ATP-dependent regulation of SK4/IK1-like currents in rat submandibular acinar cells: possible role of cAMP-dependent protein kinase

M. Hayashi, C. Kunii, T. Takahata, and T. Ishikawa

Laboratory of Physiology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Submitted 7 July 2003 ; accepted in final form 27 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SK4/IK1 encodes an intermediate conductance, Ca2+-activated K+ channel and fulfills a variety of physiological functions in excitable and nonexcitable cells. Although recent studies have provided evidence for the presence of SK4/IK1 channels in salivary acinar cells, the regulatory mechanisms and the physiological function of the channel remain unknown in these cells. Using molecular and electrophysiological techniques, we examined whether cytosolic ATP-dependent regulation of native SK4/IK1-like channel activity would involve endogenous cAMP-dependent protein kinase (PKA) in rat submandibular acinar (RSA) cells. Electrophysiological properties of tetraethylammonium (TEA) (10 mM)-insensitive, Ca2+-dependent K+ currents in macropatches excised from RSA cells matched those of whole cell currents recorded from human embryonic kidney-293 cells heterologously expressing rat SK4/IK1 (rSK4/IK1) cloned from RSA cells. In outside-out macropatches, activity of native SK4/IK1-like channels, defined as a charybdotoxin (100 nM)-blockable current in the presence of TEA (10 mM) in the bathing solution, ran down unless both ATP and Mg2+ were present in the pipette solution. The nonhydrolyzable ATP analog AMP-PNP failed to support the channel activity as ATP did. The addition of Rp-cAMPS (10 µM), a PKA inhibitor, to the pipette solution containing ATP/Mg2+ induced a rundown of the Ca2+-dependent K+ currents. Inclusion of cAMP (1 mM) into the pipette solution (1 µM free Ca2+) containing ATP/Mg2+ caused a gradual increase in the currents, the effect being pronounced for the currents induced by 0.1 µM free Ca2+. Forskolin (1 µM), an adenylyl cyclase activator, also increased the currents induced by 0.1 µM free Ca2+. In inside-out macropatches, cytosolic ATP/Mg2+ increased both the maximum current (proportional to the maximum channel activity) and Ca2+ sensitivity of current activation. Collectively, these results suggest that ATP-dependent regulation of native SK4/IK1-like channels, at least in part, is mediated by endogenous PKA in RSA cells.

Ca2+-activated K+ channel; patch clamp; human embryonic kidney-293; salivary secretion


FLUID SECRETION in salivary acinar cells is stimulated by muscarinic agonists such as ACh that are associated with an increase in cytosolic Ca2+. The currently accepted model for salivary secretion proposes that this secretory process depends on a simultaneous activation of Ca2+-dependent Cl- and K+ channels in the apical and basolateral membrane of the cells, respectively. The apical Cl- channels provide a major pathway of Cl- efflux into the lumen, and a basolateral Na+-K+-2Cl- cotransporter and Na+/H+ and / double antiporters maintain the concentration of Cl- in the cytosol above the electrochemical equilibrium. Concurrent with an increase in the apical Cl- conductance, activation of the basolateral K+ channels mediating K+ efflux is essential to maintain the electrical driving force for continuous Cl- efflux across the apical membrane. Luminal Cl- provides a driving force for the movement of Na+ across the tight junction, and the two ions then generate the necessary osmotic gradient for the movement of water across the epithelium, forming a NaCl-rich primary saliva (2).

Recent molecular and electrophysiological studies have identified the genes that encode several types of Ca2+-dependent K+ (KCa) channels in excitable and nonexcitable cells. One of these is SK4/IK1, which encodes an intermediate conductance KCa channel mainly expressed in nonexcitable cells, including secretory epithelia (15, 18, 19, 38), erythrocytes (14, 35), and lymphocytes (20, 23). Molecular evidence for the presence of SK4/IK1 in salivary glands was first provided by Jensen et al. (18), who demonstrated a strong expression of transcripts of hIK1 in human salivary glands, and has been strengthened by a recent in situ hybridization study on mouse parotid glands (27). Our recent patch-clamp study (34) has demonstrated that bovine parotid acinar cells, which are also shown to express transcripts of SK4/IK1 by RT-PCR, exhibit a type of KCa current [i.e., TEA (10 mM)-insensitive KCa currents] with the hallmark properties reported for cloned SK4/IK1 (15, 18, 19, 23, 36, 40) or native SK4/IK1-like channels (4, 10, 20), as follows: 1) time- and voltage-independent activation of the currents with a slightly inwardly rectifying current-voltage (I/V) relationship in symmetrical K+ conditions, 2) a permeability sequence of K+ = Rb+ >> Na+, NMDG+, 3) activation by submicromolar free [Ca2+] ([Ca2+]i), 4) inhibition by Ba2+, charybdotoxin (ChTX), and clotrimazole, but not by apamin, 5) activation by 1-ethyl-2-benzimidazolinone (1-EBIO), and 6) inhibition by calmodulin antagonists. At the single channel level, Nehrke et al. (27) have also shown that a heterologous expression of mSK4/IK1 cloned from mouse parotid gland displays a single channel conductance, which is very similar to a native 22 pS KCa channel. Although these recent molecular and electrophysiological studies suggest the functional expression of SK4/IK1-like channels in native salivary acinar cells, at least in several species, neither the regulatory mechanism nor the physiological function of the channel has been defined.

