High Extracellular Ca2+ Hyperpolarizes Human Parathyroid Cells via Ca2+-activated K+ Channels*

Membrane potential has a major influence on stimulus-secretion coupling in various excitable cells. The role of membrane potential in the regulation of parathyroid hormone secretion is not known. High K+-induced depolarization increases secretion from parathyroid cells. The paradox is that increased extracellular Ca2+, which inhibits secretion, has also been postulated to have a depolarizing effect. In this study, human parathyroid cells from parathyroid adenomas were used in patch clamp studies of K+ channels and membrane potential. Detailed characterization revealed two K+ channels that were strictly dependent of intracellular Ca2+ concentration. At high extracellular Ca2+, a large K+ current was seen, and the cells were hyperpolarized (–50.4 ± 13.4 mV), whereas lowering of extracellular Ca2+ resulted in a dramatic decrease in K+ current and depolarization of the cells (–0.1 ± 8.8 mV, p < 0.001). Changes in extracellular Ca2+ did not alter K+ currents when intracellular Ca2+ was clamped, indicating that K+ channels are activated by intracellular Ca2+. The results were concordant in cell-attached, perforated patch, whole-cell and excised membrane patch configurations. These results suggest that [Ca2+]o regulates membrane potential of human parathyroid cells via Ca2+-activated K+ channels and that the membrane potential may be of greater importance for the stimulus-secretion coupling than recognized previously.

Extracellular free calcium ([Ca 2ϩ ] o ) regulates the secretion of parathyroid hormone (PTH) 1 from parathyroid cells through a cell surface Ca 2ϩ -sensing receptor (CaR) (1)(2)(3). Low [Ca 2ϩ ] o stimulates the secretion of PTH, whereas high [Ca 2ϩ ] o inhibits the secretion via activation of CaR. The second messenger mediating the inhibition is the free intracellular Ca 2ϩ ([Ca 2ϩ ] i ) (4). In this respect, the parathyroid cell differs from the majority of other endocrine cells, in which secretion is stimulated by high [Ca 2ϩ ] i . The molecular mechanisms by which [Ca 2ϩ ] i regulates the secretion of PTH are not well understood.
In various endocrine cells, the membrane potential is one of the key players in the cellular signaling, and depolarization of the cell membrane triggers hormone secretion. It is generally considered that resting membrane potential in the majority of excitable and endocrine cells is determined by K ϩ conductance and that high extracellular K ϩ induces membrane depolarization and thereby hormone secretion. Interestingly, high K ϩinduced depolarization of the parathyroid cell increases PTH secretion (5). In contrast, increased [Ca 2ϩ ] o, which causes inhibition of PTH secretion, has also been shown to cause membrane depolarization (6 -8).
The majority of previous electrophysiological studies have been performed in bovine or rodent parathyroid cells, and some studies indicate that highly relevant differences may exist between species. Furthermore, only two studies have applied modern electrophysiological techniques on human parathyroid cells (9,10). It has been shown that the parathyroid cell possesses several types of K ϩ channels, although the role of these K ϩ channels is not completely understood (9 -12).
The aim of the present study was to investigate the relationship between [Ca 2ϩ ] o , [Ca 2ϩ ] i , K ϩ channels, and membrane potential in human parathyroid cells with patch clamp technique. Detailed characterization of K ϩ currents revealed a strict dependence of [Ca 2ϩ ] i . At low [Ca 2ϩ ] i , only a small K ϩ current could be detected, and the cells were depolarized, whereas elevation of [Ca 2ϩ ] i resulted in dramatic enhancement of K ϩ current, and the cells were hyperpolarized. Taken together, these findings demonstrate that extracellular Ca 2ϩ regulates membrane potential of human parathyroid cells via Ca 2ϩ -activated K ϩ channels.

EXPERIMENTAL PROCEDURES
Parathyroid Samples-Parathyroid adenoma cells were obtained from 14 patients operated on for primary hyperparathyroidism. The median age of the patients at the time of the operation was 63 years (range 27-83). The preoperative median concentration of serum calcium was 2.78 mmol/liter (range 2.48 -3.13; reference range 2.20 -2.60), and the preoperative median concentration of serum PTH was 144 ng/liter (range 57-418; reference range 12-55). The median adenoma weight was 400 mg (range 100 -2300). The routinely performed histopathological evaluation of the parathyroid samples included hematoxylin-eosin staining and Oil Red O fat staining on frozen sections. Informed consent was obtained from all patients, and the study was approved by the local ethics committee of the Karolinska Hospital.
