals <10 reads are within statistical noise and therefore scored as nonexpressed sequences. Data are averaged from triplicate experiments for each genotype and sex.

Figure 2.

Expression and permeability of Kir7.1 from murine CP. (A) A reduction in current density was observed at −80 mV after applying 20 µM of the Kir7.1 and Kir1.1 inhibitor VU590 to CPECs. VU590 prevented any potentiation of the current by P4. (B) Schematic image showing localization of the lateral ventricle in the mouse brain. (C) Immunohistochemical image from a C57BL/6N adult male showing that Kir7.1 localized to the apical side of the lateral ventricle CP (1). (D) Kir7.1 is also detected in the plasma membrane of CPECs that have been isolated from an adult female mouse and cultured for 3 h. (E and F) Representative I-V relationships (E) extracted from representative traces (F) of Kir7.1 currents recorded in whole-cell mode from lateral CPECs in response to a voltage-step protocol (from −120 mV to +40 mV, in 20-mV increments, starting from a holding potential of −40 mV) as shown. Exposing CPECs to different bath solutions shows large conductance when the cells are in a Rb⁺-based buffer, while Cs⁺- and K⁺-based buffers showed smaller but similar conductances. The Na⁺-based bath solution did not elicit any current. Insert shows the shift in reverse potentials upon changes in the bath ion composition. (G) Current densities from three CPECs exposed to the different bath solutions at −80 mV, as recorded in E. Data are means ± SEM.

Figure 3.

Potentiation of Kir7.1 by steroid hormones. (A) Representative traces recorded from female murine CPECs isolated from the lateral ventricle. A gradual increase in current was observed after applying increasing concentrations of P4 to the bath solution. (B) Half-maximal effective concentration (EC₅₀) for P4 was calculated using the average current density at −80 mV for 12 cells isolated from CPECs, as mentioned in A. (C and D) Representative traces (C) and the average current fold increase (D) of Kir7.1 at −80 mV recorded from five CPECs that were exposed to 10 µM testosterone (T), estradiol (E2), PS, and P4. Three CPEC were assessed on their modulation by 10 µM levonorgestrel (LNG). The chemical structure of the different steroids used for patch-clamp recordings is shown. The steroids were applied in different orders for each cell, with P4 always added last. (E) A representative current-clamp measurement, which shows hyperpolarization of a CPEC after applying 10 µM P4 (red) to the bath buffer. When blocking Kir7.1 activity with 100 µM VU590 a significant cellular depolarization was detected. (F) Average changes in membrane potential (mV) as measured by current-clamp recordings of three cells when applying 10 µM P4 (red bar, circles) or 100 µM VU590 + 10 µM P4 (gray bar, squares). Data are means ± SEM.

Figure S2.

Expression of Abhd2 in murine CPs. (A, B, and F) ISH shows strong expression of Abhd2 in the lateral (A, solid arrow; and B), third (A, open arrow), and fourth ventricle CP (F) of Abhd2^+/+ mice. (D, E, H, and I) The adult Abhd2^−/− mice display similar background staining when using either the antisense (D and H) or the sense (E and I) probe against Abhd2. (J) Although the antisense probe failed to bind to Abhd2 mRNA in the Abhd2^−/− animals in ISH experiments, a product lacking either Abhd2 exon 6 or both exon 6 and exon 7 was detected in the kidney of Abhd2^−/− mice. However, this mRNA product would lead to a frameshift and results in a truncated nonfunctional form of the protein. (K, K′, and M) ABHD2 protein was detected by immunohistochemistry and Western blot in the CP of Abhd2^+/+ mice (K and M), with a localization on the apical side of the epithelium (K′). (L and L′) ABHD2 was not detected in CP of Abhd2^−/− mice (L′). (M) ABHD2 was not detected in the other regions of the brain, such as the olfactory bulb (OB), cortex (COR), hippocampus (HIP), and cerebellum (CER). However, it was exclusively found in both the lateral (LCP) and the fourth (4CP) ventricle CP of Abhd2^+/+ animals. Abhd2^−/− animals display a strong expression of Kir7.1 even in the absence of ABHD2. Actin was used as a loading control.

Figure 4.