SK4/IK1 channel gating is primarily activated by cytosolic [Ca2+], the Ca2+ sensitivity being conferred to the channel by the association with calmodulin, acting as a Ca2+ sensor (6, 20). In addition, several recent studies have demonstrated that the Ca2+-dependent activation of SK4/IK1 channel is modulated by cytosolic ATP through a phosphorylation-dependent mechanism in both heterologous expression systems (8, 36, 40) and native cells (or cell line) (8, 28, 31), a modulation involving several protein kinases, including PKA, in a tissue- or cell-specific manner. Interestingly, most of these studies have consistently shown in excised membrane patches that the channel activity is regulated by ATP applied to the cytosolic surface of the membrane and that the ATP-dependent activity is either blocked or enhanced by maneuvers that either inhibit or stimulate, respectively, protein kinase activity (8, 28, 31, 36), suggesting a role of endogenous protein kinase localized at the membrane or at cytosol close to the membrane. To test the ATP dependency of the channel activity may be also useful to assess the kinase-dependent regulation of SK4/IK1-like channels in native epithelial cells.

We were interested in examining whether such ATP-dependent regulation of native SK4/IK1-like channel occurs in salivary acinar cells for three reasons. First, it has been shown in situ and in vitro that a simultaneous activation of the Ca2+-dependent pathway and the cAMP-dependent pathway produces a potentiated response in salivary glands, the fluid secretion being greater than the sum of that produced by either pathway alone (5, 16, 22, 24). Second, previous in vitro studies have demonstrated that cytosolic cAMP potentiates a Ca2+-dependent K+ efflux (most likely through a KCa channel) induced by muscarinic agonists or the Ca2+ ionophore, A-23187 in rat submandibular and parotid glands (16, 21), a potentiated response being insensitive to external TEA (10 mM), a maxi-K+ channel blocker in rat submandibular gland (16). Finally, a TEA (10 mM)-insensitive whole cell KCa conductance induced by A-23187 in rat submandibular acinar (RSA) cells dialyzed with a pipette solution containing both cAMP and ATP was shown to be significantly greater than that in RSA cells dialyzed with ATP alone, although the addition of cAMP to the pipette solution in the absence of A-23187 does not induce a TEA (10 mM)-insensitive K+ conductance (16), implying a cAMP-dependent modulation of the TEA-insensitive KCa conductance. However, the molecular mechanism of this modulation is still unknown. Although it also remains to be determined whether SK4/IK1 mediates a TEA-insensitive KCa conductance in RSA cells, these previous findings led us to hypothesize that PKA may regulate native SK4/IK1 channels in RSA cells and this might also give us a clue for an important physiological function of the channels in salivary acinar cells.

In the present study, we thus addressed two questions as to whether a TEA-insensitive KCa conductance in RSA cells would fulfill the hallmark properties of SK4/IK1 and whether cytosolic ATP would regulate the conductance via PKA. Using electrophysiological and molecular techniques, we first show that electrophysiological profiles of the TEA-insensitive KCa currents in RSA cells are quantitatively similar to those of heterologously expressed rat SK4/IK1 (rSK4/IK1) cloned from RSA cells. We then show that the activity of native SK4/IK1-like channels in excised outside-out membrane macropatches is dependent on the presence of cytosolic ATP/Mg2+, the mechanism likely involving protein phosphorylation, at least in part, via PKA. We finally demonstrate that cytosolic ATP/Mg2+ increases both the maximum current and Ca2+-sensitivity of current activation in excised inside-out macropatches. Together, these results form the first demonstration of ATP-dependent regulation of SK4/IK1-like currents in native salivary acinar cells, and might shed light on a molecular mechanism by which an increase in cytosolic cAMP potentiates a Ca2+-mediated fluid and electrolyte secretion in salivary gland.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Patch-clamp experiments. All experiments were performed in accordance with a protocol approved by the Laboratory Animal Care and Use Committee of Graduate School of Veterinary Medicine, Hokkaido University. Male Sprague-Dawley rats (230–380 g) were euthanized immediately by cervical dislocation, and the submandibular glands were rapidly removed. Isolated acini and acinar cells were prepared as described previously (34) with some modification. Briefly, small pieces of gland, trimmed of fat and connective tissue, were minced finely with scissors and incubated in a nominally magnesium- and Ca2+-free, NaCl-rich bathing solution containing collagenase (type II, 100 U/ml, Worthington, Freehold, NJ), and incubated for 30 min at 37°C in a shaking water bath. The fragments were then sieved through a 148-µm nylon mesh and subjected to gentle trituration with a pipette. The suspension was then washed twice, resuspended in a standard bathing solution (pH 7.4 adjusted with NaOH) containing (in mM) 145 NaCl, 5 KCl, 10 HEPES, 1 CaCl2, 1 MgCl2, and 10 glucose, and stored at 4°C until used.

The cell preparations described above were pipetted on to a coverslip and transferred to a chamber mounted on an inverted microscope. Current recordings were made using inside-out and outside-out configurations of the patch-clamp technique (12). Membrane patches were excised from basolateral membrane of acinar cells. Membrane currents were also recorded using the standard whole cell patch-clamp technique (12) from HEK-293 cells stably expressing rSK4/IK1 cloned from rat submandibular acinar cells. The capacitance transient current was compensated by using the Axopatch-1D amplifier. In these experiments, the whole cell capacitance and the series resistance (Rs) were 32.7 ± 1.4 pF (n = 123) and 9.7 ± 0.8 M{Omega} (n = 123), respectively. The Rs was not electronically compensated during the experiments, and the potentials reported here have not been corrected for the Rs. The whole cell currents were not corrected for leakage.

The patch-clamp pipettes, which were pulled from glass capillaries (LG16, Dagan; Minneapolis, MN) with a vertical puller (model PP-830, Narishige; Tokyo, Japan), had resistances of 1–5 M{Omega} when filled with a standard K-glutamate-rich solution described below. An Axopatch-1D patch-clamp amplifier (Axon Instruments; Union City, CA) was used to measure the membrane currents. The reference electrode was a Ag-AgCl electrode, which was connected to the bath via an agar bridge filled with a standard bathing solution. The amplifier was driven by pCLAMP6 software (Axon Instruments) to allow the delivery of voltage-step protocols with concomitant digitization of the membrane current. The membrane currents were filtered through an internal four-pole Bessel filter at 500 Hz and sampled at 2 kHz. Typically, the current-voltage (I/V) relationships were studied with the use of voltage ramps; the command voltages were varied from -80 to +50 mV over an 800-ms period every 10 s. Subsequent current analysis was performed using programs supplied with pCLAMP6 software.

