Characterization of an Inwardly Rectifying Potassium Channel in the Rabbit Superior Lacrimal Gland

George H. Herok,1'2 Thomas J. Millar,1'2 Philip J. Anderton,1 and Donald K. Martin13

+ PURPOSE. TO characterize the properties of an inwardly rectifying K (KlR) current in fresh, enzymatically isolated acinar cells from the rabbit superior lacrimal gland. METHODS. New Zealand White rabbits of both sexes were killed by injecting 45 mg/kg pentobarbital sodium, and the glands were excised. Single acinar cells were isolated enzymatically from these glands. Standard patch-clamp techniques were used to record ion currents.

RESULTS. Hyperpolarizing voltages evoked Km currents that had a conductance of 2.7 ± 0.16 nS (n = 6) in the range —50 mV to — 160 mV. The Km current was activated with steep voltage dependence on hyperpolarization, and the conductance was approximately dependent on the square root of the external K+ concentration. Increasing the pipette Ca2+ concentration from 10~9 M to 10~6 M increased the conductance to 5.3 ± 0.45 nS (n — 7). Internal substitution of K+ with various cations + + + gave the following permeability sequence: K (1.0) > Rb (0.83) > Ii (0.15). The Km current was inhibited by Ba2+ (100 jum), tetraethylammonium (10 mM), and Cs+ (5 mM) but was insensitive to 4-aminopyridine (5 mM). The single-channel conductance was 43 ± 2.7 pS (n = 11), and the relationship between between single-channel conductance (y) and external K+ concentration ([K],,) is 37 2 given by: y = 7.04[K]° (pS, r = 0.99, P < 0.05). The relationship between [K]o and zero current 2 potential (£rev) is given by: £rev = 35.5 log[K]0 - 77.8 (mV; r = 0.99, P < 0.05).

CONCLUSIONS. The Km current identified in these lacrimal acini has a similar dependence on [K]o as other inward rectifiers of excitable tissues and exocrine glands. However, this study highlights that there are interspecies variations and similarities between Km channels that could be related to their individual physiological roles. The authors' investigations suggest that one role of the Km channel in the rabbit superior lacrimal gland acinar cells is to set and stabilize the resting membrane potential. However, this Km channel may also be involved in secretion, as has been shown to occur in the sheep parotid gland. (Invest Ophthalmol Vis Sci. 1998;39:308-3l4)

nwardly rectifying currents have been described in a wide secretion in the absence of nerve stimulation and a unique variety of excitable cells, including skeletal muscle1"4 and characteristic of this salivary gland.19 5 12 + Icardiac muscle, " and nonexcitable cells, including renal It is of interest that this inwardly rectifying K (KlR) distal tubule,13 rat hepatocytes,14 lens epithelial cells,1516 rat channel has not been previously reported in other lacrimal osteoclasts,17 rabbit parietal cells,18 sheep parotid,19 and reti- glands. Therefore, in the present study, we investigated the nal Muller cells.20 In these tissues, they have been reported to properties of this channel in the rabbit superior lacrimal gland. have several physiological roles. First, the inward rectifier is involved in setting and stabilizing the resting membrane po- MATERIALS AND METHODS tential.15'21 Second, in some cells, including glial cells,22 its function is to buffer transient increases in extracellular K+ Preparation of Lacrimal Gland Cells concentration. Third, it may be involved in removing K+ to New Zealand White rabbits of both sexes (1.5-2.5 kg) were prevent its accumulation in the cell caused by increased activ- killed by injecting 45 mg/kg pentobarbital sodium (Nembutal; ity of the Na+-H+ antiporters.23 In this manner, accumulated Abbott Laboratory, Irving, TX) administered intravenously in K+ ions may then leave through the inward rectifier. Finally, in accordance with the Australian National Health and Medical the sheep parotid, an inwardly rectifying K+ channel is Research Council guidelines for animal ethics and the tenets of thought to be involved in spontaneous secretion, which is the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The superior lacrimal glands were excised and placed in a petri dish containing 5 ml Dulbecco's modified Eagle's medium supplemented with L-glutamine (0.584 g/1) and From the 'Co-operative Research Centre for Eye Research and D-glucose (4.5 g/1). The glands were cut into small pieces and Technology, University of New South Wales, Kensington, Australia; the gently centrifuged for 1 minute at 25g using a hand-operated 2Department of Biological Sciences, University of Western Sydney/ Nepean, Sydney, Australia; and the 'Department of Health Sciences, centrifuge (Model 1011; Hettich, Tuttlimgene, Germany). The University of Technology, Sydney, New South Wales, Australia. supernatant was discarded, and the glandular fragments were Submitted for publication June 9, 1997; accepted November 6, then incubated in 1 mg/ml trypsin (Type IX; Sigma Chemical, 1997. St. Louis, MO) for 8 minutes at 37°C in an oscillating water bath Proprietary interest categories: C7 (GH), C4 (TM, PA), C2 (DM). (130 oscillations/minute). Reprint requests: Donald K. Martin, Department of Health Sci- ences, University of Technology, Sydney, P.O. Box 123, Broadway, After centrifugation of the trypsin solution for 1 minute at New South Wales 2007, Australia. 25g, the supernatant was discarded and the cellular pellet was

