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1 Department of Pharmacology and Toxicology, Medical Faculty, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany
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Department of Physiological Chemistry, Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany
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Department of Anatomy, Medical Faculty, University of Tübingen, Österbergstrasse 3, D-72074 Tübingen, Germany
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Department of Anatomy, Charité, Philippstrasse 12, D-10098 Berlin
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Abstract |
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–60 mV) of the juxtaglomerular cells were examined. In order to increase the number of juxtaglomerular cells, afferent arterioles from NaCl-depleted rats were used, and > 90% of the afferent arterioles were renin positive at the distal end of the arteriole. Whole-cell and cell-attached single-channel patch-clamp experiments showed that juxtaglomerular cells are endowed with a strongly inwardly rectifying K+ channel (Kir). The channel was highly sensitive to inhibition by Ba2+ (inhibition constant 37 µM at 0 mV), but relatively insensitive to Cs+ and, with 142 mM K+ in the pipette, had a single-channel conductance of 31.5 pS. Immunocytochemical studies showed the presence of Kir2.1 but no signal for Kir2.2 in the media of the afferent arteriole. In PCR analyses using isolated juxtaglomerular cells, the mRNA for Kir2.1 and Kir2.2 was detected. Collectively, the results show that Kir2.1 is the dominant component of the channel. The current carried by these channels plays a decisive role in setting the membrane potential of juxtaglomerular cells.
(Received 25 June 2004;
accepted after revision 28 July 2004;
first published online 5 August 2004)
Corresponding author U. Russ: Department of Pharmacology and Toxicology, Medical Faculty, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany. Email: ulrich.russ{at}uni-tuebingen.de
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Introduction |
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Control of renin secretion from juxtaglomerular cells is complex, involving intrarenal mechanisms (e.g. the macula densa and the baroreceptor mechanisms) and extrarenal signals (e.g. from sympathetic nerves and circulating hormones; for review see Kurtz, 1989; Hackenthal et al. 1990; Osswald & Quast, 1995). An intriguing feature of the control of renin secretion is the ‘Ca2+ paradox’, i.e. the observation that manoeuvres that decrease the cytoplasmic Ca2+ concentration ([Ca2+]i) increase the rate of renin secretion from juxtaglomerular cells and vice versa (Kurtz, 1989; Hackenthal et al. 1990). Depolarization of juxtaglomerular cells leads to an increase of the cytoplasmic free calcium concentration (Russ et al. 1999) and to a decrease of the renin secretion (review in Kurtz, 1989). In non-pressurized afferent arterioles, the resting membrane potential of juxtaglomerular cells is –60 mV (Fishman, 1976 (two populations with –70 and –35 mV); Bührle et al. 1985; Russ et al. 1999; review in Kurtz, 1989); isolated juxtaglomerular cells from rat kidney are more depolarized (resting membrane potential –32 mV, Friis et al. 2003). In a pioneering study, Kurtz & Penner (1989), using mouse glomeruli with the distal part of the afferent arteriole attached, demonstrated a strongly inwardly rectifying K+ conductance in juxtaglomerular cells, the activity of which determined the resting membrane potential. However, a more detailed electrophysiological characterization of this channel and of its molecular composition has not yet been undertaken.
In this study we have addressed these questions. In order to increase the number of renin-positive cells, intact afferent arterioles from NaCl-depleted rats were used. In particular the sensitivity of the Kir channel in juxtaglomerular cells to Ba2+ and Cs+ and the single-channel conductance were determined; in addition, immunocytochemical and PCR experiments were performed to elucidate the molecular composition of the channel. We show that, also in rat juxtaglomerular cells, the activity of this channel determines membrane potential. The membrane potential of juxtaglomerular cells is directly or indirectly (i.e. via the cytoplasmic Ca2+ concentration) involved in the regulation of renin secretion (Kurtz, 1989; Persson, 2003) which, in turn, determines the activity of the renin–angiotensin system and thereby regulates blood pressure and electrolyte homeostasis.