Preparation of Cells-Pieces of parathyroid adenomas were transported immediately after surgery to the laboratory at room temperature in sterile tubes. The tissue pieces were placed in sterile plates containing serum-free keratinocyte medium (Keratinocyte-SFM, Invitrogen) containing 5 ng/ml epidermal growth factor, 50 l/ml bovine pituitary extract, 100 units/ml penicillin G sodium, and 100 g/ml streptomycin sulfate (Invitrogen). The Ca 2ϩ concentration of this medium is 0.09 mM. This serum-free medium has been shown previously to be suitable for culturing human parathyroid cells for a considerable time, enabling physiological studies (13). Possibly remaining adipose or connective tissue was macro-* This study was supported by the Biomed BMH4-97-2225, the Swedish Medical Research Council, the Swedish Cancer Foundation, the Torsten and Ragnar Söderberg Foundations, the Nilsson-Ehle Foundation, the Robert Lundberg Foundation and, the Novo Nordisk Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ To whom correspondence should be addressed: Dept. of Surgical Sciences, Karolinska Institutet, Karolinska Hospital P9:03, SE-171 76 Stockholm, Sweden. Tel.: 46-8-517-73727; Fax: 46-8-33-15-87; E-mail: lars-ove.farnebo@kirurgi.ki.se. 1 The abbreviations used are: PTH, parathyroid hormone; CaR, Ca 2ϩsensing receptor; pF, picofarads. scopically removed so that only a small piece of homogenous parathyroid tissue remained. Single parathyroid cells were then plated into Nunclon™ Surface plates (NUNC™, Nalge Nunc International, Roskilde, Denmark) using the following procedure. First, one droplet of medium was added into a plate. The tissue was then held with forceps in contact with medium when mincing the tissue with a sterile scalpel. This enabled the cells to fall with gravity. The plates were then incubated for 1 h at 37°C before filling the plates with additional medium. The plates were incubated at 37°C until studied by patch clamp technique. The majority of the experiments were carried out within 2-24 h after the surgery, and no experiments were performed later than 36 h after surgery.
Immunostaining-To demonstrate the parathyroid identity of the cells, immunocytochemistry was performed using an antibody raised against the N-terminal part of the human PTH (amino acids 1-34, NovoCastra, Newcastle upon Tyne, UK, clone OP-4, mouse IgG 1 , diluted 1:600). Single parathyroid cells were plated as described above with the difference that glass slides instead of Nunclon™ Surface plates were used. Slides with cells were rehydrated, and endogenous peroxidase activity was suppressed with 0.6% H 2 O 2 . A standard retrieval technique was used prior to incubation with the PTH antibody. The slides with cells were heated in citrate buffer, pH 6.0, for 20 min at 200 watts in a household microwave oven to intensify the immunostaining. The primary antiserum was incubated overnight, and antigen-antibody binding was visualized according to the avidin-biotin peroxidase complex method, following the instructions of the manufacturer (Vectastain Elite kit, Vector Laboratories, Burlingham, CA). Diaminobenzidine was used as chromogen, and counterstaining was done with Mayer's hematoxylin. This showed that the cells were immunoreactive against PTH, thus demonstrating their parathyroid origin.
Electrophysiology-Single-and whole-cell currents were recorded with the patch clamp technique (14), using an Axopatch 200 patch clamp amplifier (Axon Instruments Inc., Foster City, CA). Whole-cell currents were recorded using the conventional or perforated patch wholecell configuration of the patch clamp technique, and whole-cell currents are expressed as total current divided by cell capacitance, if not stated otherwise. Single channel studies were performed using inside-out and cell-attached configurations. Current traces are displayed according to the convention, upward deflections denoting outward currents. All experiments were performed at room temperature (22-24°C).