Downloaded from iovs.arvojournals.org on 09/28/2021 IOVS, February 1998, Vol. 39, No. 2 Potassium Channel in Rabbit Lacrimal Gland 309

rinsed with 1 mg/ml soybean trypsin inhibitor (Type 1-S; The free Ca2+ in the pipette solutions was adjusted using Sigma). The cellular pellet was then transferred to a 25-ml different amounts of EGTA27 to achieve concentrations of Erlenmyer flask and incubated in the standard NaCl-bathing 1.0 X 10"6 M (1.54 mM Ca2+, 1.73 mM EGTA) or 1.0 X 10"9 solution (see Solutions and Chemicals) containing 200 U/ml M (no added Ca2+, 5 mM EGTA). All chemicals used in the collagenase (Type II; Worthington Biochemical, Freehold, NJ) study were obtained from Sigma except 4-aminopyridine, and 600 U/ml hyaluronidase (Type V; Sigma) for 48 minutes at which was obtained from Fluka, Buchs, Switzerland. All chem- 37°C in the oscillating water bath. The enzyme solution was icals used in the experiments were of AR grade or higher. filtered through a 100-/u,m, nylon-mesh filter and centrifuged for 2 minutes at 25g. The cellular pellet was washed twice with the standard NaCl bath solution containing 2 mg/ml bovine RESULTS serum albumin (Sigma). After each wash, the cells were centrifuged for 2 minutes at 25g, and the supernatant was dis- The resting cell membrane potential under physiological con- carded. After the second wash, the cells were resuspended in ditions (KCl-rich pipette solution and NaCl-rich bath solution) 6 ml Dulbecco's modified Eagle's medium containing 10% fetal was — 40 ± 2 mV (n = 17). This was similar to the resting bovine serum (P.A. Biologicals, Trace Biosciences, Sydney, membrane potential of —37 ± 8 mV (n = 50) obtained by Australia). All of the results described in this study were re- Kikkawa28 using intracellular electrodes. Figure 1A shows the corded from these freshly isolated acinar cells. steady state, whole-cell, current-voltage (I-V) relationship from a sample of 12 single rabbit superior lacrimal gland acinar Patch-Clamp Recording and Analysis cells before and after the addition of 10 mM tetraethylammonium (TEA) to the extracellular bath solution. This caused a Whole-cell and single-channel currents were recorded by stan- 24 shift of the reversal potential from -39 ± 2 mV to -1 ± 2 mV. dard patch-clamp techniques at room temperature The total membrane conductances for inward currents was (20-25°C). The channel cvirrents were amplified and filtereda t reduced by 18% and for outward currents by 72%, which 1 kHz (-3 dB) using an Axopatch 200A and CV201A headstage suggested that TEA-sensitive currents comprised the majority (Axon Instruments, Foster City, CA) and sampled online by a of the outward currents and was a minor component of the microcomputer (IBM 486-compatible) using commercial soft- inward currents. ware and associated analog-to-digital hardware (pClamp 5.5 1/Labmaster TL-1-125; Axon Instruments and Scientific Solu- Figure IB shows the effect of TEA on single-channel cur- tion, Foster City, CA). Patch pipettes (4-10 Mil) were fabri- rent responses to voltage steps over time. Addition of TEA (10 cated from thin-walled borosilicate glass (Vitrex Microhemat- mM) to the pipette solution totally inhibited single channel activity. Similarly, the Km current showed inhibition by the ocrit tubes; Modulohm I/S, Denmark). The reference electrode + 2+ was an electrode of Ag-AgCl immersed in a KC1 solution (140 addition of either Cs (5 mM) or Ba (100 /xM) to the pipette mM) that was connected to the bath by an agar bridge. In solution, with