Wild-type and Abhd2^−/− murine CPECs show similar potentiation of Kir7.1 by progesterone (P4). (A and B) Immunohistochemical staining of the lateral ventricle CP of ABHD2 in Abhd2^+/+ (A) and Abhd2^−/− (B) adult male mice. (C and D) Immunocytochemical staining of cultured CPECs shows the presence of Kir7.1 in cells of both genotypes (Abhd2^+/+ and Abhd2^−/−), although the latter lack ABHD2. (E) Representative recordings from adult male CPECs from Abhd2^−/− mice show potentiation of the Kir7.1 current similar to that of cells from Abhd2^+/+ animals when 10 µM P4 was added to the bath solution. Whole-cell recordings were performed using Cs⁺ bath and pipette solutions, as indicated. (F) The fold change of I_Kir7.1 at −80 mV was calculated from recordings of 6 female and 10 male wild-type cells and 8 female and 16 male Abhd2^−/− cells, as shown. Although the Abhd2 knockout female cells show a slightly smaller current fold increase, the difference is not statistically significant when compared with that of wild-type cells.

Figure 5.

Potentiation of Kir7.1 by progesterone in HEK293 and CPECs is independent of G protein–coupled receptor signaling. (A) Representative traces recorded from HEK293 cells transfected with a pIRES2-EGFP/Kir7.1 construct show a fivefold potentiation of the current after addition of 10 µM P4 to the bath. The cells transfected with the empty vector (upper panel) do not respond to P4 or display I_Kir7.1. (B) Representative I_Kir7.1 from an isolated adult female lateral CPEC is similarly activated by 10 µM P4 in the presence of 1 mM GDPβS in the pipette. (C) Immunohistochemical staining of recombinant Kir7.1 in HEK293 cells transfected with the pIRES2-eGFP/Kir7.1 construct. (D) Western blotting detects both the mature glycosylated and the shorter immature product of Kir7.1 seen in HEK293 cells transfected with the pIRES2-EGFP/Kir7.1 construct. The cells transfected with the empty vector do not express Kir7.1. Actin was used as a loading control. (E) Graph depicting the fold change in the inward I_Kir7.1 after exposure to 10 µM P4 recorded from transfected HEK293 cells and CPECs, with “e” representing endogenous Kir7.1 expression as in CPECs. Statistical significance was calculated using the paired t test and the significance of changes is indicated as follows: **, P ≤ 0.01. Data are means ± SEM.

Figure S3.

Cumulative current densities of recordings from HEK293, RPE, and CP cells. (A) Cumulative current densities recorded at −80 mV and +80 mV for all recordings shown in this study. Data are means ± SEM. Current densities recorded from HEK293 cells transfected with a recombinant Kir7.1 or with the empty vector, as well as from CPECs and RPE cells. Currents were recorded in the presence or absence of P4, GDPβS, or VU590 as indicated. HEK293 cells transfected with the empty vector were used as a control. (B) Whole-cell recordings from CPECs depict the presence of two ion channels, Kir7.1 and TRPM3. Representative traces from CPECs elicited in response to a voltage ramp from −80 mV to +80 mV from a holding potential of 0 mV. Under the condition where Kir7.1 activity is inhibited by 20 µM VU590, an additional conductance can be stimulated by an application of 50 µM PS (green), a TRPM3 channel agonist. Application of 10 µM P4 (red) in the absence of VU590 and PS results in Kir7.1 activation on the same CPEC. (C) Cumulative current densities recorded at −80 mV and +80 mV in the control condition, in the presence of VU590 and/or PS or P4. Data are means ± SEM.

Figure 6.

Potentiation of Kir7.1 by progesterone in RPE cells. (A) Representative whole-cell recordings from RPE cells in response to 10 µM P4. RPE was isolated from a 1-mo-old female mouse. A significant increase in the inward current density occurred at −80 mV, while the outward current recorded at +80 mV did not change. (B) Immunocytochemical staining of mouse RPE cells detects Kir7.1. (C) The fold change in the inward I_Kir7.1 after exposure to 10 µM P4 recorded from RPE cells. Statistical significance was calculated using the paired t test and the significance of changes is indicated as follows: **, P ≤ 0.01. Data are means ± SEM. (D) Representative inward current amplitudes obtained at −80 mV from the experiments shown in A and plotted against time show repetitive responses to P4 in female RPE cell. The time of applications of 10 µM P4 to the bath solution is indicated by red bars above. Addition of Kir7.1 inhibitor VU590 is shown by the black bar.