The pipette potential was corrected for the liquid junction potentials between pipette solution and the external solution, and between the external solution and the agar bridge, as described elsewhere (1, 25).

Except where indicated, the compositions of the standard pipette and bath solution in outside-out patch and whole cell experiments were as follows: the pipette solution (pH 7.4 adjusted with KOH) contained (in mM) 100 K-glutamate, 10 KCl, 2 ATP, 3 MgCl2, 10 HEPES, 10 glucose, and 10 EGTA. For an ATP-free solution, equimolar MgCl2 (2 mM) was also removed from the solution. The [Ca2+]i of the pipette solution were calculated from an equation that takes into account the concentrations of Mg2+, Ca2+, EGTA (96% purity), ATP and pH (29), and the appropriate amount of CaCl2 was added to the solution. The free Mg2+ concentrations of the solution were ~1 mM. The bath solution (pH 7.4 adjusted with KOH) contained (in mM) 150 K-glutamate (or KCl in whole cell experiments), 10 HEPES, 1 CaCl2, 1 MgCl2, 10 TEA-Cl, and 10 glucose. In inside-out patch experiments, the composition of the standard pipette solution was as follows: the pipette solution (pH 7.4 adjusted with KOH) contained (in mM) 150 K-glutamate, 10 HEPES, 1 CaCl2, 1 MgCl2, 10 TEA-Cl, and 10 glucose. The bath solution (pH 7.4 adjusted with NaOH) contained (in mM) 110 Na-glutamate, 2 (or 0) ATP, 3 (or 1) MgCl2, 10 glucose, 10 HEPES, and 10 EGTA. When the free Ca2+ concentrations of the bath solution were varied between 10-5 and <10-9 M, the appropriate amount of CaCl2 was added to the solution as described above. The pH of the pipette and bath solutions used in the present study was adjusted to 7.4.

Patch-clamp experiments were performed at room temperature (~20°). Bath solution changes were accomplished with the use of a gravity feed from reservoirs. The results were reported as means ± SE of several independent experiments (n), where n refers to the number of cells patched.

Statistical significance was evaluated using the two-tailed paired and unpaired Student's t-test or ANOVA as appropriate. Differences between means were considered to be statistically significance at a value of P < 0.05.

ATP, adenosine-5'-({beta},{gamma}-imido)triphosphate (AMP-PNP), cAMP, clotrimazole, TEA-Cl, HEPES, EGTA, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) HCl, calmidazolium chloride (compound R24571 [GenBank] ), 1,9-dideoxyforskolin, and trifluoperazine dihydrochloride were obtained from Sigma (St. Louis, MO), forskolin and staurosporine were from Wako Chemicals (Osaka, Japan), Rp-cAMPS was from Calbiochem (San Diego, CA), 1-EBIO was from Tocris (Avonmouth, UK), and ChTX and apamin were from Peptide Institute (Osaka, Japan). Other chemicals employed were of reagent grade.

Data fitting. The relationship between the K+ current and [Ca2+]i at each membrane voltage was fit to the Hill equation

(1)
where Y is K+ channel current, Ymax is the maximum current (proportional to the maximum channel activity, which is defined as the product of overall open probability of active channel and the number of active channel), Kd is the apparent dissociation constant, and nH is the Hill coefficient. In the context of this equation, the Hill coefficient controls the steepness of the relation between K+ channel activation (current) and [Ca2+]i.

To analyze titration curves for Ba2+ inhibition of the K+ current (IK), the ratio I/Io measured in the presence (I) of the blocker to that in its absence (Io) was described by the following equation

(2)
where Ki is the inhibitory constant of the blocker, A is the concentration of the blocker, and n is pseudo Hill coefficient. In the case of a voltage-dependent block, Ki(V) is the voltage-dependent inhibitory constant, which has been expressed by Woodhull (39) as a Boltzmann relationship with respect to the voltage

(3)
where Ki(0) is the inhibitory constant at 0 mV, Z is a slope parameter, and F, V, R, and T have their conventional thermodynamic meanings. Z is equal to the product of the actual valence of the blocking ion z and the fraction of the membrane potential (or electrical distance) ({delta}) acting on the ion.

Cloning of SK4/IK1 from rat submandibular cells. mRNA was extracted from rat submandibular cells prepared as described above using TRIzol reagent (Life Technologies, Grand Island, NY) and BioMag mRNA purification kit (Polysciences, Warrington, PA) following the manufacturer's instructions. First-strand cDNA was generated from mRNA using SuperScriptII RT (Life Technologies). The specific oligonucleotide primers for PCR for rSK4/IK1 were 5'-GTCCCAGGGTGAGCAGAACA-3' (nt 26–45, sense) and 5'-GGCACCCCGCACCTTACTC-3' (nt 1887–1869, antisense), which were derived from the published sequences of the rat SK4/IK1 (GenBank accession no. NM_023021 [GenBank] ). The size of the expected fragment was 1,862 bp. The PCR reaction was performed with TaKaRa LA Taq (Takara Bio, Otsu, Japan). PCR products were gel excised, purified, cloned into the pGEM-T Easy vector (Promega, Madison, WI), sequenced, and found to correspond to the sequence of rSK4/IK1. The PCR conditions were the following: denaturation 94°C/30 s; annealing 60°C/30 s; and extension 72°C/3 min; 35 cycles. As a control, {beta}-actin-cDNA was amplified using 5'-GACTACCTCATGAAGATCCT-3' (sense) and 5'-CCACATCTGCTGGAAGGTGG-3' (antisense), and 510-bp product was obtained.