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Methods |
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Animal experimentation was conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and German Law on the Protection of Animals. Sprague-Dawley rats (250–500 g) were used. To induce NaCl-depletion, animals were treated once with furosemide (10 mg kg–1, I.P.) and kept on a NaCl-depleted diet (C1036, Altromin, Lage, Germany) for at least 2 weeks after furosemide application. Control animals were not treated with furosemide and were kept on a standard diet (C1324, Altromin).
Immunocytochemistry
The anti-renin antibody was produced by immunization of rabbits with a polypeptide comprising the amino acids 303–319 from rat renin conjugated to KLH-protein and the purified serum was used. Polyclonal antibodies against the less conserved C-terminus of the Kir2.1, Kir2.2 and Kir6.1 protein were raised in rabbits, and purified serum (Kir2.1) or affinity-purified antibodies (Kir2.2, Kir6.1) were used (Prüss et al. 2003; Thomzig et al. 2001, 2003). Tissue preparation for immunocytochemistry was done according to Pfaff et al. (1999). The animals were anaesthetized by intraperitoneal injection of thiopental sodium (Byk Gulden, Konstanz, Germany; 60 mg kg–1) on the day of the experiment. After anaesthesia, the left cardiac ventricle was exposed through subcostal incision of the abdominal cavity and subsequent opening of the pericardium. The tip of a perfusion system was placed into the left ventricle, and the arterial system was perfused for 1 min with phosphate-buffered saline (PBS; 120 mM NaCl, 16 mM Na2HPO4, 2.9 mM KH2PO4) to clear the kidneys of blood and subsequently for 10 min with 150 ml of a fixation solution (4% paraformaldehyde and 3% sucrose in PBS). Both solutions were at room temperature. Kidneys were removed, cut into slices (3 mm in thickness) displaying cortex and outer and inner medulla, and incubated for 1 h in the fixation solution at 4°C. After rinsing 4 times in PBS for 15 min, kidney slices were cryoprotected by 30% sucrose in PBS for 12 h at 4°C, frozen in isopentane precooled by liquid nitrogen, and stored at –80°C until further use. Cryosections of approximately 16 µm were made at –20°C and transferred onto gelatin-coated glass slides. The following steps were performed at room temperature. After preincubation for 45 min in PBS containing 4% normal goat serum, 1% bovine serum albumin, and 0.25% Triton X-100 in PBS, sections were incubated for 4 h with the respective antibody (anti-Kir2.1 was diluted 1: 50; anti-Kir2.2, 1: 25; anti-Kir6.1, 1: 50; anti-renin 1: 100). After washing the sections three times for 5 min with PBS, they were incubated for 1.5 h with Alexa 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA; final concentration, 13 µg ml–1). Sections were then washed twice for 10 min in PBS and mounted in FluorSave (Calbiochem, San Diego, CA, USA) as a fading retardant. For immunocytochemical reference, cryosections of rat brain were prepared and stained with all Kir antibodies. Cryosections were analysed on a confocal microscope (LSM 410, Carl Zeiss, Oberkochen, Germany), using an Argon laser for excitation at a wavelength of 488 nm and appropriate filter sets. Optical section thickness for confocal fluorescence detection was set at approximately 0.8 µm, and non-confocal transmitted images were captured simultaneously in a separate detector. Five rats were used for tissue preparation and 38 (Kir2.1), 16 (Kir2.2) and 36 (Kir6.1) slices were evaluated.
