Cell Calcium (1987) 8, 377-383 ‘i;a Longman Group UK Ltd 1987

Ca-;"- AND VOLTAGE-DEPENDENT K" CONDUCTANCE IN DISPERSED PARATHRYOID CELLS

Antonio Castellano, Elizabeth Pintado* and Jose L6pez-Barneo

Departamentos de Fisiologia y (*)Bioquimica, Facultad de Medicina, Avda. Sanchez Pizjuan, 4, 41009, Sevilla, Spain (reprint requests to J L-B).

The membrane ionic conductances of dispersed parathyroid cells kept in primary culture were studied using the "whole-cell" and "inside-out excised patch" variants of the patch-clamp technique. The major component of the total current was a voltage-dependent outward K,+ current without an appreciable inward current. The amplitude of the K' current was markedly reduced when free internal Ca"+ was buffered by addition of 10 mM EGTA. Recordings of single-channel current in excised membrane patches revelaled the presence of K' channels with large unitary conductance (200 pS in symmetrical 130 mM K' solutions) which were also activated by depolarization when internal CaL-' concentration was about lo-s-1O'-'. M. At any membrane voltage these channels were closed most of the time at internal Ca" concentrations lower than lo--10 M. These results demonstrate the existence of a Ca.",+- and voltage- dependent K' permeability in parathyroid cells which may participate in the unusual membrane potential changes induced by alterations of external Ca2' and, possibly, in the regulation of parathormone secretion.

The inverse relationship existing between external Cai'* concentration and parathyroid hormone (PTH) secretion is well studied both in viva (1) and in vitro (2,3). This response is different from that observed in other secretory systems where Cad*' influx is required for exocytosis (4). From an electrical viewpoint parathyroid cells are also unusual since they are fully polarized at low external Ca"' and depolarized when Ca"" increases (5,6). The relationship between the membrane potential and external CaX-' i- =, very steep and a change of CaZ+ from 1 to 2 mK, which is known causes a strong inhibition of secretion (2,3), produces a depolarization of almost 40 mV (6). Although valuable information about the regulation of PTH secretion by internal Ca"“ and other mediators has been recently obtained (7-lo), the membrane ionic permeabilities and the possible relation between the electrical activity

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and secretion in parathyroid cells are still unknown. We have studied the ionic conductance6 existing in dialyzed parathyroid cells under voltage-clamp and found that CL++ and voltage-dependent K+ channels mediate the dominant voltage-dependent current of these cells. This K’ conductance represents a link between cell metabolism and membrane ionic permeability and may contribute to the generation of the membrane potential changes induced by alterations in external Ca”” and to the regulation of secretion.

Although some preliminary recordings were obtained in bovine parathyroid cells most experiments were done on cells obtained from rats due to the availability of this animal species and because most previous electrophysiological work on the parathyroid gland has been done in rodents (5,6). The current described here was found to be similar in both preparations. The preparation of bovine cells followed a procedure previously reported (ll), thus only a brief description of the method used to dissociate rat parathyroid cells is given here. Six to eight glands were removed from Wistar rats, either sex, weighing loo-150 g and placed for 5 min in a Ca”+ and Mgp+ -free solution containing 140 mM NaCl, 5 mM KCl, 10 mK Hepes and 0,5 mM BGTA. Afterwards each gland was cut into two or three pieces and changed to a solution of the same composition but without BGTA added and with 2 mM Mgcl~, 5 mM glucose, 3 mg/ml collagenase and 1 mg/ml trypsin. After 5-10 min the tissue was washed twice with a solution containing albumin (1 mg/ml) and resuspended in Ham F-10 culture medium with 5% BSA and antibiotics added. Mechanical dispersion of the cells was done in the culture medium using fire polished Pasteur pipettes. Cells were plated on cover slips treated with polylysine and placed in a CO2 incubator. Total and single- channel ionic currents were measured in cells lo-24 hr after plating using the “whole-cell” and “inside-out excised patch” variants of the patch-clamp technique (12). Successful recordings have been obtained from 32 cells of 11 different cell preparations. The composition of solutions used in each experiment are indicated in the figure legends. Current collected by the patch pipette was recorded with an amplifier built in our laboratory, digitalized and stored on an IBM-PC computer. Because input resistance of the cells was between 2-5 Gohm, only a tiny leakage current was present and therefore lineal subtraction was not used. Series resistance and electrode capacitance were electronically compensated as indicated in (13).