Solutions-The extracellular solution contained 138 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl 2 , 5 mM HEPES-NaOH, and 2.6 mM CaCl 2 . The pH of this solution was 7.4. The standard intracellular solution consisted of 125 mM KCl, 1 mM MgCl 2 , 10 mM EGTA, 25 mM KOH, and 5 mM HEPES-KOH at pH 7.15. The free Ca 2ϩ concentration of this solution is calculated to be Ͻ10 nM using the MaxChelator software version 2.10, which uses the method described by Bers et al. (15). A total of 100 l of 1 M CaCl 2 was added to 10 ml of intracellular solution to produce the required free Ca 2ϩ concentration of 10 M. In the perforated patch experiments, the pipette solution contained 10 mM KCl, 76 mM K 2 SO 4 , 10 mM NaCl, 1 mM MgCl 2 , 10 mM HEPES-NaOH at pH 7.35 (KOH), and 200 g/ml amphotericin B dissolved in dimethyl sulfoxide (Me 2 SO). The final concentration of Me 2 SO was less than 0.1%. All chemicals were of analytical grade and obtained from Sigma.
Data Analysis-For analysis of mean current, the channel recordings were filtered at 2 kHz (Ϫ3 db value, 8-pole Bessel filter; Frequency Devices, Haverhill, MA), digitized at 10 kHz and stored in a computer, using an Axon Instrument analogue digital converter (TL-1). The data are presented as mean values Ϯ S.E. Channel activities were compared using Student's t test or analysis of variance for multiple groups, and p values less than 0.05 were regarded as significant.

RESULTS
To further characterize the function of K ϩ channels in parathyroid cells, a series of patch clamp experiments were performed on human parathyroid adenoma cells. The configurations applied were whole-cell, cell-attached, excised patch, and perforated patch. The findings are summarized in Figs. 1-4.
The Activity of K ϩ Current in Human Parathyroid Adenoma Cells Depends on [Ca 2ϩ ] i -In the whole-cell configuration, human parathyroid cells were voltage-clamped at Ϫ80 mV and subsequently depolarized according to the protocol presented in Fig. 1 Fig. 1, A and B). The K ϩ current was almost completely blocked by the addition of extracellular 10 mM tetraethylammonium (data not shown). When [Ca 2ϩ ] i was clamped, changes in [Ca 2ϩ ] o did not alter K ϩ currents (p ϭ 0.56; Fig. 1C (Fig. 1C). Taken together, this indicates that human parathyroid adenoma cells display K ϩ channels, which have Ca 2ϩ binding site/sites located on the inside of the cell membrane, and that these channels are activated by [Ca 2ϩ ] i . Since the internal milieu of the cell is modulated in this configuration with the possible loss of crucial intracellular signal transduction molecules, we continued the characterization of the nature and function of the K ϩ channels in the following experiments.
Human Parathyroid Adenoma Cells Have at Least Two Types of Ca 2ϩ -activated K ϩ Channels-In a previous study on bovine cells, a big conductance 175-pS K ϩ -selective Ca 2ϩ -activated channel maximally activated at ϳ160 nM [Ca 2ϩ ] i has been reported (11). In human parathyroid adenoma cells, a 35-pS FIG. 2. Two types of K ؉ channels are seen in excised membrane patches. A, the voltage protocol used, in which the freshly excised membrane patches were initially depolarized to ϩ80 mV and then repolarized with 20 mV stepwise every 2 s. Intracellular K ϩ solutions in bath and pipette were used. As shown in B, at low free Ca 2ϩ concentrations, in the bath (corresponding to [Ca 2ϩ ] i ), few K ϩ channels are seen at ϩ80 mV, whereas at Ϫ80 mV, no K ϩ channel openings are recorded. As shown in C, this is more obvious at the expanded time scale. The top traces from B are further expanded in i and ii, respectively. The addition of Ca 2ϩ (10 M) induced a marked increase of channel activity at both ϩ80 mV and Ϫ80 mV. D, in enlargement, two Ca 2ϩ -activated K ϩ channels. The large conductance channel is marked K BK , and the low conductance channel K SK . As shown in E, the current-voltage relation for K ϩ currents displays an outwardly rectifying behavior. In B-D, the dashed line and arrowhead indicate zero current line. and a small 12-pS K ϩ channel have been described that are both sensitive to [Ca 2ϩ ] i (9). Here we used the excised patch configuration to study single K ϩ channels and to further characterize the Ca 2ϩ -activated K ϩ channels seen in the whole-cell configuration. In Fig. 2, single K ϩ channels from human parathyroid adenoma cells are displayed. In the presence of low Ca 2ϩ in the bath solution, i.e. [Ca 2ϩ ] i , only few K ϩ channels were seen in parathyroid adenoma cells (Fig. 2, B and C). Single channels were only possible to evaluate at low [Ca 2ϩ ] i and ϩ80 mV, and the single channel conductances of these channels were 168 Ϯ 10 pS and 