Electrophysiology
Animals were killed by cervical dislocation, exsanguinated and the kidneys removed. A freshly isolated kidney was transferred in a Hepes-buffered physiological salt solution (PSS) containing (mM): NaCl 142, KCl 2.8, MgCl2 1, CaCl2 1, D(+)-glucose 11, buffered with Hepes (10 mM) and titrated to pH 7.4 with NaOH at 37°C. The kidney was decapsulated, cut longitudinally into two halves and the isolated cortex was minced with a razor blade. Glomeruli were prepared by modifications of published methods (Kurtz & Penner, 1989; Metzger & Quast, 1996; Russ et al. 1999). The minced tissue was incubated for 50 min in 20 ml PSS with 20 mg collagenase A (Roche Diagnostics, Mannheim, Germany) at 37°C under gentle shaking. The suspension was then passed through stainless steel sieves of mesh 200 and 125 µm and the final material collected on a 63 µm sieve. Microscopic inspection showed that the preparations consisted mainly of glomeruli and some contamination with tubular fragments; about 10% of the glomeruli contained a remnant of the afferent arteriole up to 100 µm in length.
Glomeruli were transferred to the recording chamber equipped with a poly-L-lysin-coated cover slide. Electrophysiological recordings were taken from cells in the distal afferent arteriole close to the entrance into the glomerulus (Russ et al. 1999). The patch-clamp technique was used in the whole-cell (at 37°C) and cell-attached (at room temperature) configurations as described by Hamill et al. (1981). Bath solution was PSS in the whole-cell current-clamp and most cell-attached experiments or a high-K+ solution (mM: KCl 142, NaCl 2.8, MgCl2 1, CaCl2 1, D(+)-glucose 11, Hepes 10, titrated to pH 7.4 with NaOH) in the whole-cell voltage-clamp and some cell-attached experiments. Patch pipettes were drawn from borosilicate glass capillaries (GC 150, Harvard Apparatus Ltd, Edenbridge, UK) and heat polished using a horizontal microelectrode puller (Zeitz, Augsburg, Germany). For whole-cell current-clamp experiments pipettes were filled with (mM) potassium glutamate 132, NaCl 10, MgCl2 2, Hepes 10, EGTA 1, Na2ATP 1, titrated to pH 7.2 with NaOH. For whole-cell voltage-clamp experiments the pipette solution was slightly changed (mM: potassium glutamate 126, NaCl 10, MgCl2 4, Hepes 10, EGTA 1, Na2ATP 3, titrated to pH 7.2 with NaOH). Potentials given in these experiments were corrected for a liquid junction potential of 10 mV (Neher, 1992). Single-channel activity was recorded using the cell-attached configuration with the high-K+ solution in the pipette. Here, sampling frequency was 2000 Hz and the data were filtered with 1000 Hz. To compute the histograms showing the frequency distribution of the current amplitude, 10 s of the recording were analysed. A Gaussian function with two to four components was fitted to the data. For presentation of current traces, sample frequency was reduced to 200 Hz and filtered correspondingly.
Pipette resistance was always 3–5 M
. Data were recorded with an EPC 9 amplifier using Pulse software (HEKA, Lambrecht, Germany). Results are given as mean ± standard error of the mean.
PCR
RT-PCR was performed using RNA from isolated juxtaglomerular cells from rat kidney, prepared as described by Albinus et al. (1998), from rat kidneys and from brain. Total RNA was extracted using the peqGOLD RNAPure System (Peqlab, Erlangen, Germany) according to the manufacturer's protocol. The yield was determined spectrophotometrically at 260 nm and the integrity of the purified RNA was analysed by agarose gel electrophoresis. First strand cDNA was synthesized from 1 µg total RNA in a solution of 20 µl containing random primer (final concentration 10 mM; Promega, Mannheim, Germany), 1 mM dNTP, 250 ng oligo dT Primer (Promega), 5 mM MgCl2, 20 units (U) RNAase inhibitor (Promega), 125 U AMV-reverse transcriptase (Peqlab) and 1 x PCR buffer (Pan Biotech, Aidenbach, Germany). The solution was incubated for 10 min at room temperature, then for 60 min at 42°C and finally for 3 min at 95°C. The PCR reaction mixture (50 µl) contained 15 µl of this cDNA solution, dNTP (0.2 mM), 2.5 U PANTMScript Taq polymerase (Pan Biotech), 1 x PCR buffer, and Kir2.x specific primers (50 nM). The primers were designed to amplify a 326 bp from the Kir2.1 and a 836 bp sequence from Kir2.2 channels, respectively (Kir2.1 sense nucleotides (nt) 864–883, and anti-sense, nt 1170–1189, gene accession no. GA L48490; Kir2.2 sense, nt 419–438, and anti-sense, nt 1235–1254, GA X78461; Bradley et al. 1999). PCR was performed for 2 min at 94°C, followed by 32 cycles of 1 min at 94°C, 30 s at 61°C and 1 min at 72°C, and finished with a final 10 min extension at 72°C. The reaction products were analysed by agarose gel electrophoresis and photographed. Cyclophillin (sense nt 159–178, anti-sense nt 397–415; GA M25637; 257 bp) was used as an internal control; for cyclophillin mRNA detection, 5 µl cDNA solution were used.