On the basis of the complete sequence information of rSK4/IK1, we amplified from total mRNA three overlapping PCR fragments by RT-PCR with high-fidelity DNA polymerase (Pfu-Turbo, Stratagene, La Jolla, CA) using the following primers: 5'-GTCCCAGGGTGAGCAGAACA-3' (nt 26–45; sense), 5'-TCCATCATGAAGTTGTGCAC-3' (nt 1047–1028; antisense); 5'-GGAACTGGCATCGGACTCAT-3' (nt 242–261; sense); 5'-CAGTTCAGTCAGGGCATCCA-3' (nt 1363–1344; antisense); 5'-GGTGGCCCGGAAGCTGGA-3' (nt 988–1005; sense); and 5'-GGCACCCCGCACCTTACTC-3' (nt 1887–1869; antisense).

The fragments were subcloned into the pGEM-T Easy vector and sequenced on both strands. The first fragment EcoR I (in vector)/MluI (nt 26–664; start codon at nt 143) was subcloned into pCI-neo (Promega) plasmid linearized by EcoR I and MluI, the construct being linearlized by MluI and SmaI (in vector), into which the other two fragments were assembled by double ligation of fragments MluI/EcoR I (nt 664–1232) and EcoR I/SmaI (nt 1232–1740).

HEK-293 cells stably expressing rSK4/IK1. HEK-293 cells stably expressing rSK4/IK1 were generated by transfecting cells with a plasmid construct encoding the rSK4/IK1 and a neomycin resistance gene contained within the mammalian expression vector pCI-neo using lipofectamine (Life Technologies). G418 (0.8 mg/ml)-resistant colonies were purified and tested for rSK4/IK1 expression by whole cell patch clamp. Parental HEK-293 cells or the cells stably transfected with vector alone did not exhibit SK4/IK1-like currents (data not shown). The cells were then maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml)/streptomycin (0.1 mg/ml), and 0.2 mg/ml G418. The cells were grown at 37°C in a water-saturated 5% CO2-95% air atmosphere, and passaged once or twice weekly. For patch-clamp experiments, the cells were seeded on a cover glass and patched 2–6 days after being seeded.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Electrophysiological properties of TEA-insensitive KCa currents in macropatches from RSA cells. Previous whole cell patch-clamp studies have shown that RSA cells exhibit a TEA-insensitive KCa conductance (16, 17). Using excised membrane macropatch recording techniques, as described previously for bovine parotid acinar cells (34), we first examined whether the conductance in RSA cells would fulfill the hallmark properties of SK4/IK1. Figure 1A shows representative traces of currents elicited by 400-ms voltage steps from outside-out macropatches excised from the basolateral membrane of RSA cells. The pipette contained a standard K-glutamaterich solution with a 1 µM [Ca2+]i, and the bath contained a standard K-glutamate-rich solution with TEA (10 mM) to block the maxi-K+ channel. Under these experimental conditions, the evoked currents whose amplitudes were variable between each experiment were reasonably stable and characterized by a time- and voltage-independent activation, and had a slightly inwardly rectifying I/V relation in symmetrical K+ conditions (Fig. 1B). When extracellular [K+] ([K+]o) was reduced from 154.3 to 5 mM, its reversal potential (Erev) was shifted toward a negative membrane potential, suggesting that the currents were mainly mediated by K+. The conclusion was strengthened in experiments, where the dependence of Erev on [K+]o was examined (Fig. 1C). Erev of the TEA-insensitive current was shifted from -57.2 ± 4.2 mV (n = 6) to +9.0 ± 2.2 mV (n = 9) with a slope of 33.7 ± 1.9 mV (n = 9) per decade when [K+]o was varied between 1 and 154.3 mM. The ion selectivity of the currents was tested by measuring Erev in the presence of various monovalent cations in the bath. When extracellular K+ (154.3 mM) was replaced with equimolar Na+ or NMDG+ (Fig. 1D), its reversal potential was largely shifted toward a negative membrane potential. In contrast to Na+ or NMDG+, Rb+ was equally permeable to K+, so that in nine cells, the average shift in Erev was -1.4 ± 0.5 mV (n = 9) (Fig. 1D). With an assumption that the current was only carried by these monovalent cations, we calculated the permeability ratio (PX/PK) from the shift in Erev ({Delta}Vrev) when external K+ is replaced by monovalent cation X (13); that is, from

(4)



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. A: representative traces of tetraethylammonium (TEA)-insensitive Ca2+-activated K+ currents in an outside-out macropatch excised from basolateral membrane of a rat submandibular acinar (RSA) cell. The pipette was filled with a standard K-glutamate-rich solution containing 1 µM free Ca2+ and the bath contained a K-glutamaterich solution with 10 mM TEA. The cell was held at +2 mV and stepped for 400 ms to potentials ranging between -78 and +42 mV in 10-mV steps. B: instantaneous current-voltage (I/V) relations of TEA-insensitive Ca2+-activated K+ currents. Currents were elicited by 800-ms voltage ramps from -78 to +52 mV in the bath solution containing 5 or 154.3 mM K+. Vm, membrane potential. C: semilogarithmic plot of the reversal potential of the TEA-insensitive Ca2+ (1 µM)-activated currents as a function of extracellular [K+] ([K+]o). The continuous line shows the linear regression fit to the data. Values are means ± SE of 6–9 experiments. D: representative instantaneous I/V relations of the TEA-insensitive Ca2+ (1 µM)-activated currents obtained from an outside-out macropatch with Rb+, Na+, or NMDG+ substituted for K+ (154.3 mM) in the bathing solution. Ramp command voltages were the same as shown in B.

