Stimulation of Ca²⁺ -sensing receptor inhibits the basolateral 50-pS K channels in the thick ascending limb of rat kidney

Abstract

We used the patch-clamp technique to study the effect of changing the external Ca²⁺ on the basolateral 50-pS K channel in the thick ascending limb (TAL) of rat kidney. Increasing the external Ca²⁺ concentration from 1 mM to 2 or 3 mM inhibited the basolateral 50 −pS K channels while decreasing external Ca²⁺ to 10 μM increased the 50-pS K channel activity. The effect of the external Ca²⁺ on the 50-pS K channels was observed only in cell-attached patches but not in excised patches. Moreover, the inhibitory effect of increasing external Ca²⁺ on the 50-pS K channels was absent in the presence of NPS2390, an antagonist of Ca²⁺-sensing receptor (CaSR), suggesting that the inhibitory effect of the external Ca²⁺ was the result of stimulation of the CaSR. Application of the membrane-permeable cAMP analogue increased the 50-pS K channel activity but did not block the effect of raising the external Ca²⁺ on the K channels. Neither inhibition of phospholipase A2 (PLA₂) nor suppression of cytochrome P450-ω-hydroxylation-dependent metabolism of arachidonic acid was able to abolish the effect of raising the external Ca²⁺ on the 50-pS K channels. In contrast, inhibition of phospholipase C (PLC) or blocking protein kinase C (PKC) completely abolished the inhibition of the basolateral 50-pS K channels induced by raising the external Ca²⁺. We conclude that the external Ca²⁺ concentration plays an important role in the regulation of the basolateral K channel activity in the TAL and that the effect of the external Ca²⁺ is mediated by the CaSR which stimulates PLC-PKC pathways. The regulation of the basolateral K channels by the CaSR may be the mechanism by which extracellular Ca²⁺ level modulates the reabsorption of divalent cations.

1. Introduction

The TAL is responsible for reabsorption of 20-25% filtered Na load by a two-step process [1]: Na and Cl enter the cells through the apical type II Na/K/Cl cotransporter (NKCC2) [2] and leave the cells through basolateral Na-K-ATPase and basolateral Cl channels [3,4], respectively. Although Na and Cl transport across the apical membrane through NKCC2 are an electroneutral process, both apical K channels and basolateral K channels play an important role in the regulation of NKCC2 activity. While apical K channels are responsible for K recycling which is essential for maintaining NKCC2 activity [5], basolateral K channels determine the driving force for Cl diffusion across the basolateral membrane thereby indirectly affecting NKCC2 activity [6].

The CaSR is highly expressed in the TAL and plays an important role in the regulation of NaCl transport and NKCC2 activity in the TAL [7,8]. Gain-function-mutation of the CaSR has been reported to cause type V Bartter's syndrome, characterized by hypokalemia, hyperreninemia and hyperaldosteronism [9-11]. Our previous study has shown that activation of the CaSR by increasing the external Ca²⁺ inhibited the apical 70 pS channels [12] via stimulating PLA₂ in the TAL [13]. The inhibition of apical K channel activity and apical K recycling induced by stimulation of the CaSR is a possible mechanism by which the CaSR regulates NKCC2 activity in the TAL. However, it is also possible that the CaSR may modulate NKCC2 activity by regulating the basolateral K channel activity which could affect Cl movement in the TAL. The notion that the CaSR may regulate the basolateral K channel activity is also suggested by the report that the CaSR has been shown to interact with inwardly-rectifying K channels 4.1 (Kir. 4.1/KCNJ10), a possible component of the basolateral K channels in the TAL [14-16]. Therefore, the aims of this study are to examine the role of the external Ca²⁺ in the regulation of the basolateral 50-pS K channels in the TAL and to investigate the pathway by which the external Ca²⁺ regulates the K channels.