Ionic currents recorded in parathyroid cells dialyzed with a high K+ solution and with equivalent Cl”- concentrations in both sides of the membrane are illustrated in Fig. 1. Depolarizing voltage steps elicited outward currents without an appreciable component of inward current. The direction and kinetic of this current suggest that it is a K--current; in addition, it totally disappeared when internal KC1 was substituted by CsCl, The amplitude of the K+ current was larger and the activation kinetic faster as the size of the voltage pulse increased. On large depolarizations there was some inactivation during the 50 ms pulse. This

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Elg;ure 1. Whole-cell currents recorded in parathyroid cells with a patch clamp pipette. A. Superimposed current recordings generated by voltage steps to the membrane potential values indicated in each trace from a holding potential of -70 mV. Capacity currents recorded before and after breaking into the cell are illustrated in B. These last recordings were elicited by a positive pulse of 10 mV. The solutions used in this experiment had, in mK: external solution (130 NaCl, 2.7 KCl, 2 Mgcl~, 0.5 CaCL, 10 Hepes) and internal solution (140 KU, 2 M&la, 10 Hepes, 0.5 EGTA). pH was 7.4 and temperature 20-220 C. C Sr D. Same experimental protocol in a different cell but with an internal solution that had 10 mM EGTA. E, Normalized current amplitude as a function of the membrane potential for traces shown in A (dots) and C (stars).

K+ current was, as shown in Pig. 1 (A B C), also dependent of the internal Caz+ concentration. The average peak current amplitude elicited by voltage pulses to t50 mV was 1.451tO.5 nA (mean*SD, n=4) in an internal solution without Ca. 2+ added but with 0.5 mM EGTA. In this solution the estimated free Caz+ was about 10'-w-10-9 M assuming a contaminating calcium concentration of lO_~~ M (14). The peak current amplitude in presence of 10 mM EGTA (estimated free Cazz+ lower than 10‘-Jc' M) was 0.18rtO.08 nA (n=Q). Ionic currents shown in Figs. 1 A h C were obtained from cells with a similar size as indicated by the values of membrane capacitance calculated from the capacity transients recorded in each case after breaking Into the cells: 2.14 pF in the cell dialyzed with 0.5 mM BGTA (B) and 2.36 pF in the cell dialyzed with 10 mM EGTA CD). The average membrane capacitance of parathyroid cells was 2.9iO.9 pF (n=14) which, assuming a membrane capacitance of 1 pF/cm" (15), results in an average cell diameter of 10 pm, This value is similar to the one directly measured under the microscope and equivalent to values previously reported for rat parathyroid cells (16). Normalized current vs. voltage plots corresponding to the traces of Figs. 1 A & C are shown in Fig. 1E. These curves do not have the N-shape typical of Ca"+- dependent K' currents observed in cells with functional calcium channels (17) which further suggests the absence of Ca"+ influx during depolarization in parathyroid cells. The properties of the macroscopic currents recorded in parathyroid cells and the shape of the I-V plot in the range of -20 to t70 mV are similar to Ca"+-activated K' currents recorded In chromaffin (17') and adrenocortical cells (Tabares and

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w2. Single-channel currents recorded from inside-out excised patches of parathyroid cells. A. Current recorded at the membrane potentials indicated on the left of each trace. The closed (cl and open (0) states of the channel are also indicated in the figure. The compositicn of solutions in this experiment was, in mM: solution in the external surface of the membrane (130 KCl, 2 MgCla, 0.1 CaClz, 10 Hepes) and solution in the internal surface of the membrane (140 NaCl, 2:7 KCl, 2 MgCls:,, 10 Hepes). pH was 7.4 and temperature 20-220 C. B. Current recorded in the same membrane patch after switching to an internal solution of the same composition but with 4 mM EGTA added. C. Single- channel current amplitude as a function of the patch membrane potential in asymmetrical (filled symbols) and symmetrical (130 mM, open symbols) K+ concentrations. The continuous line is the theoretical I-V curve of a K+ selective channel in asymmetrical (130 mM/2.7 mM) K+ concentrations.