 

The sequence of the relative monovalent cation permeabilities for the TEA-insensitive KCa currents was estimated to be K+ (1; n = 14) {approx} Rb+ (0.95 ± 0.02; n = 9) >> Na+ (0.08 ± 0.01; n = 14) = NMDG+ (0.07 ± 0.01; n = 8).

The TEA-insensitive KCa currents were blocked by the addition of ChTX or clotrimazole to the bath solution (Fig. 2A). ChTX (100 nM) and clotrimazole (1 µM) decreased KCa currents at -78 mV to 32.7 ± 2.0% (n = 40, P < 0.001) and 38.1 ± 3.3% (n = 6, P < 0.001) of the control values, respectively, whereas apamin (100 nM) failed to inhibit the currents [99.8 ± 1.6% (n = 6, P = 0.92) at -78 mV]. The degree of inhibition by ChTX and clotrimazole of the TEA-insensitive KCa currents may have been underestimated, because it was difficult to completely eliminate a background current. We also found that the KCa currents were reversibly inhibited by external Ba2+ in a concentration- and a voltage-dependent manner with a Kd (0) of 311.1 ± 75.6 mM and a slope of 0.61 ± 0.04 (n = 5) (Fig. 2B). Furthermore, we found that external 1-EBIO (100 µM), which has been shown to increase SK4/IK1-like currents (4), increased the amplitude of the ChTX-sensitive currents induced by 0.1 µM [Ca2+]i to 307.0 ± 144.7% (n = 9, P < 0.001) of the control value (Fig. 2C), whereas it did not further increase of the amplitude of the currents activated by 1 µM free Ca2+ (106.6 ± 5.0%, n = 8, P = 0.24), suggesting that 1-EBIO shifted the Ca2+ dependency of channel activation without changing the maximum level.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2. A: effect of external charybdotoxin (ChTX; 100 nM) or clotrimazole (CLT; 1 µM) on TEA-insensitive Ca2+ (1 µM)-activated K+ currents in an outside-out macropatch. Representative instantaneous I/V relationships in the absence and presence of blockers are shown. Ramp command voltages were the same as shown in Fig. 1B. Control I/V relation was measured before the addition of ChTX to the bath solution. The effect of ChTX was completely reversible. Control I/V relation for clotrimazole was thus almost the same as the one for ChTX (not shown). B: effect of external Ba2+ (0.1, 1, 3, and 10 mM) on Ca2+ (1 µM)-activated K+ currents in outside-out patches. Representative I/V relationships in the absence and presence of blocker obtained from an outside-out patch are shown. Inset: voltage dependence of dissociation constant (Kd) values for Ba2+ block of Ca2+ (1 µM)-activated K+ currents. Data were fitted with an equation derived from Eqs. 2 and 3 (see MATERIALS AND METHODS). Kd values were plotted on a semilogarithmic scale. Values are means ± SE of 5 experiments. C: effect of external 1-ethyl-2-benzimidazolinone (1-EBIO; 100 µM) on TEA-insensitive KCa currents activated by 0.1 (left) or 1 µM free Ca2+ (right) in outside-out macropatches. Bar graphs showing ChTX (100 nM)-sensitive current at -78 mV in the absence (C) or presence of 1-EBIO. In these experiments, SK4/IK1-like currents activated by 0.1 µM free Ca2+ were extremely large. Values are means ± SE of 9 (left) or 8 (right) experiments. *P < 0.001. D: effects of the calmodulin antagonists calmidazolium (Cal; 10 µM), trifluoperazine (TFP; 100 µM), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (50 µM) on TEA-insensitive, ChTX-sensitive KCa currents (at -78 mV) activated by 1 µM free Ca2+ in outside-out macropatches. Values are means ± SE of 5 or 6 experiments.

 

The Ca2+ sensitivity of SK4/IK1 channel gating is shown to be conferred to the channel by the association with calmodulin (6, 20). Our recent study also shows that calmodulin antagonists such as trifluoperazine, W-7, calmidazolium inhibit native SK4/IK1-like currents in bovine parotid acinar cells (34). However, we unexpectedly found that external application of these antagonists had little, if any, effects on TEA-insensitive, ChTX-sensitive KCa currents in RSA cells, so that calmidazolium (10 µM), trifluoperazine (100 µM), and W-7 (50 µM) reduced the currents at -78 mV to 83.9 ± 7.7% (n = 5, P = 0.1), 95.3 ± 25.1% (n = 6, P = 0.87), and 84.0 ± 6.9% (n = 5, P = 0.07) of the control values, respectively (Fig. 2D).

Molecular identification of rSK4/IK1 in RSA cells. We confirmed our recent RT-PCR analysis (34) that RSA cells express a transcript of rSK4/IK1 (Fig. 3), subsequently amplified 3 overlapping PCR fragments using the specific primers with high-fidelity DNA polymerase (Pfu-Turbo) and found that the nucleotide sequence of the coding region for the rat submandibular rSK4/IK1 was 1,278 bp in length and encoded a 425-amino acid protein. Sequence comparison of rSK4/IK1 cloned from rat submandibular acinar cells with that from rat distal colon (NM_023021 [GenBank] ) and from aortic smooth muscle rSK4/IK1 (AF190458 [GenBank] ) showed seven nucleotide differences, resulting in four amino acid changes and perfect identity, respectively.



View larger version (72K):
[in this window]
[in a new window]
 
Fig. 3. RT-PCR analysis of SK4/IK1 in RSA cells. PCR products were resolved by 1% agarose gel electrophoresis, and an amplicon (1,862 bp) for SK4/IK1 was detected by staining with ethidium bromide. As a control, {beta}-actin cDNA was amplified. No DNA fragment was amplified with the template without RT treatment.