2. Material and methods

2.1. Preparation of the TALs

Sprague-Dawley rats of either sex (5-6 weeks) were purchased from the animal facility of the second affiliated hospital of Harbin medical university (Harbin, China). The animals were kept on a normal chow and free-access to water before use. Rats were killed by cervical dislocation (body weight is less than 90g), and the kidneys were removed immediately. The kidney was cut into 1mm thick slices with a razor blade and the kidney slices were incubated in an HEPES buffer solution containing collagenase type IA (1 mg/ml) (Sigma, St Louis, MO, USA) at 37°C for 45-60 minutes. After the collagenase treatment, the TALs were isolated under a dissecting microscope. The dissection buffer solution contained (in mM) 140 NaCl, 5 KCl, 1.8 MgCl_2, 1.8 CaCl_2, and 10 HEPES (pH=7.4). The isolated tubule was transferred onto a 5×5-mm cover glass coated with polylysine (Sigma) to immobilize the tubule. The cover glass was placed in a chamber mounted on an inverted microscope (Nikon, Japan), and the tubules were superfused with HEPES-buffered NaCl solutions.

2.2. Patch-clamp technique

We used an Axon 200B patch-clamp amplifier to record channel currents. The currents were low-pass filtered at 0.2 kHz and digitized by an Axon interface (Digidata 1322). Data were analyzed using the pClamp software system 9 (Axon Instruments, Burlingame, CA). Channel activity was defined as NP_o, a product of channel open probability (P_o) and channel number (N). The NP_o was calculated from data samples of 90-s duration in the steady state as follows:

NP_o=Σ (1t₁+2t₂+ ^… +it_i) where t_i is the fractional open time spent at each of the observed current levels.

2.3. Chemicals and experimental solution

The pipette solution contained (in mM) 140 KCl, 1.8 MgCl₂, and 10 HEPES (pH=7.4). The bath solution for cell-attached patches was composed of (in mM) 140 NaCl, 5 KCl, 1.0 CaCl_2, 1.8 MgCl₂, and 10 HEPES (pH=7.4). AACOCF3 (Arachidonyltrifluoromethyl Ketone), calphostin C, U73122, NPS2390, dibutyryl-cAMP (db-cAMP) and 17-octadecynoic acid (17-ODYA) were purchased from Sigma (St. Louis, MO).

2.4. Statistics

Data are shown as mean±SEM. We used t-tests to determine the significance of the difference between the control and experimental periods. If the P value is <0.05, the difference is considered to be significant.

3. Results

We and others previously demonstrated that the inwardly-rectifying 50-pS K channels are highly expressed in the TAL and that the 50-pS K channels might be a main type of the K channel in the basolateral membrane of the TAL [17,18]. Therefore, the present study focused on studying the regulation of the 50-pS K channel by the external Ca²⁺. To examine the role of the external Ca²⁺ in regulating the basolateral 50-pS K channels in the TAL, we analyzed the channel activity in cell-attached patches by changing the external Ca²⁺ (bath) from 1 mM (as a control value) to either a higher or a lower than 1 mM Ca²⁺. Figure 1 is a single-channel recording showing that increasing the external Ca²⁺ from 1 mM to 2 mM inhibited the 50-pS K channel activity and significantly decreased NP_o from 0.32±0.11 to 0.10±0.04 within 1 min ( n=8). The inhibitory effect of 2 mM Ca²⁺ on the channel activity was reversible because wash-out by 1 mM Ca²⁺-containing bath solution restored NP_o to 0.28±0.06 (N=8)within 1-2 min. Moreover, further increasing the external Ca²⁺ to 3 mM caused an additional inhibition of the basolateral K channels. Figure 2 is a channel recording demonstrating the effect of increasing external Ca²⁺ to 3 mM on the 50-pS K channel. It is apparent that raising the external Ca²⁺ from 1 mM to 3 mM almost completely blocked the 50-pS K channel and decreased NP_o from 0.37±0.15 to 0.02±0.01 (N=8). Again, the effect of 3 mM Ca²⁺ on the channel activity was reversible and the wash-out by 1mM Ca²⁺-containing solution increased NP_o to 0.34±0.08 (N=8). The inhibitory effect of Ca²⁺ on the 50-pS K channels was observed only in cell-attached patches but not in inside-out patches, suggesting that the effect of increasing the external Ca²⁺ on the 50-pS K channel was indirect. Moreover, raising external Ca²⁺ from 1 mM to 2 or 3 mM also decreased the current amplitude by 80±8% and 60±10%, respectively. This suggests that stimulation of CaSR depolarized the basolateral cell membrane potential.