L6pez-Barneo, unpublished) after wash-out of calcium channels. As also noted in these other preparations, EGTA was rather inefficient to block the K' current and an appreciable outward current was seen with an estimated Ca"-' concentration lower than 10.-'c' N. Fig. 2 shows high gain recordings of current flowing through an inside-out membrane patch excised from a parathyroid cell in contact with an external solution containing 130 mM K-' and a solution with 2.7 mN K" bathing the internal surface of the membrane. Membrane current switched between two discrete levels due to the opening and closing of the channel included in the patch. Although sub-states of intermediate amplitude were observed, most steps of intermediate current amplitude probably represented events lasting less than 3-5 ms that were partially filtered due to the slow sampling rate used to digitalize the analog signal. When the internal solution had no Ca" added, which resulted in a free Ca'+ concentration close to lo--", M (la), transitions from the closed to the open state

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appeared more frequently as the membrane potential was progressively made more negative (compare for example traces at 0 and -60 mV). Single-channel current amplitude also became larger as the membrane was gradually polarized and the driving force for K' ions increased. Exposure of the same membrane patch to an internal solution with 4 mM EGTA added (B, estimated free Ca.2+ concentration of about 10'--"z' 1) caused an almost permanent closure of the channel and brief openings only appeared at positive membrane potentials. The current-voltage plot for this experiment is shown in Fig. 2C (filled symbols). In this figure the current-voltage relationship of the same channel recorded in a different patch in symmetrical (130 mM) K' solutions is indicated by the open symbols. The equilibrium potential was 0 mV and the value of the single-channel conductance was near 200 pS. In Fig. 2C it is also represented the theoretical current-voltage curve of a K.+ selective channel (continuous line) calculated by the Goldman, Hodgkin 8 Katz ecuation (19) with asymmetrical (130 mM/ 2.7 mM) K' concentrations. Comparision of the theoretical curve and the experimental data (filled symbols) shows a clear deviation from independence which, although with less extent, has been also observed in the large Ca"'+-activated K-' channels of rat muscle (19) and chromaffin (20) cells. The deviations from independence may be due to blockade by Na' ions which were highly concentrated in the solution in contact with the internal surface of the membrane (19,20), however, it may also reflect an abnormal Na+ permeability of the channels in parathyroid cells. A theoretical curve calculated with a PN~/PL ratio of 0.3 (not included in the figure) superimposed almost perfectly the dotted line drawn over the experimental data points (filled symbols).

These results demonstrate that the major voltage-dependent ionic conductance in parathyroid cells is a Ca'+- and voltage-activated K' conductance. These cells have K+ channels of large unitary conductance which resemble the Ca*"*'--activated K" channels found in several electrically excitable cells (17-20) although some differences may exist. Variabilities in the unitary conductance and kinetic properties of this channel type among different tissues have been observed (17,19,20). Parathyroid cells are unexcitable (6) and cytosolic Ca'" concentration, as measured with quin 2, directly depends on the level of external Ca-"'-+ (8,9). The free CaS+ concentrations estimated in our experiments and those measured with fluorescent probes are not totally equivalent but the available information can support the idea that the Ca-“'-activated K-' channels may participate in the depolarizing response of parathyroid cells to an elevation of external Car'+ and other divalent cations (5,6). The average internal Ca" concentration measured in parathyroid cells after equilibration with low (0.5 raM) external Ca-:+ is about 2 x lo'-' M (8,9), that is a concentration at which most K' channels may be open and therefore generate a large resting potential. Intracellular recordings have shown that at this external Ca"-' concentration parathyroid cells have a membrane potential of about -75 mV (6) and that they are highly permeable to K' ions (5,6). Exposure to high external Ca'i:" is known to induce a large increase of the average internal Caz'+ concentration (8,9). It is conceivable to hypothesize that in this situation the internal Ca=+ levels near the membrane may reach values of several tens of FM that it is known can inactivate (18) or block (21) some K" channels and thus produce membrane depolarization,