 

We subsequently generated HEK-293 cells stably expressing rSK4/IK1 cloned from RSA cells and performed whole cell current measurements to examine whether biophysical and pharmacological properties of artificially expressed rSK4/IK1 currents match those of native currents. Figure 4A shows instantaneous I/V relations and traces of whole cell currents evoked by 400-ms voltage steps obtained from a HEK-293 cell stably transfected with rSK4/IK1. Consistent with kinetics of native currents (Fig. 1, A and B), voltage step commands evoked time-independent currents, which had a slightly inwardly rectifying I/V relationship under symmetrical K+ conditions and a nearly linear I/V relation in physiological Na+/K+ gradient (Fig. 4A). Although we found that TEA (1 or 10 mM) reduced rSK4/IK1 currents to 94.2 ± 0.5% (n = 5) or 74.3 ± 1.2% (n = 5) of the control level at -76 mV, respectively, we characterized electrophysiological properties of rSK4/IK1 currents in detail in the presence of external TEA (1 or 10 mM), a similar experimental condition where native currents were characterized. Biophysical and pharmacological properties of artificially expressed rSK4/IK1 currents were confirmed to almost match those of native currents (Figs. 1D, 2A, 2B, 2C, and 7C), based on its Ca2+-dependendent activation (Fig. 4B), monovalent cation permeability ratios of K+ (1; n = 7) {approx} Rb+ (0.90 ± 0.02; n = 7) >> Na+ (0.01 ± 0.00; n = 7), activation by 1-EBIO (100 µM) (Fig. 4C) and inhibition by clotrimazole (1 µM) and ChTX (100 nM), but not by apamin (100 nM) (Fig. 4D). We also found that the KCa currents were reversibly inhibited by external Ba2+ in a concentration- and a voltage-dependent manner with a Kd (0) of 95.4 ± 75.1 mM and a slope of 0.49 ± 0.04 (n = 4) (Fig. 4E), these values being similar to those of native currents.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4. Electrophysiological and pharmacological properties of whole cell currents recorded from human embryonic kidney (HEK)-293 cells stably transfected with rat SK4/IK1 (rSK4/IK1) cloned from RSA cells. A: instantaneous I/V relations of whole cell currents recorded from a HEK-293 cell expressing rSK4/IK1. Currents were elicited by 800-ms voltage ramps from -88 to +42 mV or from -86 to +44 mV in the bath solution containing 5 mM K+ or 150 mM K+, respectively. The pipette was filled with a standard K-glutamate-rich solution containing of 10 µM free Ca2+ and the bath contained a KCl-rich solution. Inset: representative traces of whole cell currents in the same cell as shown in A. The cell was held at -6 mV and stepped for 400 ms to potentials ranging between -106 and +34 mV in 10-mV steps. B: dependency of the whole cell rSK4/IK1 current activation on free [Ca2+] ([Ca2+]i) in the pipette solution. Whole cell current amplitudes normalized to cell capacitance (pA/pF) at -76 mV are plotted against [Ca2+]i in the pipette solution. The bath contained a KCl-rich solution having 10 mM TEA. Values are means ± SE of 3–5 experiments. C: [Ca2+] response for rSK4/IK1 currents in the absence (C) and presence of 1-EBIO (E). The current measured at -76 mV in the presence of 1-EBIO was normalized to the current in the absence of the drug. Values are means ± SE of 3–5 experiments. Inset: representative traces of the ChTX (100 nM)-sensitive currents before and after addition of 1-EBIO (100 µM) to the bathing solution. [Ca2+]i was 300 nM. D: effect of external clotrimazole (1 µM), ChTX (100 nM), or apamin (100 nM) on whole cell rSK4/IK1 currents activated by 10 µM free Ca2+. Average current at -106 mV in the presence of the blocker was expressed as a percentage of control current. Values are means ± SE of 6 or 7 experiments. The pipette and the bath solutions (containing 1 mM TEA) were the same as in A. Inset: representative traces before and after addition of ChTX or CLT to the bath solution. Control I/V relation was measured before addition of ChTX to the bath solution. Effect of ChTX was completely reversible. Control I/V relaion for clotrimazole was thus almost the same as the one for ChTX (not shown). E, top: representative instantaneous I/V relationships in the absence and presence of external Ba2+ obtained from a HEK-293 cell expressing rSK4/IK1. Bottom: voltage dependence of Kd values for Ba2+ block of Ca2+ (10 µM)-activated K+ currents. Data were fitted with an equation derived from Eqs. 2 and 3 (see MATERIALS AND METHODS). Kd values were plotted on a semilogarithmic scale. Values are means ± SE of 4 experiments. F, top: effect of TFP (100 µM), on whole cell rSK4/IK1 currents activated by 10 µM free Ca2+. Representative I/V relationships in the absence and presence of the blocker are shown. Bottom, summary of the effects of the calmodulin antagonists (10 µM Cal, 100 µM TFP, and 50 µM W-7) on ChTX-sensitive whole cell rSK4/IK1 currents at -76 mV. Values are means ± SE of 4–6 experiments.

 


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 7. Ca2+-dependent activation of TEA-insensitive K+ currents in an inside-out macropatch in the presence (A) and absence (B) of cytosolic ATP. Currents were elicited by 800-ms voltage ramps from -78 to +52 mV. The pipette contained a K-glutamate-rich solution having 10 mM TEA, and the bath contained a Na-glutamate-rich solution containing ATP [2 mM (A) or 0 mM (B)]/Mg2+ having different [Ca2+]i (<1 nM, and 0.1, 1, and 10 µM). The instantaneous current-voltage relation in the presence of pCa >9 represents the baseline, leak current component and is displaced positively (A). The addition of 2 mM ATP to the bathing solution (0 mM ATP) containing 1 µM [Ca2+]i increased the current (B). C: [Ca2+] response for TEA-insensitive K+ currents measured at -78 mV normalized by the response to Ca2+ (10 µM) is plotted as a function of [Ca2+]i. The pipette contained a K-glutamate-rich solution having 10 mM TEA, and the bath contained Na-glutamate-rich solution having 2 mM ATP ({bullet}) or no ATP ({circ}). The open circles are concealed by the solid circles at the lowest and highest [Ca2+]. Data were fit with the Hill equation (Eq. 1). Values are means ± SE of 7 experiments. D: effect of cytosolic ATP (2 mM) on TEA-insensitive K+ currents activated by a saturating [Ca2+] (10 µM). Instantaneous I/V relationship was first measured in the absence of and then in the presence of cytosolic ATP. Bar graphs show inward current amplitudes at -78 mV. Values are means ± SE of 13 experiments. The pipette contained a K-glutamate-rich solution having 10 mM TEA, and the bath solution was Na-glutamate rich solution containing 10 µM free Ca2+. *P < 0.05.