Fig. 1.

A channel recording shows the effect of raising the external Ca²⁺ from 1 mM to 2 mM on the activity of the 50-pS K channel in the basolateral membrane of the TAL. The experiments were performed in cell-attached patches. The pipette solution contained (in mM) 140 KCl, 1.8 MgCl₂, and 10 HEPES (pH=7.4) and the bath solution was composed of (inmM) 140 NaCl, 5 KCl, 1.0 CaCl₂, 1.8 MgCl₂, and 10 HEPES (pH=7.4). Three parts of the trace indicated by numbers are extended to show the fast time resolution. The channel closed level is indicated by letter “C” and the holding potential was 0 mV. A bar graph in the bottom of the figure summarizes the results from 8 experiments.

Fig. 2.

A channel recording demonstrates the effect of raising external Ca²⁺ from 1 mM to 3 mM on the activity of the 50-pS K channel in the basolateral membrane of the TAL. The experiments were performed in cell-attached patches. Three parts of the trace indicated by numbers are extended to show the fast time resolution. The channel closed level is indicated by letter “C” and the holding potential was 0 mV. A bar graph in the bottom of the figure summarizes the results from 8 experiments.

While an increase in the external Ca²⁺ inhibited the 50-pS K channels in the TAL, a decrease in the external Ca²⁺ activated the basolateral K channels. Figure 3 is a recording showing that a decrease in the external Ca²⁺ from 1 mM to 10 μM Ca²⁺ significantly stimulated the 50-pS K channel in the TAL and increased the channel activity from 0.38±0.15 to 0.78±0.25 (N=8). Again the stimulatory effect of decreasing external Ca²⁺ was observed only in cell-attached patches.

Fig. 3.

A channel recording demonstrates the effect of decreasing the external Ca²⁺ from 1 mM to 10 μM on the activity of the 50-pS K channel in the basolateral membrane of the TAL. The experiments were performed in cell-attached patches. Two parts of the trace indicated by numbers are extended to show the fast time resolution. The channel closed level is indicated by letter “C” and the holding potential was 0 mV. A bar graph in the bottom of the figure summarizes the results from 8 experiments.

After demonstrating that the alteration of the external Ca²⁺ modulates the basolateral K channel activity, we suspect that the effect of the external Ca²⁺ on the basolateral 50-pS K channel is mediated by the CaSR which is expressed in the basolateral membrane of the TAL [19]. This hypothesis was tested by examining the effect of raising the external Ca²⁺ from 1 to 3mM on the basolateral 50-pS K channel in the presence of 5 μM NPS 2390, an agent which blocks the CaSR [20]. Application of NPS2390 slightly increased the basal level of the 50-pS K channel and the mean NP_o was 0.40±0.14 (N=6). Moreover, in the TAL treated with NPS2390, increasing the external Ca²⁺ failed to inhibit the 50 pS K channel (NP_o, 0.36±0.17). This suggests that the effect of increasing the external Ca²⁺ on the 50-pS K channel was the result of stimulation of the CaSR.