single-channel current responses identical with experiments in which the salt bridge was not used, the pipette those shown in Figure IB. In comparison, the Km current was potential was corrected for the liquid junction potentials be- insensitive to 4-aminopyridine (1 mM and 5 mM) with no tween the pipette solution and bath solution.25 In both the inhibition of single channel activity observed. The steady state, whole-cell, current-voltage relationship whole-cell and the cell-attached configurations, positive cur- + rent was defined as positive ions moving out of the headstage of single rabbit superior lacrimal acinar cells with Cs (5 mM) and leaving the pipette. This positive current in the whole-cell added to the extracellular bath solution is shown in Figure 2. configuration corresponds to the outward membrane current The Km current was inhibited by Cs"*" (5 mM), and the reversal and was shown by an upward deflection in all traces. Con- potential shifted from -37.4 ± 0.5 mV to -26.8 ± 0.5 mV versely, positive current in the cell-attached configuration cor- in = 3). responds to the patch inward current and is represented by The slope conductance of the whole-cell Km current in- upward deflections in single-channel traces. Because it has creased from 2.7 ± 0.16 nS to 5.2 ± 0.45 nS (n = 6, Fig. 3) after 26 an increase in the free Ca2+ concentration from 1.0 X 10~9 M been shown in rat lacrimal glands that furosemide (1 mM) 6 inhibits inward CF current activity, it was added to the extra- to 1.0 X 10~ M. cellular bath solution during whole-cell K+ current recordings. Figure 4A shows representative cell-attached recordings at Results are reported as the mean ± SEM, with n representing various voltages in a single rabbit superior lacrimal gland acinar the number of observations in parentheses. Statistical compar- cell, under physiological conditions. An I-V curve representing isons were performed using Student's £-test, with the appropriate the single-channel amplitude as a function of voltage for the + corrections (Bonferroni) when used for multiple comparisons. cell described in Figure 4A is shown in Figure 4B. K currents recorded from the rabbit superior lacrimal gland were shown to have a single-channel conductance of 43 ± 2.7 pS (n = 11). Solutions and Chemicals Figure 5B shows the current-voltage relationship of the Km The standard pipette solution (pH 7.4) contained (in mM): KC1 channel in cell-attached patches in rabbit superior lacrimal + + (140), MgCl2 (1.13), Hepes (10), and EGTA (5). The K pipette gland cells in which the K concentration was varied among 5, concentration was varied in some single-channel experiments. 20, 70, and 140 mM by the substitution of Na+ for K+. Rep- In experiments that required the K+ concentration in the resentative cell-attached recordings, at a pipette potential of 50 pipette solution to change, we used an equimolar substitution mV for each of the four pipette K+ concentrations, are shown of NaCl for KC1. The standard bath solution (pH 7.4) contained in Figure 5A. The relationship between single-channel conduc- + (in mM): NaCl (145), MgCl2 (1.13), KC1 (5), CaCl2 (1), glucose tance and pipette K concentration (Fig. 6) is described by the (10), and Hepes (10). In the ion substitution experiments, the Michaelis-Menten equation using a Km of 19-2 mM and a K"*~ in the pipette solution was substituted with an equimolar maximum single-channel conductance (Vmax) of 46.1 pS. A amount of Rb+ or Li+. log-log plot of the single-channel conductance (y), as a func-