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Results |
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In a preliminary set of experiments we used neutral red to identify juxtaglomerular cells in afferent arterioles as described by Fishman (1976). However with this dye, which stains acidic cellular compartments, we found red vesicles in almost all cells of the afferent arteriole (see supplementary material available online only). More specifically the distribution of renin-secreting cells in rat kidney was characterized using a polyclonal antibody serum raised against rat renin. Figure 1A shows a typical section from a kidney slice prepared from a rat kept on a NaCl-depleted diet. The picture on the left shows the fluorescence signal from the renin antibody. In the overlay of the bright field pictures and the fluorescence image on the right one can see the glomerulus in the centre with the afferent arteriole leading to the right. This overlay picture clearly identifies the terminal part of the afferent arteriole, i.e. the juxtaglomerular cells, as the major origin of the renin signal. Figure 1B illustrates the distribution of renin-positive vessel segments along afferent arterioles from NaCl-depleted and control rats. Close to the entrance of the afferent arteriole into the glomerulus, the probability of finding renin-positive cells was 93% for NaCl-depleted rats and 76% for control rats, respectively. With increasing distance from the glomerulus, there was a strong decrease in the number of renin-positive cells in both cases. NaCl depletion led to an increase in the number of renin-positive vessels and in the length of the renin-positive region (P < 0.01, Mann–Whitney rank sum test).
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For the experiments, bulgy roundish cells in the distal afferent arteriole were selected close to the entrance into the glomerulus, a region where in almost all vessels renin-secreting cells are located (see above; Fig. 1; Russ et al. 1999). These cells are clearly distinguished from smooth muscle cells which have a spindle-like appearance and a spiral arrangement around the vessel (Bührle et al. 1984; Hackenthal et al. 1990). First, the sensitivity of the membrane potential to Ba2+ was determined in whole-cell current-clamp experiments. Resting membrane potential was –63 ± 6 mV (n = 6). Addition of Ba2+ (100, 300 and 1000 µM) to the bath depolarized the cell by 6 ± 1, 10 ± 1 and 16 ± 1 mV, respectively (Fig. 2A; n = 6). Washout of Ba2+ restored the resting membrane potential.
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0.8, 1.4, 2.5, 5.0, 8.7 and 14.5 µM at –130, –110, –90, –70, –50 and –30 mV holding potential, respectively. Assuming a one-site model for Ba2+ block (Liu et al. 2001) and setting IC50 = Kd, a semilogarithmic plot of these data (Fig. 2D) gives a straight line and allows interpretation of the voltage dependence according to Woodhull (1973):
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denotes the fraction of the electric field sensed at the Ba2+ binding site. Linear regression of the data gave for Kd(0) a value of 37 µM; from the slope, was calculated to 0.39. Positive to –10 mV a prominent current component was visible which was almost insensitive to 1 mM Ba2+ (Fig. 2B and C).