Stimulation of the CaSR in the TAL has been shown to activate PLA₂ thereby increasing release of arachidonic acid which inhibits the apical K channels in the TAL [12,13]. Therefore, we examined whether the inhibitory effect of increasing the external Ca²⁺ on the 50-pS K channels was also the results of activation of PLA₂. Figure 5 is a typical channel recording from 8 similar experiments in which the effect of 3 mM Ca²⁺on the 50-pS K channel was examined in the presence of the PLA₂ inhibitor, AACOCF3 [21]. From inspection of Fig. 5, it is apparent that the inhibition of PLA₂ failed to abolish the effect of raising the external Ca²⁺ on the 50-pS K channels in the TAL because 3 mM Ca²⁺ decreased NP_o from 0.47±0.11 to 0.08±0.05 in the presence of 5 μM AACOCF3 (N=8). Moreover, raising the external Ca²⁺ to 3 mM could still inhibit the 50-pS K channels in the presence of 17-ODYA (5 μM), an agent blocking cytochrome P450-ω-hydroxylation-dependent metabolism of arachidonic acid [22](Fig. 6). This suggests that the inhibitory effect of raising the external Ca²⁺ on the 50 pS K channels was not mediated by cytochrome P450-ω-hydroxylation-dependent metabolism of arachidonic acid.

Fig. 5.

A channel recording demonstrates the effect of raising external Ca²⁺ from 1 mM to 3 mM on the activity of the basolateral 50-pS K channel in the presence of or in the absence of AACOCF3 (5 μM), an inhibitor of phospholipase A2. The arrow indicates the change in the external Ca²⁺. The experiments were performed in cell-attached patches of the isolated TAL. Three parts of the trace indicated by numbers are extended to show the fast time resolution. The channel closed level is indicated by letter “C” and the holding potential was 0 mV.

Fig. 6.

A bar graph summarizing the experiments in which the effect of raising the external Ca²⁺ from 1 mM to 3 mM on basolateral 50-pS K channels in the TAL was examined under control conditions or in the presence of AACOCF3 or 17-ODYA (5 μM), respectively. The experiments were performed in cell-attached patches and the experimental numbers were at least 7.

The CaSR is a G-protein couple-receptor and activation of the CaSR has been shown to inhibit adenylate cyclase and to decrease cAMP generation [23,24]. Since the 50-pS K channels were stimulated by cAMP [18], we examined whether the effect of raising the external Ca²⁺ on the 50-pS K channel was the result of decreasing cAMP Figure 7 is a recording showing the effect of 3 mM Ca²⁺ on the basolateral 50-pS K channels in the presence of 100μM dibutyryl-cAMP (db-cAMP.) Although application of db-cAMP increased the channel activities from 0.33±0.12 to 0.51±0.10 (N=6), stimulation of PKA failed to block the inhibitory effect of 3 mM Ca²⁺on the 50-pS K channel. In the presence of db-cAMP, increasing the external Ca²⁺ from 1 mM to 3 mM decreased channel activities from 0.51±0.10 to 0.04±0.02 (N=6). Therefore, the inhibitory effect of raising the external Ca²⁺ on the 50-pS K channel was not due to decreasing cAMP.

Fig. 7.

A channel recording demonstrates the effect of raising the external Ca²⁺ from 1 mM to 3 mM on the activity of the basolateral 50-pS K channel in the presence of 100 μM db-cAMP. The experiments were performed in cell-attached patches of the isolated TAL. Three parts of the trace indicated by numbers are extended to show the fast time resolution. The channel closed level is indicated by letter “C” and the holding potential was 0 mV. A bar graph in the bottom of the figure summarizes the results from 6 experiments.

Stimulation of the CaSR has been shown to activate PLC and to stimulate PKC [25,26], we next examined whether PLC-PKC pathways were responsible for mediating the effect of raising the external Ca²⁺ on the 50-pS K channels. Figure 8 is a representative recording from 4 experiments in which the effect of 3 mM Ca²⁺on 50-pS K⁺ channel was tested in the presence of U73122 (10 μM). Fig. 8 clearly demonstrated that inhibition of PLC abolished the effect of increasing Ca²⁺ on the 50-pS K channel activity (Control 0.53±0.12, 3 mM Ca²⁺, 0.50±0.10). Wash-out of U73122 restored the inhibitory effect of raising the external Ca²⁺ on the 50-pS K channels. Furthermore, inhibition of PKC mimicked the effect of U73122 and abolished the effect of raising the external Ca²⁺. Fig 9 summarizes the results of experiments in which the effect of 3 mM external Ca²⁺ on the 50-pS K channels was examined in the presence of calphostin C (100 nM). Inhibition of PKC completely abolished the effect of increasing the external Ca²⁺ on the K channel activity (Control 0.50±0.10, 3 mM Ca²⁺, 0.45±0.10) (N=5). Taken together, results strongly suggest that the effect of stimulation of the CaSR on the basolateral K channels is the result of activation of PLC-PKC pathways in the TAL.