Downloaded from iovs.arvojournals.org on 09/28/2021 310 Herok et al. IOVS, February 1998, Vol. 39, No. 2

10 pA

400 ms -2.00

-500-1- FIGURE 1. (A) Steady state, whole-cell, current /- Vrelations of single rabbit superior lacrimal gland acinar cells before (•, mean of 12 experiments) and after (O, mean of 8 experiments) the addition of 10 mM tetraethylammonium (TEA) to the extracellular bath solution. In each experiment the data were recorded under physiological conditions; that is, the pipette contained a KCl-rich solution (140 mM) and the extracellular bath contained a NaCl-rich solution. The vertical bars represent the SEM when they were larger than the symbols. (B) The effect of TEA (10 mM) on the inwardly rectifying K+ channel in cell-attached recordings. Current responses were to voltage steps between —60 mV and 80 niV from a holding potential of 0 mV. Similar results were obtained with Cs+ (5 mM) and Ba2+ (100 /xM). The data were recorded under physiological conditions with the different blocking agents added to the pipette. Control recordings of the inwardly rectifying K+ channel activity in cell-attached patches not exposed to TEA are shown in Figure 4A.

+ + + + + tion of the pipette K concentration ([K ]o), is described by permeability sequence of K (1.0) > Rb (0.83) > Li (0.15). the equation, y = 7.04[K]£37 pS (r2 = 0.99, P > 0.05). The The numbers in parentheses indicate the numerator in the + extrapolated Ert,v values versus log [K ]o are described by the fraction P^/PK, where X is the test ion. 2 equation, £rev = 35.51og[K]0 - 77.8 mV (r - 0,99, P > 0,05). The Etcv was determined by extrapolating the linear portion of the single-channel /-V curves shown in Figure 5B. DISCUSSION Ion substitution experiments were performed in which the pipette K+ was replaced with the cations Rb+ and Li+. The Using cell-attached, patch-clamp techniques, this study demon- channel was highly selective for potassium ions, with the strated the existence of inwardly rectifying K+ currents in

—200

-Moo FIGURE 2. Steady state, whole-cell, current-voltage relationships of single rabbit superior lacrimal gland cells in the voltage range between —160 mV and —20 mV before (•, mean of three experiments) and after (O, mean of three experiments) the addition of 5 mM Cs+ to the extracellular bath solution. The vertical bars represent the SEM when they were larger than the symbols.

Downloaded from iovs.arvojournals.org on 09/28/2021 IOVS, February 1998, Vol. 39, No. 2 Potassium Channel in Rabbit Lacrimal Gland 311

T200

-MOOO pA

FIGURE 3- Steady state, whole-cell, current-voltage relationships of single rabbit superior lacrimal gland cells in the voltage range between —160 mV and —20 mV with differing free Ca2+ concentrations in the pipette solution: 10~9 M (•) and 10~6 M (O). The vertical bars represent the SEM when they were larger than the symbols.

rabbit superior lacrimal gland acinar cells. The Km current we tance is approximately dependent on the square root of the identified in these rabbit lacrimal acini satisfies the criteria for external K+ concentration. A similar current has not been identifying an inward rectifier: The channels open with steep reported in the rat lacrimal gland, although an inwardly recti- voltage dependence on hyperpolarization and the conduc- fying, nonspecific cation channel with a single-channel con-

A B

-60 mV -100

-Vp(mV) -50 mV

-40 mV

-30 mV

**vr>»v*»*MH~^^ "*" 3OmV

5pA| 400 ms + FIGURE 4. (A) Inwardly rectifying K single channels recorded from a cell-attached patch from a rabbit superior lacrimal gland acinar cell. Single-channel activity was recorded using the standard pipette and bath solutions described in Materials and Methods. Representative channel activity is shown at the pipette potentials of —60, —50, —40, —30, 0, 30, and 70 mV. For each trace, the solid horizontal arrow indicates the zero current level at which there are no channels open. (B) Single-channel I-V relationship for the cell shown in A. The single-channel amplitude is plotted against the negative of the pipette potential