Membrane potential and whole-cell current were rather insensitive to Cs+. In current-clamp experiments with PSS in the bath, Cs+ (1 mM) showed no effect; at 5 mM, a small depolarization by 2 mV was seen (n = 3; data not shown). In voltage-clamp experiments with high-K+ bath solution, Cs+ (50 µM) partially inhibited the current at –130 and –110 mV; at potentials positive to –90 mV, no inhibition was seen (n = 4; Fig. 3). Additionally, two other K+ channel blockers, tedisamil and ciclazindol, were tested in whole-cell voltage-clamp experiments with high-K+ solution in the bath (data not shown). In two patches, 10 µM tedisamil showed no or a very small effect on the inwardly rectifying current; at 100 µM, a slow inhibition was seen which reached about 30% after 10 min (n = 3) and which was partially reversible. Ciclazindol was somewhat more potent; 10 and 100 µM reversibly inhibited about 10 and 50% of the current, respectively (n = 3). Inhibition by both drugs was restricted to inward currents (i.e. at negative potentials) and was independent of voltage within this range.
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= 31.5 ± 0.6 pS (n = 11).
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180 pS. Further experiments were conducted in which the K+-rich solution used for filling of the pipette was also present in the bath. Figure 6 shows recordings from juxtaglomerular cells from a rat fed on the standard diet. At negative clamp potentials, observations were very similar to those shown in Fig. 4; however, the reversal potential was shifted to –15 mV (Fig. 6). At clamp potentials positive to +40 mV, the large-conductance channel was regularly seen and channel activity increased continuously with increasing depolarization up to +100 mV (higher potentials were not applied). At potentials positive to +80 mV, single-channel currents deviated from Ohmian behaviour (Fig. 6) and these values were not used for calculation of the single-channel conductance, which gave a value of 177 pS. The reversal potential of the channel, if calculated from the data up to +80 mV, was almost identical to that obtained for the 31.5 pS inwardly rectifying channel (Fig. 6). The differences between the reversal potentials in K+-rich and Na+-rich bath solution (Fig. 4) point to a depolarization of the cells by 50 mV if the K+-rich solution is applied. No obvious differences were observed in this series of experiments between control animals (15 patches from 4 rats) and rats fed on a Na+-restricted diet (10 patches from 2 rats).
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Using polyclonal antibodies against Kir2.1 and 2.2, the localization of these channel proteins was analysed in slices from rat kidney; rat brain slices were stained as a control. Figure 7 shows that the Kir2.1 antibody gave a strong signal in the juxtaglomerular region of the afferent arteriole; furthermore, Kir2.1 was also found in the proximal part of the tubulus (data not shown). For Kir2.2, no signal was detected in the kidney. Using an antibody against Kir6.1, the pore-forming subunit of KATP channels in vascular smooth muscle cells, the juxtglomerular region of the afferent arteriole was strongly labelled, similar to the observations with the Kir2.1 antibody. All three antibodies gave clear and typical signal patterns in brain tissue slices (supplementary material which can be found online) similar to those obtained by other groups (Prüss et al. 2003).
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Discussion |
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Identity of cells selected for electrophysiological experiments and electrical coupling
The tunica media of the distal part of the afferent arteriole contains both smooth muscle and juxtaglomerular cells; hence, the first question arising is that of the identity of the cells examined. Juxtaglomerular cells can be metaplastically transformed to smooth muscle cells and vice versa (Cantin et al. 1977; Sequeira Lopez et al. 2001) and differences in the channel equipment of the two cell types may occur. As an example, vascular smooth muscle cells are endowed with voltage-dependent Ca2+ channels whereas juxtaglomerular cells are not (Kurtz et al. 1990; Scholz & Kurtz, 1995); however, evidence for the existence of such channels on rat isolated juxtaglomerular cells has recently been presented (e.g. Friis et al. 2003). In experiments using sharp microelectrodes, no differences in membrane potential between smooth muscle and granulated cells in the afferent arteriole were detected, nor were there different responses to several neurotransmitters and hormones between the cell types (Taugner & Hackenthal, 1989).