48 Ϯ 4 pS, respectively (Fig.  2D). The channel open times for the big and small conductance channels were 2.1 Ϯ 0.9 and 9.8 Ϯ 7.0 ms. Both K ϩ channels appear to be voltage-sensitive since they were activated at low [Ca 2ϩ ] i and ϩ80 mV (Fig. 2C). The addition of high [Ca 2ϩ ] i solution enhanced K ϩ channel activity dramatically at both ϩ80 mV and Ϫ80 mV (Fig. 2, B and C). Current-voltage relation for K ϩ currents in inside-out recordings displayed an out-  Fig.  2A (with the difference that the steps were run from Ϫ80 mV to ϩ80 mV in the pipette) was applied. C, in expanded current traces, a small negative shift of the membrane potential at 2.6 mM [Ca 2ϩ ] o since symmetrical K ϩ solutions were used. In i, zero current was measured at pipette potential 0 mV, equivalent with a membrane potential of 0 mV, whereas in ii, zero current was at Ϫ20 mV, indicating a membrane potential of Ϫ20 mV. In B and C, arrowheads indicate zero current, and i and ii represent current traces at 0.5 and 2.6 mM [Ca 2ϩ ] o , respectively. wardly rectifying behavior (Fig. 2C). These experiments demonstrate that there are at least two types of K ϩ channels present in the parathyroid adenoma cell membrane, both of which are activated by [ In Fig. 3, the cell-attached configuration was used. An excess of K ϩ was added to extracellular solution to decrease the influence of endogenous membrane potential on K ϩ currents. In the extracellular solution, [K ϩ ] was 50 mM, whereas the pipette solution contained 150 mM K ϩ . Pipette potential was held at 0 mV. At 2.6 mM [Ca 2ϩ ] o , K ϩ channel activity was seen, whereas lowering of [Ca 2ϩ ] o to 0.5 mM resulted in significant decrease of channel activity. Intermediate [Ca 2ϩ ] o caused recovery of channel activity, but not to the same extent as seen in the presence of 2.6 mM (Fig. 3A). Base-line drift seen in low [Ca 2ϩ ] o is likely to be due to a change of membrane potential and was noted in all recordings. Since 50 mM K ϩ in extracellular solution was used, this approach allows estimation of membrane potential even in the cell-attached configuration, in which no access to the cell interior is obtained. At 0.5 mM [Ca 2ϩ ] o , zero current was measured at pipette potential 0 mV, equivalent with a membrane potential of 0 mV. At 2.6 mM [Ca 2ϩ ] o , zero current was at Ϫ20 mV, indicating a membrane potential of Ϫ20 mV (Fig. 3C). This small membrane potential shift is expected since extracellular K ϩ concentration had been adjusted to a total of 50 mM. Taken together, these experiments show that [Ca 2ϩ ] o modulates K ϩ channel activity in intact parathyroid cells and furthermore show that membrane potential varies upon changes of [Ca 2ϩ ] o .
Further support of this observation was gained by using the perforated patch configuration. Using this technique, the cells were current-clamped, and membrane potential was measured while changing the [Ca 2ϩ ] o . As expected and in agreement with our results in the whole-cell and cell-attached configurations, the K ϩ currents increased significantly from 5.6 Ϯ 5.6 pA/pF to 33.0 Ϯ 6.9 pA/pF when [Ca 2ϩ ] o was elevated from 0.5 mM to 2.6 mM (*, p Ͻ 0.05; n ϭ 6; measured at ϩ70 mV, Fig. 4B). At low [Ca 2ϩ ] o , the cells were depolarized, whereas an increase of [Ca 2ϩ ] o lead to hyperpolarization of the cell membrane (Fig.  4C). Compiled data showed that in the presence of low [Ca 2ϩ ] o , the cell membrane was depolarized to Ϫ0.1 Ϯ 8.8 mV, whereas following increase of [Ca 2ϩ ] o to 2.6 mM, the cells were hyperpolarized to Ϫ50.4 Ϯ 13.4 mV (***, p Ͻ 0.001; n ϭ 12; Fig. 4). DISCUSSION Two important elements influencing the stimulus-secretion coupling in various excitable and endocrine cells are [Ca 2ϩ ] i and membrane potential. The parathyroid cell is an exceptional secretory cell since increases in [Ca 2ϩ ] i are associated with inhibition of PTH secretion. This contrasts the general principle by which elevated [Ca 2ϩ ] i stimulates secretion. The parathyroid cell is also remarkable because high [Ca 2ϩ ] o , which inhibits PTH secretion, has been postulated to cause depolarization of the parathyroid cells (6 -8). Therefore, further studies are obviously needed to understand the relation between [Ca 2ϩ ] i , membrane potential, and PTH secretion. Another reason for renewed investigation of the role of the membrane potential is a most interesting finding of Oetting et al. (16), which seems to have gone unnoticed by other investigators. This group showed that permeabilization of parathyroid cells using electroshock revealed classical stimulus-secretion coupling, i.e. augmented PTH secretion in response to increased Ca 2ϩ , when membrane potential was absent (16).