Fig. 8.

A channel recording demonstrates the effect of the raising external Ca²⁺ from 1 mM to 3 mM on the activity of the basolateral 50-pS K channel in the presence of or in the absence of U73122 (10 μM). The arrow indicates the change in the external Ca²⁺. The experiments were performed in cell-attached patches of the isolated TAL. Five parts of the trace indicated by numbers are extended to show the fast time resolution. The channel closed level is indicated by letter “C” and the holding potential was 0 mV.

Fig. 9.

A bar graph summarizing the experiments in which the effect of raising the external Ca²⁺ from 1 mM to 3 mM on basolateral 50-pS K channels in the TAL was examined under control conditions or in the presence of 10 μMU73122 (N=6) or 100 nM calphotin C (N=5), respectively. The experiments were performed in cell-attached patches.

4. Discussion

In the present study, we have demonstrated that raising the external Ca²⁺ inhibited the basolateral 50-pS K channels in the TAL by a PKC-dependent mechanism. Two lines of evidence suggest that the effect of the external Ca²⁺ on the basolateral 50-pS K channels was the result of stimulating the CaSR: 1) the inhibitory effect of raising the external Ca²⁺ on the 50-pS K channels was observed only in cell-attached patches but not in excised patches; 2) the effect of 3 mM external Ca²⁺ on the 50 pS-K channel was absent in the presence of the CaSR antagonist. However, NPS2390 did not significantly increase the K channel activity, it is possible that the CaSR at 1 mM external Ca2+ may be in a less active state. In this regard, we suspect that activation of the K channels induced by lowering external Ca²⁺ to 10 μM may not be completely due to inhibition of CaSR. Using in situ hybridization, single tubule RT-PCR and immunocytochemical staining, it was convincingly documented that the CaSR is highly expressed in the TAL [19]. Moreover, it is possible that the CaSR might directly interact with the basolateral 50 pS K channels in the TAL. This speculation is supported by reports from several studies. First, the basolateral 50-pS K channels in the TAL might be the hetero-tetramers of Kir4.1/Kir5.1 because the basolateral K channels in the distal tubules with similar biophysical properties as those of the 50-pS K channel in the TAL are composed of Kir4.1 and Kir5.1 [14,27]. Second, immunostaining experiments demonstrated that Kir4.1 and Kir5.1 were expressed in the basolateral membrane of the TAL [28,29]. Finally, the study using immunoprecipitation performed in the cells transiently transfected with Kir4.1 and the CaSR has demonstrated that Kir4.1 was associated with the CaSR [16]. Therefore, the present study provides the physiological relevance regarding the role of the CaSR in the regulating basolateral K channels in the native tubules. Relevant to our finding is the report that expression of CaSR inhibited K currents in HEK293 cells transfected with Kir4.1 by stimulating the endocytosis of the K channels [30].