Unambiguous identification of a juxtaglomerular cell in electrophysiological experiments may be difficult; however, two points suggest that generally juxtaglomerular cells were selected. First, only roundish protruding cells were used for patch clamping, and these are more likely to be juxtaglomerular cells than the spindle-like smooth muscle cells. Second, afferent arterioles from salt-depleted rats were used; in this case, > 90% of the afferent arterioles were renin positive at the vessel pole (Fig. 1; see also Taugner & Hackenthal, 1989). The experiments identifying Kir (Figs 4 and 5) were performed using 11 cells and gave reproducible results; it is therefore extremely unlikely that by chance in these 11 cases exclusively vascular smooth muscle cells were studied. This observation was confirmed by a second set of experiments with afferent arterioles from control rats (15 patches) and NaCl-depleted rats (10 patches).
A further complexity arises from the fact that cells in the distal part of the afferent arteriole are strongly electrically coupled (Kurtz & Penner, 1989; Russ et al. 1999), resulting in synchronized electrophysiological behaviour of the tissue. Hence, if a juxtaglomerular cell was selected, neighbouring smooth muscle cells may have contributed to the observed response. Whereas this may play a role for the results from experiments in the whole-cell mode (Figs 2 and 3, Ba2+ and Cs+ effects), it does not apply for the single-channel experiments performed in the cell-attached mode (Figs 4–6) and in which the current–voltage relationship and the single-channel conductance of Kir2 were determined (see below).
Juxtaglomerular cells from rats on NaCl-depleted versus normal diet
A NaCl-depleted diet was used in order to increase the number of renin-positive cells (Fig. 1). It is well known that NaCl restriction induces adaptive changes in the expression of ion channels and transporters (see, e.g. Masilamani et al. 2002). Therefore, side-by-side comparison of juxtaglomerular cells from rats with and without dietary NaCl restriction were made in single-channel experiments. No difference between the two preparations was found, demonstrating that the prominent appearance of Kir is not a consequence of the NaCl-restricted diet. Taking the evidence presented above together, we feel that the presence of Kir.2 on juxtaglomerular cells integrated in the rat afferent arteriole is established beyond reasonable doubt.
Isolated juxtaglomerular cells
At 2.8 mM K+ in the bath, the juxtaglomerular cells prepared from rat kidney have a resting membrane potential of –32 mV (Friis et al. 2003) and a similar value may be guessed for isolated mouse juxtaglomerular cells from published current–voltage relationship data (Friis et al. 1999, 2002). From these values it is obvious that there is no major contribution from a strongly inwardly rectifying K+ channel to the membrane potential, and one can only speculate why the channel is (essentially) inactive. It is possible that the procedure required for cell isolation might reduce the activity of the inwardly rectifying K+ channels. Alternatively, the integrity of the tissue, in particular coupling to neighbouring juxtaglomerular and smooth muscle cells via gap junctions to allow exchange of cellular metabolites, might be critical for the appearance of a prominent inwardly rectifying current. In addition, the channel, if it was active, may have been difficult to detect under the conditions used by Friis et al. (2003). According to the Goldman-Hodgkin-Katz current equation, inward potassium currents are rather small if physiological K+ concentrations (i.e. 2.8 mM) are used (Friis et al. 2003) rather than the high-K+ solution (142 mM) used here for the experiments in the voltage-clamp mode (Figs 2B–D and 3–6). The increase of the inwardly rectifying current with high K+ in the bath compared with standard PSS can be seen pretty well in Fig. 3.
Electrophysiological characterization
Inwardly rectifying K+ current.