 

In contrast to the striking similarities of pore properties between cloned and native currents, we found that the expressed rSK4/IK1 currents were significantly inhibited by external application of calmodulin antagonists. Trifluoperazine (100 µM) and W-7 (50 µM) significantly reduced the current to 61.0 ± 5.4% (n = 5; P < 0.02) and 69.9 ± 6.3% (n = 6; P < 0.04) of the control values, respectively. The inhibition by these drugs was not dependent on both membrane potential and current density (data not shown). In contrast to these antagonists, calmidazolium (10 µM) had a minor effect on the currents, so that current amplitudes after addition of the drug were 85.2 ± 5.9% (n = 4; P = 0.23) of the control values (Fig. 4F).

Regulation of native SK4/IK1-like channel by cytosolic ATP. To examine whether cytosolic ATP regulates the native SK4/IK1-like currents in RSA cells, we next examined the effect of removal of ATP from the pipette solution on the currents. The SK4/IK1-like channel activity in outside-out patches was evaluated as external ChTX-sensitive currents in the presence of TEA (10 mM) in the bathing solution. Figure 5, A and B, depicts an example of excised outside-out patch experiments using the pipette solution with and without ATP, respectively, and demonstrates that native currents ran down unless ATP was present in the pipette solution. As also summarized in Fig. 5C, the currents [initial current amplitude; -265.2 ± 73.1 pA at -78 mV (n = 9)] decreased in a time-dependent manner (i.e., rundown of channel activity) when ATP was not included in the pipette solution, whereas the initial levels of currents [-755.2 ± 135.9 pA at -78 mV (n = 13)] were kept in the presence of ATP in the pipette solution. In separate outside-out macropatch experiments, we also confirmed that residual TEA-insensitive KCa currents in the absence of cytosolic ATP still possessed similar biophysical and pharmacological properties to those of the currents in the presence of ATP/Mg2+ mentioned above (data not shown).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5. A and B: stability of TEA-insensitive, ChTX-sensitive KCa currents (SK4/IK1-like currents) at -78 mV in outside-out patches obtained from RSA cells. Two independent experiments using a K-glutamaterich pipette solution (1 µM free Ca2+) having ATP (2 mM) (A) or no ATP (B) are shown. Bath solution was K-glutamate- or Na-glutamate-rich as indicated and contained TEA (10 mM). C: effect of removal of ATP (2 mM) from the pipette solution (1 µM free Ca2+) on SK4/IK1-like currents. ChTX-sensitive inward current amplitudes at -78 mV in the presence ({circ}) or absence ({bullet}) of ATP in the pipette solution are shown. When ATP was not included in the pipette solution, an appearance of currents attributable to a nonselective cation conductance often prevented a stable current recording. Values are means ± SE of 7–13 experiments. D: effect of removal of Mg2+ from the pipette solution on SK4/IK1-like currents in outside-out macropatches. Time course of normalized currents at -78 mV in the absence ({bullet}) or presence ({circ}) of Mg2+ (1 mM free Mg2+) in the pipette solution (1 µM free Ca2+) having ATP (2 mM) is shown. Values are means ± SE of 7–13 experiments. E: effect of inclusion of AMPPNP into the pipette solution on SK4/IK1-like currents in outside-out macropatches. The pipette contained a K-glutamate-rich solution (1 µM free Ca2+) having 0.2 mM AMP-PNP ({bullet}) or 0.2 mM ATP ({circ}). Values are means ± SE of 3–8 experiments. F: effect of staurosporine (1 µM) on SK4/IK1-like currents in outside-out macropatches. Time course of normalized currents at -78 mV in the presence ({bullet}) or absence ({circ}) of staurosporine in the pipette solution (1 µM free Ca2+) having ATP (2 mM)/Mg2+ is shown. Values are means ± SE of 7–13 experiments.

 

To determine whether ATP-dependent regulation of channel activity may involve protein phosphorylation, we first examined the effect of removal of Mg2+ from the pipette solution containing ATP (2 mM) on the stability of the currents and found rundown of the KCa currents (Fig. 5D), so that the ChTX-sensitive currents significantly decreased within 15 min to 28.0 ± 7.9% (n = 7, P < 0.05) of the initial values (-704.3 ± 141.6 pA at -78 mV: n = 11). We next tested whether the nonhydrolyzable analogue AMP-PNP could be substituted for ATP. In control experiments, where the pipette solution contained ATP (0.2 mM), the currents slightly decreased and reached a steady-state level [71.9 ± 4.8% (n = 7) of the initial values (-269.9 ± 50.0 pA at -78 mV)] within 6 min. When AMP-PNP (0.2 mM) was included into the pipette solution, the currents decreased within 6 min to 43.6 ± 9.7% (n = 6) of the initial values (-141.3 ± 35.7 pA at -78 mV), which were significantly different from those observed with ATP (0.2 mM) (P < 0.05) (Fig. 5E). We also found that inclusion of a nonspecific protein kinase inhibitor, staurosporine (1 µM) into the pipette solution having ATP/Mg2+ caused a rundown of the currents (Fig. 5F). In the presence of staurosporine, the currents gradually decreased to 28.8 ± 9.6% (n = 7, P < 0.05) of the initial values (-325.6 ± 118.6 pA at -78 mV) within 15 min. These results together imply a possible involvement of a protein phosphorylation in the ATP regulation of the native SK4/IK1-like channel in RSA cells.