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The data presented in this report gives for the first time information about the ionic currents of parathyroid cells and demonstrate the existence of Ca2+-activated K+ channels in their membrane. Future experimental work must be directed to establish the possible relationships existing between external and internal Ca"+, membrane ionic permeability and secretion and to elucidate the general scheme of stimulus-secretion coupling in these unusual secretory cells which is still very incomplete.

Support for this study was provided by grants from CAICYT (3464- 83), Spanish-USA Joint Committee (CCB8504-002) and FISSS (8411080).

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3. Sherwood, L.M., Herman, I. and Basset, C.A. (1970). Parathyroid hormone secretion in vitro: regulation by calcium and magnesium ions. Nature, 225, 1056-1058.

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5, Bruce, B.R. and Anderson, N.C. (1979). Hyperpolarization in mouse parathyroid cells by low calcium. Am. J. Physiol., 236(l), C15-C21.

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7. Getting, M., Le Boft, W., Swiston, L., Preston, J. and Brown, E. (1986). Guanine nucleotides are potent secretagogues in permeabilized parathyroid cells. FEBS Lett. 208, 99-104.

8. Nemeth,, B.F. and Scarpa, A. (1986). Cytosolic Ca" and the regulation of secretion in parathyroid cells. FEBS Lett. 203, 15-19.

9. Shoback, D., Thatcher, J., Leombruno, R, and Brown, E. (1983). Effect of extracellular Ca"+ and MgZ+ on cytosolic Ca'* and PTH release in dispersed bovine parathyroid cells. Endocrinology, 113, 424-426.

10. Muff, R. and Fischer, J.A. (1986). Parathyroid hormone secretion does not respond to changes of free calcium in electropermeabilized bovine parathyroid cells, but is stimulated with phorbol ester and cyclic AMP. Biochem. Biophys. Res. Commun. 139, 1233-1238.

11. Wallace, J. and Scarpa, A. (1982). Regulation of parathyroid hormone secretion j_n vitro by divalent cations and cellular metabolism. J. Biol. Chem. 252, 4108-4117.

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12. Hamill, O.P., Marty, A., Beher, E,, Sakmann, B. and Sigworth, F. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Archiv. 381, 85-100.

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14. Martell, A.E. and Smith, R.M. (1974). Critical stability constants, vol. 1; Amino Acids. Plenum Press, New York.

15. Cole, K.S. (1968). Membranes, Ions and Impulses, University of California Press, Berkeley.

16. Roth, S.I. and Raisz, L.G. (1964). Effect of calcium concentration on the ultrastructure of rat parathyroid in organ culture. Lab. Invest. 13, 331-345.

17. Marty, A. and Neher, E. (1985). Potassium channels in cultured bovine adrenal chromaffin cells. J. Physiol., 367, 117-141.

18. Tabares, L., L6pez-Barneo, J. and de Miguel, C. (1985). Calcium- and voltage-activated potassium channels in adrenocortical cell membranes. Blochim. Biophys. Acta, 814, 96-102.

19. Blatz, A. and Magleby, K.L. (1984). Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle. J. Gen. Physiol. 84, 1-23.

20. Yellen, G. (1984). Ionic permeation and blockade in Ca'+-activated K' channels of bovine chromaffin cells. J. Gen. Physiol. 84, 157-186.

21. Latorre, R., Vergara, C. and Moczydlowski, E. (1983). Properties of a Ca"-activated K' channel in a reconstituted system. Cell Calcium, 4, 343-357.

received 28 April 1987 revised version received 8 July 1987

accepted 27 July 1987

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