Voltage-dependent Ba2+ block. The IC50 value of 2 µM, determined for Ba2+ at a clamp potential of –100 mV, is in excellent agreement with results from native Kir2.x channels in rat cerebral arteries (Quayle et al. 1993), rat coronary arteries (Robertson et al. 1996), guinea-pig cardiomyocytes (Liu et al. 2001), parotid acinar cells (Hayashi et al. 2003) and trabecular meshwork cells (Llobet et al. 2001). As a voltage-independent value for sensitivity to Ba2+ block, the Kd value extrapolated to 0 mV, Kd(0), is generally used. The value of 37 µM determined here is also in good agreement with values obtained in arterial smooth muscle cells (38.1 µM, Quayle et al. 1993; 21.1 µM, Robertson et al. 1996) and other tissues (Llobet et al. 2001; Hayashi et al. 2003). Finally, the value for the fraction of field sensed by Ba2+ at its binding site, = 0.39, is in reasonable agreement with those determined in arterial smooth muscle cells (0.51 Robertson et al. 1996; 0.55 Quayle et al. 1993). Regarding the channel block by Ba2+, this agreement shows that there is no difference between the inwardly rectifying K+ channel in juxtaglomerular cells and arterial smooth muscle cells; in the latter cells, Kir2.1 is dominant (Bradley et al. 1999). Four Kir2 clones have been described (Kubo et al. 2002) and their Ba2+ sensitivity varies considerably (values at –100 mV: Kir2.1, 3.24 µM; Kir2.2, 0.51 µM; Kir2.3, 10.26 µM; Kir2.4, 235 µM; Liu et al. 2001). Although these values were determined at 60 mM K+ in Xenopus oocytes and direct comparison with the results of this study must be made with care, it is conspicuous that the best agreement is found for Kir2.1.
Unitary conductance. In this study, a value of 31.5 pS was determined for the single-channel conductance of native channels in juxtaglomerular cells. Cloned Kir2 differ in their single-channel conductances, which are listed as 23, 34, 13 and 15 pS for Kir2.1, Kir2.2, Kir2.3 and Kir2.4, respectively (high symmetrical K+, Kubo et al. 2002). The best agreement is found with Kir2.2 and the comparison seems to exclude Kir2.3 and Kir2.4 from being a major component of Kir2 in juxtaglomerular cells. However, reported values for Kir2.1 vary from 20 to 30 pS (Lopatin & Nichols, 2001), probably due to different expression systems and recording conditions. Furthermore, it was observed that the single-channel conductance values of Kir2.1 in the same preparation ranged from 2 to 33 pS, showing prominent scattering of the single-channel conductance for unknown reasons (Picones et al. 2001). With guinea pig Kir2.1 expressed in HEK293 cells, Liu et al. (2001) found a value of 30.6 ± 2.5 pS, which is very close to the value determined here and which was recorded under conditions similar to ours (mammalian cells, room temperature, cell-attached configuration, Hepes-buffered pipette solution with 140 mM K+, 1 mM Ca2+ and 1 mM Mg2+). Hence, the value determined here is well within the range reported for Kir2.1.
Other properties. Kir2.1 and 2.2 differ in their Cs+ sensitivity and their inactivation properties. First, at –60 mV, Kir2.2 and 2.3 are almost completely blocked by 50 µM Cs+ (Takahashi et al. 1994; Morishige et al. 1994) whereas inhibition is very small for Kir2.1 (Bradley et al. 1999 and references therein). Figure 3 shows no inhibition by Cs+ (50 µM) at –60 mV. Second, Kir2.2 strongly inactivates at hyperpolarizing potentials whereas Kir2.1 does not (Takahashi et al. 1994). In Fig. 2B, there was a small inactivation of the current at the beginning of the experiment which was no longer seen at the end after washout of Ba2+. Taken together, these points exclude a major contribution of Kir2.2 channels to the inwardly rectifying K+ current in juxtaglomerular cells. Kir2.1 and 2.2 subunits can form heteromultimers (Kubo et al. 2002; Preisig-Müller et al. 2002); however, the weak inactivation and Cs+ sensitivity of the current and the uniform single-channel conductance strongly suggest that Kir2.1 underlies the observed current in juxtaglomerular cells, although a (very) minor contribution of heteromultimers cannot be excluded.