Downloaded from iovs.arvojournals.org on 09/28/2021 312 Herok et al. IOVS, February 1998, Vol. 39, No. 2

5mM

Vm (mV)

70mM

-L-6 10pA[ pA

+ FIGURE 5- (A) Representative cell-attached recordings of inwardly rectifying K (XIR) at a pipette potential of —50 mV for different pipette (extracellular) K+ concentrations. For each trace, the solid horizontal arrow indicates the zero current level at which there are no channels open. (B) Single-channel I-V relationship of the Km channel in cell-attached patches for pipette (extracellular) K+ concentrations of 140 mM (•, n = 11), 70 mM (O, n = 8), 20 mM(l,n = 5), and 5 mM (•, n = 5). The vertical bars represent the SEM when they were larger than the symbols. The single-channel amplitude is plotted against the transmembrane potential (Fm) calculated by subtracting the pipette potential (Vp) from the average membrane potential (—40 mV) reported in Results.

ductance of 25-30 pS,29'30 which is equally selective for Na+, exposed to TEA and Cs+. The resting membrane potential in K"1", and Cs+, has been recorded. We have not observed this the rabbit lacrimal gland under physiological conditions was type of nonspecific cation current in rabbit lacrimal acinar —40 ± 2 mV (n = 17), which was similar to the resting cells. This interspecies variation suggests that the mechanism membrane potential of —37 ± 8 mV (n = 50) obtained by of secretion differs between the rat and the rabbit and suggests Kikkawa28 using intracellular electrodes. The addition of TEA further that a model of secretion based on other species is not to the bath solution shifted the reversal potential from —40 mV totally applicable to the human model. to —1 mV, and, similarly, extracellular application of Cs+ The KlR current that we report contributes the minor caused the membrane potential to shift from -37.4 ± 1 mV to component of the total inward current (18%) in rabbit lacrimal -26.8 ± 0.5 mV (n = 3). acinar cells. It appears that the main physiological role for this Single-channel recording methods revealed a 43-pS chan- current in rabbit lacrimal acinar cells is to stabilize the resting nel that displayed inward rectification, which is within the membrane potential. The evidence for this centers on changes range of conductance (22- 47 pS) reported for similar channels observed in the resting membrane potential when cells were 2+ in other tissues. The Km current we report here is Ca 19 activated, whereas the KlR in sheep parotid and rat hepato- cytes14 is Ca2+ insensitive. However, there is a conflicting 50 report31 that demonstrates the presence of Ca2+-activated in- + wardly rectifying K channels in rat hepatocytes. The A",R 40 current is inhibited by TEA, whereas in the sheep parotid19 and mouse lens epithelial cells15 the inwardly rectifying K+ current was insensitive to TEA. However, in the mouse lens epithelial 30 cells, TEA (10 mM) reduced the inward current at -160 mV to 64 ± 5% (n = 4) of its control value. In this study TEA (10 mM) •55 20 added to the extracellular bath caused the reversal potential to shift from —40 mV to —1 mV. The Km current is insensitive to 10 4-aminopyridine (5 mM), which is similar to the reports of the inwardly rectifying K+ current in mouse lens epithelial cells.15 The divalent cation Ba2+ is a characteristic blocker of inwardly rectifying K+ current in a variety of tissues, including sheep 0 20 40 60 80 100 120 140 160 19 15 parotid, mouse lens epithelial cells, murine macrophage K+ Concentration (mM) cell line J774.1,32 starfish egg cells,33 frog skeletal muscle,4'34 calf Purkinje cells,35 and rat osteoclasts.17 In this study, Ba2+ FIGURE 6. Plot of conductance as a function of pipette (extra- + totally inhibited Km single-channel activity in rabbit superior cellular) K concentration in cell-attached patches. The curve + is a nonlinear least-squares fit of the Michaelis-Menten equa- lacimal gland cells. The Km current is also sensitive to Cs , and 2 tion giving values of Km = 192 mM and F11VIX = 46.1 pS (r = this sensitivity is similar to the current found in other spe- 0.95). cies.7-15-17'19