It is well established that the CaSR plays a role in the regulation of epithelial transport in the TAL [31,32] which is responsible not only for reabsorption of 20-25% of filtered NaCl load but also is involved in reabsorbing divalent cations such as Ca²⁺ and Mg²⁺ [33]. The physiological significance of the present study is to provide a novel mechanism by which the CaSR regulates the membrane transport in the TAL by controlling the activity of the basolateral K channels. Activation of basolateral K channels in the TAL is expected to increase the negativity of the cell membrane potential thereby augmenting the driving force for Cl exit across the basolateral membrane. In contrast, decreasing basolateral K channel activity depolarizes the cell membrane potential thereby diminishing the driving force for Cl exit across the basolateral membrane. Consequently, inhibition of Cl exit leads to an increase in intracellular Cl concentration which suppresses the interaction of With-No-Lysine kinase 3 (WNK3) and Step20-related Prolin/Alanine-Rich kinase (SPAK) [34]. The interaction between WNK3 and SPAK is required for the phosphorylation of NKCC2 thereby increasing NKCC2 activity. Therefore, an increase in the intracellular Cl concentration is expected to decrease the phosphorylation of NKCC2 by SPAK thereby inhibiting the activity of NKCC2. Accordingly, the regulation of the basolateral 50-pS K channel by the external Ca²⁺ should play a role in the modulation of transepithelial Na transport and concentrating ability in the TAL. Since the active reabsorption of NaCl in the water-impermeable TAL is essential for urinary concentrating mechanism, inhibition of NaCl reabsorption in the TAL should result in a decrease in concentrating ability. Indeed, it has been reported that hypercalcemia impairs urinary concentrating ability [35].

In addition, the TAL is also responsible for reabsorption of divalent cations such as Ca²⁺ and Mg²⁺ [31]. The reabsorption of divalent cations in the TAL is through the paracellular pathway and driven by the luminal positive voltage which is generated by apical K recycling and basolateral Cl exit [33]. Because basolateral K channels are participated in generating the cell membrane potential which affects Cl diffusion across the basolateral membrane in the TAL, inhibition of basolateral K channels by high external Ca²⁺ is expected to decrease lumen positive transepithelial voltage. Therefore, a high external Ca²⁺-induced inhibition of the basolateral 50-pS K channels would reduce the transport of divalent cations in the TAL. Indeed, it has been reported that an increase in plasma Ca²⁺ /Mg²⁺ level enhanced urinal Ca²⁺/Mg²⁺ excretion [36,37]. We hypothesize that regulation of transepithelial divalent cation transport by the CaSR is achieved, at least partially, by modulating the basolateral K channel activity.

Stimulation of the CaSR has been shown to inhibit the apical 70-pS K channel in the TAL by activating PLA₂ and enhancing cytochromep450-ω–hydroxylation-dependent metabolism of arachidonic acid [12,13]. However, neither AACOCF3 nor 17-ODYA was able to abolish the inhibition of basolateral 50-pS K channels induced by raising the external Ca²⁺. This suggests that cytochromeP450-ω–hydroxylation of arachidonic acid was not responsible for the inhibition of the 50-pS K channel induced by stimulation of the CaSR. On the other hand, the observation that the effect of raising the external Ca²⁺ on the basolateral 50-pS K channel was blocked by calphostin C suggests that the effect of stimulation of CaSR was mediated by PKC. Therefore, results suggest that the two different signaling pathways or G-proteins are coupled to the CaSR responsible for the regulation of the apical 70-pS and basolateral 50-pS K channels in the TAL, respectively. However, it is unlikely that inhibition of the apical K channels by the external Ca²⁺ is mediated by activation of the CaSR in the apical membrane, since the CaSR is overwhelmingly expressed in the basolateral membrane [19]. Alternatively, two types of the CaSR coupled with two different G-proteins in the basolateral membrane are responsible for regulating apical and basolateral K channels. This speculation was supported by the observation that 5 mM external Ca²⁺ was required for inhibition of the apical K channel, an effect was not blocked by inhibition of PKC [12]. In contrast, 3 mM external Ca²⁺ could almost completely inhibit the basolateral 50-pS K channels and the inhibitory effect was blocked by calphostin C. Relevant to our view is the report that the CaSR activating PLA₂ is coupled to G-protein G_qα [38] whereas the CaSR activating PLC is mediated by G-proteins, Gα_I or Gα_q [39].