One reason for the contradictory findings may be that previous results were obtained by applying impalement of single parathyroid cells by microelectrodes or indirectly using a voltage-sensitive dye (6 -8). Much effort has also been put into experiments searching for K ϩ channels with characteristics that could explain the paradoxical effect of [Ca 2ϩ ] o on the membrane potential (10 -12, 17). However, no confirmation of the effect of [Ca 2ϩ ] o on the membrane potential of single cells has been obtained using modern patch clamp technique. On the contrary, some uncertainty regarding the effect of [Ca 2ϩ ] o on the membrane potential has been expressed in more recent studies (9,12).
The present study shows that there are at least two types of K ϩ channels present in human parathyroid adenoma cells. Detailed characterization of these channels reveal that both are activated by [Ca 2ϩ ] i and that the channels are voltage-dependent. The finding that [Ca 2ϩ ] o can alter K ϩ currents only via its effect on [Ca 2ϩ ] i indicates that K ϩ channels present in the parathyroid cells must have Ca 2ϩ binding site/sites located on the inside of the cell membrane. The intriguing question is then the relationship between CaR and the Ca 2ϩ -sensitive K ϩ channels. Previous investigations have shown that the activation of CaR is coupled to Ca 2ϩ -sensitive K ϩ channels in human lens and murine osteoblastic cells (18,19). In bovine parathyroid cells, the effect of [Ca 2ϩ ] o , supposedly via activation of CaR, has been shown to have both an activating and a blocking effect on different K ϩ channels, and the possible role of each channel in the stimulus-secretion coupling is not known (12,17). Our results are consistent with the findings of Kanazirska et al. (12), indicating that the K ϩ currents are indirectly activated by increased levels of [Ca 2ϩ ] o and linked to an intracellular transduction pathway involving an increase in [Ca 2ϩ ] i . We further show that at high extracellular Ca 2ϩ , a large K ϩ current is seen, and the cells are hyperpolarized, whereas lowering of extracellular Ca 2ϩ results in a dramatic decrease in K ϩ current and depolarization of the cells. The finding that human parathyroid adenoma cells are depolarized in low [Ca 2ϩ ] o is compatible with the study by Dempster et al. (5), who showed that high K ϩ increases hormone secretion from the parathyroid cells. Also in line with our results is the study by Oetting et al. (16), which showed that the inhibiting effect of [Ca 2ϩ ] i on PTH secretion disappeared when membrane potential was absent. Together these data clearly indicate that the stimulation of PTH secretion is coupled to the depolarization as in other endocrine cells. In insulin-producing ␤-cells, the stimulation of insulin secretion is induced by the closure of K ATP channels, depolarization of the ␤-cell plasma membrane, and opening of the voltage-gated the Ca 2ϩ -channels (20). In rat lactrotrophs, erg-like inward-rectifying K ϩ current and Ca 2ϩ -activated K ϩ current are coupled to the depolarization stimulating prolactin secretion (21), whereas in rat corticotrophs, corticotropin-releasing hormone triggers membrane depolarization via a protein kinase A-dependent closure of K ϩ channels, ultimately leading to the release of adrenocorticotropin (22). In conclusion, we propose that the molecular mechanism for inverted relation between [Ca 2ϩ ] i and PTH secretion is the Ca 2ϩ -activated K ϩ channel, which controls the membrane potential.