Downloaded from iovs.arvojournals.org on 09/28/2021 IOVS, February 1998, Vol. 39, No. 2 Potassium Channel in Rabbit Lacrimal Gland 313

The single-channel conductance of the Km channels we inward rectifier, which can serve as a vehicle for the outward + report here has dependence on [K]o similar to that of other movement of K ions from the cell as mentioned above. inward rectifiers observed in exocrine glands19 and other tis- In conclusion we have identified a Ca2+-activated in- sues.1415'32 However, there is some interspecies variation in wardly rectifying K+ channel in the rabbit superior lacrimal the degree to which conductance varies by a particular power gland with a single conductance of 43 pS, which is dependent + 048 + dependency on [K ]o. For example, [K] in guinea pig on the extracellular K concentration. The current is blocked ventricular myocytes,7 [K]055 in sheep parotid cells,19 [K]056 by TEA, Ba2+, and Cs+ but is insensitive to 4-aminopyridine. in the murine macrophage cell line J774.1,32 and [K]037 in the From our data, the primary role of this inward rectifier seems rabbit superior lacrimal gland from this study. These small to set and stabilize the resting membrane potential. differences in the degree with which conductance varies by a + particular power dependency on [K ]o may not be closely related to the functional role of the channel. For example, in References 32 murine macrophages and the rabbit lacrimal gland, the KlR 1. Aimers W. Potassium conductance changes in skeletal muscle and channel is responsible for stabilizing the cell membrane poten- the potassium concentration in the transverse tubules. / Physiol tial, whereas in the sheep parotid,19 the inwardly rectifying K+ (Lond). 1972;225:33-56. channel is not involved in stabilizing the cell membrane poten- 2. Leech CA, Staniield PR. Inward rectification in frog skeletal muscle tial and its role requires further investigation. fibres and its dependence on membrane potential and external potassium. / Physiol (Lond). 1981;310:295-309. Many physiological processes are dependent on tempera- 3. DeCoursey TE, Dempster J, Hutter OF. Inward rectifier current ture. For example, the dynamic process of regulatory volume noise in frog skeletal muscle. J Physiol (Lond). 1984;349:299-327. increase in rat lacrimal gland acini36 does not occur in HEPES- 4. DeCoursey TE, Hutter OF. Potassium current noise induced by buffered solutions at 20cC but is present in these solutions at barium ions in frog skeletal muscle. / Physiol (Lond). 1984;349: 37°C. This observation suggests that the active NaK2Cl cotrans- 329-351. 5. Carmeliet E. Induction and removal of inward-going rectification in porter is inactive at the lower temperature. Although we char- sheep purkinje fibers. / Physiol (Lond). 1982;327:285-308. acterized KiR at room temperature, the probable role of Km is 6. Hume JR, Giles W. Ionic currents in single isolated bullfrog atrial in setting and stabilizing the membrane potential. Such a role cells./ Gen Physiol. 1983;81:153-194. depends on ion diffusion processes rather than active transport 7. Sakmann B, Trube G. Conductance properties of single inwardly mechanisms and is less likely to be greatly affected by changes rectifying potassium channels in ventricular cells from guinea-pig in temperature. Indeed, reports of the single-channel current of heart. J Physiol (Lond). 1984;347:64l-657. CIC-0 chloride channels show only a weak dependence on 8. Sakmann B, Trube G. Voltage-dependent inactivation of inward- 37 rectifying single-channel currents in the guinea-pig heart cell mem- temperature. Similarly, the condvictance of inwardly rectify- brane. / Physiol (Lond). 