Also, we speculate that the activation of the CaSR-induced inhibition of basolateral 50-pS K channels may take place in the close vicinity or that the activation of PKC by the CaSR might be compartmentalized. This notion is based on the following observations: 1) PKC and cytochrome P450-ω-hydroxylation-dependent metabolites of arachidonic acid were able to inhibit the basolateral 50 pS-K channels and the apical 70-pS K channels in the TAL [12,40,41]; 2) the inhibitory effect of raising the external Ca²⁺ on the basolateral 50-pS K channels was not affected by suppressing cytochrome P450-ω-hydroxylation of arachidonic acid while the effect of the external Ca²⁺ on the apical K channel was not abolished by blocking PKC. Our speculation is also supported by the finding that the CaSR has been shown to physically interact with Kir4.1 [16]. Accordingly, the effect of PKC on the basolateral 50-pS K channel is compartmentalized in response to stimulation of the CaSR. The possibility that CaSR regulates the basolateral K channels in a compartmentalized environment through a physical or functional association may also explain the previous observation that inhibition of PKC failed to block the effect of raising the external Ca²⁺ on the apical 70-pS K channels [12].

Because alteration of either apical K or basolateral K channel activity could affect NKCC2 activity, the CaSR could regulate Na and divalent cation transport in the TAL by either modulating apical or basolateral K channel activity. However, we hypothesize that the CaSR-mediated regulation of the membrane transport in the TAL is mainly though controlling the basolateral K channel activity rather than through apical K channels (Fig. 10). This notion is supported by the observation that alteration of the external Ca²⁺ from 1 mM to 2 mM, a physiological range of extracellular Ca²⁺, significantly inhibited the 50-pS K channels in the basolateral membrane of the TAL. In contrast, inhibition of apical K channels requires a high and less physiological relevant concentration of the external Ca²⁺ [12]. Fig. 10 is a cell scheme illustrating the role of basolateral K channels in mediating the effect of stimulation of the CaSR on divalent cation transport in the TAL. Under physiological conditions, the basolateral K channels maintain the cell membrane potential such that it could sustain a constant Cl diffusion across the basolateral membrane. An increase in the external Ca²⁺ (hypercalcemia) activates the CaSR thereby inhibiting basolateral K channels and decreasing the driving force for Cl diffusion across the basolateral membrane. Inhibition of Cl exit is expected to hyperpolarization of transepithelial voltage (V_te). A less positive V_te would diminish the reabsorption of divalent cations such as Ca²⁺ and Mg²⁺. We conclude that hypercalcemia inhibited while hypocalcemia increased the basolateral K channel activity in the TAL, an effect is mediated by the CaSR linking to activation of PLC-PKC pathways. The regulation of basolateral K channel activity by the CaSR may be the mechanism by which plasma concentration of divalent cations modulate their reabsorption.

Fig. 10.

A cell scheme illustrating the role of basolateral K channels in mediating the effect of stimulation of the CaSR on the divalent cation transport under control conditions (left panel) and during hypercalcemia (right panel). A red solid line, a blue solid line and a blue dotted line represent an active, a less active and an inactive status, respectively. Note that the association between the CaSR and the basolateral K channels may be functional rather than physical binding. Abbreviations: CaSR (Ca²⁺-sensitive receptor), 20-HETE (20-hydroxyeicosatetraenoic acid), V_te (transepithelial voltage), AA (arachidonic acid).

Fig. 4.

A single channel recording shows the effect of raising external Ca²⁺ from 1 mM to 3 mM on the 50-pS K channel in the presence of NPS2390 (5 μM), an antangost of the CaSR. The experiments were performed in a cell-attached patch and the channel closed level is indicated by C. Three parts of the trace indicated by numbers are extended to show the fast time resolution. The channel closed level is indicated by letter “C” and the holding potential was 0 mV. The arrow indicates the addition of the corresponding chemicals.

Highlights.

The patch-clamp experiments were performed in the thick ascending limb (TAL). > Stimulation of Ca²⁺-receptor by raising external Ca²⁺ inhibits basolateral K channels in the TAL. >The basolateral K channel activity determines transepithelial voltage (V_te). > V_te provides the driving force for transepithelial Ca²⁺ absorption in the TAL. > Regulation of Ca²⁺ absorption in the TAL may be achieved by modulating basolateral K channels.