Tedisamil and ciclazindol are K+ channel blockers with limited specificity. Tedisamil is a bradycardic and anti-anginal agent possessing class III anti-arrhythmic activity and has been reported to block several potassium currents including the voltage activated current (IKv), the calcium-activated potassium current (IK,Ca), as well as the ATP-sensitive current IK,ATP at concentations < 10 µM (Barrett et al. 2001 and references therein). Ciclazindol is an inhibitor of IK,ATP at concentrations < 10 µM and of IKv at concentrations up to 100 µM (Green et al. 1996). We found here that at 100 µM, these drugs inhibited the inwardly rectifying K+ current (Kir2.1) by 30 and 50%, adding to the low selectivity profile of these compounds.
Depolarization-activated channel.
In cell-attached patches a big (180 pS) channel was often observed at potentials positive to +40 mV (K+-rich buffer in the bath) or +80 mV (Na+-rich buffer in the bath). Although this channel was not characterized in detail, the high conductance in a solution where K+ and Cl– are the dominating small ions on both sides of the membrane, together with the reversal potential, which agreed with that of the Kir2 channel, point to a potassium channel. Moreover, the high conductance and the fact that the depolarization threshold required for channel activation was reduced when cells were bathed in the high-K+, Ca2+-containing solution point to the calcium-sensitive voltage-gated K+ channel of large conductance BKCa (KCa1.1). The presence of a cAMP-sensitive splice variant of this channel in isolated juxtaglomerular cells has recently been shown by Friis et al. (2003). In our preparation, this channel does not contribute to membrane potential at rest since no activity was detected in cell-attached patches at negative membrane potentials.
Immunocytochemistry and PCR
Collectively, the electrophysiological results showed a dominating role of Kir2.1 in the inwardly rectifying current in juxtaglomerular cells. This conclusion is supported by the detection of Kir2.1 but not Kir2.2 in the distal part of the afferent arteriole using specific antibodies (Fig. 7); controls in brain showed that the Kir2.2 antibody was functional. On the other hand, in isolated juxtaglomerular cells, there were signals for Kir2.1 and Kir2.2 mRNA in the PCR analyses (Fig. 8) pointing at a discrepancy between mRNA and protein levels. However, PCR analysis was not quantitative and the amount of Kir2.2 protein may have been below the detection level of the antibody.
Physiological importance of the inward current for juxtaglomerular cells
Following on from the work of Kurtz & Penner (1989), we have characterized here an inwardly rectifying K+ conductance in rat juxtaglomerular cells. Electrophysiological, immunocytochemical and PCR experiments show that, as in vascular smooth muscle, Kir2.1 is the dominating or even exclusive channel subtype underlying this conductance. Block of this channel by Ba2+ (this study) or angiotensin II (Kurtz & Penner, 1989) strongly depolarizes the cell, showing that this conductance is a determinant of the resting membrane potential of juxtaglomerular cells. In the rat at least, juxtaglomerular cells are endowed with L-type voltage-dependent Ca2+ channels (Friis et al. 2003) and the cytoplasmic Ca2+ concentration is a major (negative) regulator of renin secretion (reviews in Kurtz, 1989; Skøtt et al. 1991; Osswald & Quast, 1995; Persson, 2003). Hence, the inwardly rectifying conductance in juxtaglomerular cells is directly involved in the control of renin secretion and thereby in homeostasis of salt and water and the control of blood pressure.
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Supplementary material |
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DOI: 10.1113/jphysiol.2004.070359
http://jp.physoc.org/cgi/content/full/jphysiol.2004.070359DC1
and contains two supplementary figures.
This material can also be found at:
http://www.blackwellpublishing.com/products/journals/suppmat/tjp/tjp478/tjp478sm.htm
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References |
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TopAbstractIntroductionMethodsResultsDiscussionSupplementary materialReferences |
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