1984;347:659-683. ing K+ whole-cell currents in guinea pig ventricular myo- 38 9. Giles WR, Shibata EF. Voltage clamp of bull-frog cardiac pacemaker cytes is weak (Ql0 of 1.28), and the conductance of adeno- cells: a quantitative analysis of potassium currents. / Physiol sine triphosphate-dependent K+ channels in rat ventricular (Lond). 1985;368:265-292. 39 myocytes is relatively weak OQ10 of 1.4-1.6). 10. Kurachi Y. Voltage-dependent activation of the inward-rectifier Previous authors have reported functional roles of the potassium channel in the ventricular cell membrane of guinea-pig heart. J Physiol (Lond). 1985:366:365-385. inwardly rectifying K+ channels apart from setting and stabi- + 11. Matsuda H. Open-state substructure of inwardly rectifying potas- lizing the membrane potential. Inwardly rectifying K chan- sium channels revealed by magnesium block in guinea-pig heart nels may buffer transient increases in extracellular K+ as has cells. J Physiol (Lond). 1988;397:237-258. been suggested in glial cells.20'22 It has also been shown in 12. Matsuda H, Matsuura H, Noma A. Triple-barrel structure of in- + + + salivary glands that, when stimulated, cells lose K+ to their wardly rectifying K channels revealed by Cs and Rb block in surroundings.40'41 However, the sheep parotid is the only guinea-pig heart cells. / Physiol (Lond). 1989:413:139-157. salivary gland that has an inward rectifier,19'42 and it has been 13- Hunter M, Oberleithner H, Henderson RM, Giebisch G. Whole-cell 19 potassium currents in single early distal tubule cells. Am J Physiol. suggested by Ishikawa et al. that, in the sheep parotid, the 1988;255:F699-F703. NaK2Cl cotransporter plays no part in secretion as opposed to 14. Henderson RM, Graf J, Boyer JL. Inward-rectifying potassium chan- other salivary glands in which K+ uptake occurs through the nels in rat hepatocytes. Am J Physiol. 1989;256:G1028-G1035. cotransporter,43'45 and, hence, K+ uptake occurs through the 15. Cooper K, Rae JL, DeweyJ. Inwardly rectifying potassium current inward K+ rectifier. It has been suggested19 that the inward in mammalian lens epithelial cells. Am J Physiol. 1991;26l:C115- rectifier in the sheep parotid is involved with spontaneous C123. 16. Rae JL. Ion channels in ocular epithelia. Invest Ophthalmol Vis Sci. secretion by the salivary glands. Current models of secretion 1993:34:2608-2612. are based on the efflux of Cl~ across the luminal membrane 17. Sims SM, Dixon JS. Inwardly rectifying K+ current in osteoclasts. + being balanced by the efflux of K through channels in the Am J Physiol, 1989;256:C1277-C1282. basolateral membrane. It has been shown that some inward 18. Sakai H, Okada Y, Morii M, Takeguchi H. Anion and cation chan- rectifiers do allow some outward current flow,7'21 and this may nels in the basolateral membrane of rabbit parietal cells. Pflugers be sufficient to drive Cl~ efflux across the luminal membrane Arch. 1989:414:185-192. leading to spontaneous secretion. In other cells, it has been 19- Ishikawa T, Wegman EA, Cook DI. An inwardly rectifying potassium channel in the basolateral membrane of sheep parotid secre- suggested that the inward K+ rectifier is involved in removing + tory cells. / Membr Biol. 1993:131:193-202. K from the cell to prevent its accumulation in the cell in 20. Brew H, Gray PTA, Mobbs P, Attwell D. Endfeet of retinal glial cells + response to Na -dependent transport processes such as the have higher densities of ion channels that mediate K+ buffering. Na+-H+ exchange.23 As H+ is extruded by the cell, Na+ ions Nature. 1986;324:466-469- accumulate within the cell. This activates the Na+-K+ pump, 21. Hirst GDS, Edwards FR. Sympathetic neuroeffector transmission in which extrudes Na"*" ions and leaves an excess of K4" ions in arteries and arterioles. Physiol Rev. 1989;69:546-604. the cell. These K+ ions may then leave the cell through the 22. Barres BA, Chun LLY, Corey DP. Ion channels in vertebrate glia. Annu Rev Neurosci. 1990;13:44l-474.

Downloaded from iovs.arvojournals.org on 09/28/2021 314 Herok et al. IOVS, February 1998, Vol. 39, No. 2

23. Wang W, White S, Geibel J, Giebisch G. A potassium channel in the 34. Standen NB, Stanfield PR. A potential and time-dependent block- apical membrane of rabbit thick ascending limb of Henle's loop. age of inward rectification in frog skeletal muscle fibers by barium AmJPhysiol. 199O;258:F244-F253- and strontium./Physiol (Lond). 1978;280:l69-191. 24. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved 35. DiFrancesco D, Ferroni A, Vistentin S. Barium induced blockade of patch-clamp techniques for high resolution current recording the inward rectifier in calf purkinje fibers. Pflugers Arch. 1984; from cells and cell-free membrane patches. Pflugers Arch. 1981; 402:446-453. 391:85-100. 36. Douglas IJ, Brown PD. Regulatory volume increase in rat lacrimal 25. Barry PH. JPCalc, a software package for calculating liquid junction gland acinar cells./Membr Biol. 1996;150:209-217. potential corrections in patch-clamp, intracellular, epithelial and 37. Pusch M, Ludewig U, Jentsch TJ. Temperature dependence of fast bilayer measurements and for correcting junction potential mea- and slow gating relaxations of CIC-0 chloride channels. / Gen surements. / Neurosci Methods. 1994;51:107-l 16. Physiol. 1997; 109:105-116. 26. Evans MG, Marty A, Tan YP, Trautmann A. Blockage of Ca-activated 38. Mitsuiye T, Shinagawa Y, Noma A. Temperature dependence of Cl conductance by furosemide in rat lacrimal glands. Pflugers the inward rectifier K+ channel gating in guinea-pig ventricular Arch. 1986;406:65-68. cells. Jpn J Physiol. 1997;47:73-79. 27. Findlay I, Dunne MJ, Petersen OH. ATP-sensitive inward rectifier + 39- McLarnon JG, Hamman BN, Tibbits GF. Temperature dependence and voltage and calcium-activated K channels in cultured pancreatic islet cells.JMembr Biol. 1985;83:l69-175. of unitary properties of an ATP-dependent potassium channel in 28. Kikkawa T. Secretory potentials in the lacrimal gland of the rabbit. cardiac myocytes. BiophysJ. 1993;65:2013-2020. Jpn J Ophthalmol. 1970;l4:25-40. 40. Burgen ASV. The secretion of potassium in saliva. J Physiol (Lond). 1956;132:2O-39. 29. Vincent P. Cationic channels sensitive to extracellular ATP in nit + lacrimal cells. / Physiol (Lond). 1992;449:313-331. 41. Wright RD, Blair-West JR. The effects of K channel blockers on 30. Marty A, Tan YP, Trautmann A. Three types of calcium-dependent ovine parotid secretion depend on the mode of stimulation. Exp channel in rat lacrimal glands. / Physiol (Lond). 1984;357:293- Physiol. 1990;75:339-348. 325. 42. Petersen OH, Gallacher DV. Electrophysiology of pancreatic and 31. Bear CE, Petersen OH. L-Alanine evokes opening of single Ca2+- salivary acinar cells. Annu Rev Physiol. 1988;50:65-80. activated K+ channels in rat liver cells. Pflugers Arch. 1987;4l0: 43. Pirani D, Evans LAR, Cook DI, Young JA. Intracellular pH in the rat 342-344. mandibular salivary gland: the role of Na-H and C1-HCO3 antiports 32. McKinney LC, Gallin EK. Inwardly rectifying whole-cell and single in secretion. Pflugers Arch. 1987;408:178-184. + + channel K currents in the murine macrophage cell line J774.1./ 44. Turner RJ, George JN, Baum BJ. Evidence for a Na /K /Cl~ co- Membr Biol. 1988;103:4l-53- transport system in basolateral membrane vesicles from the rabbit 33. Hagiwara S, Miyazaki S, Moody W, Patlak J. Blocking effects of parotid./Membr Biol. 1986;94:l43-152. + barium and hydrogen ions on the potassium current during anom- 45. Turner RJ, George JN. CF-HCO3~ exchange is present with Na - alous rectification in the starfish eggs. / Physiol (Lond). 1978;279: K+-C1~ cotransport in rabbit parotid acinar basolateral mem- 167-185. branes. AmJPhysiol. 1988;254:C391-C396.

Downloaded from iovs.arvojournals.org on 09/28/2021