We next addressed the question as to whether the ATP-dependent regulation of native SK4/IK1-like currents is mediated by endogenous cAMP-dependent protein kinase. To this end, we examined the effect of a protein kinase A inhibitor, Rp-cAMPS, on the currents (Fig. 6A). The addition of Rp-cAMPS (10 µM) to the pipette solution (1 µM free Ca2+) containing ATP (2 mM)/Mg2+ caused a rundown of the currents, so that the currents decreased to 27.8 ± 9.6% of the initial values [-110.2 ± 37.8 pA (n = 7, P < 0.01) at -78 mV] within 6 min. However, in control experiments, where the same concentration of cAMP (10 µM), instead of Rp-cAMPS, was added to the pipette solution having ATP/Mg2+, the currents were 91.3 ± 16.1% of the initial values [-234.4 ± 99.5 pA (n = 4) at -78 mV] at 6 min (Fig. 6A).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6. A: effect of addition of Rp-cAMPS to the pipette solution containing ATP/Mg2+ on SK4/IK1-like currents in outside-out macropatches. The pipette (1 µM free Ca2+) contained a K-glutamaterich solution having ATP/Mg2+ with 10 µM cAMP ({circ}) or 10 µM Rp-cAMPS ({bullet}). Values are means ± SE of 4–7 experiments. B: effect of internal cAMP on SK4/IK1-like currents in outside-out macropatches. The pipette contained a standard K-glutamate-rich solution (1 µM free Ca2+) having cAMP (1 mM) and ATP/Mg2+ ({bullet}) or having cAMP (1 mM) and no ATP ({circ}). Values are means ± SE of 7–11 experiments. C: effect of cAMP (1 mM) on SK4/IK1-like currents activated by 0.1 µM free Ca2+ in outside-out macropatches. The pipette contained a standard K-glutamate-rich solution (ATP/Mg2+ and 0.1 µM free Ca2+) having cAMP (1 mM) ({bullet}) or no cAMP ({circ}). Values are means ± SE of 4 experiments. D: effect of external forskolin (1 µM) on SK4/IK1-like currents activated by 0.1 µM (left) or 1 µM free Ca2+ (right) in outside-out macropatches. In these experiments, SK4/IK1-like currents activated by 0.1 µM free Ca2+ were extremely large. ChTX-sensitive inward current amplitudes at -78 mV before and after the addition of forskolin are shown. Values are means ± SE of 5 experiments. *P < 0.01.

 

We further examined in outside-out patches whether addition of a higher concentration of cAMP (1 mM) to the pipette solution containing ATP/Mg2+ would increase the currents evoked by 1 µM free Ca2+. As shown in Fig. 6B, cAMP gradually increased KCa currents activated by 1 µM Ca2+ to 157.2 ± 54.2% (n = 8) of the initial level of the currents (-445.5 ± 151.8 pA at -78 mV) at 9 min. However, in control experiments, where cAMP (1 mM) was included into the pipette solution having 1 µM free Ca2+ without ATP, we instead observed a decrease of the currents to 24.7 ± 6.7% (n = 9) of the initial level (-72.0 ± 12.9 pA at -78 mV) within 6 min (Fig. 6B), suggesting that cAMP itself has no effect on the channel activity. The increase in KCa currents in the presence of both cytosolic cAMP (1 mM) and ATP (2 mM) was more pronounced when the currents were activated by a lower [Ca2+]i (0.1 µM) and the mean peak currents were 307.5 ± 36.5% of the initial level (-38.0 ± 12.2 pA at -78 mV) of currents (n = 4; P < 0.001) (Fig. 6C). We also found that addition of forskolin (1 µM), an activator of adenylyl cyclase, to the bath solution caused an increase in SK4/IK1-like currents induced by 0.1 µM free Ca2+, but not by 1 µM free Ca2+, so that the mean peak currents were 129.9 ± 5.5% of the initial level (-424.6 ± 75.0 pA at -78 mV) of currents (n = 5; P < 0.01) (Fig. 6D). However, 1,9-dideoxyforskolin (1 µM) failed to enhance SK4/IK1-like currents induced by 0.1 µM free Ca2+ (n = 3, data not shown).

In most inside-out macropatches, native KCa currents were not observed or ran down and did not reach a steady-state level under various experimental conditions, and nonselective cation currents were often activated by an increase in cytosolic Ca2+, preventing characterizing native SK4/IK1 currents in this configuration in detail. However, some inside-out macropatches satisfied the criteria that the currents declined but a new steady-state level was achieved. Using these inside-out macropatches, we were able to examine the effect of ATP on the Ca2+ sensitivity of the currents. In these experiments, the pipette solution contained 10 mM TEA to block maxi-K+ channel activity. We also ensured that the currents were mainly carried by K+ in these experiments by substituting Na+ for K+ in the bath solution and in some experiments that the currents were blocked by addition of clotrimazole (1 µM) to the bathing solution (n = 4, data not shown). Figure 7, A and B, demonstrates that the currents in the inside-out macropatches are activated by cytosolic Ca2+ concentration in the presence and absence of cytosolic ATP, respectively, and also that addition of ATP to the bath solution having no ATP increases the currents activated by 1 µM free Ca2+ (Fig. 7B). As also shown in Fig. 7C, cytosolic ATP appeared to shift the Ca2+ sensitivity of the currents. A quantitative analysis (see MATERIALS AND METHODS) showed the Ca2+ dependence to have an apparent Kd of 1.35 ± 0.18 µM and a Hill coefficient of 2.61 ± 0.33 (n = 6) at -78 mV in the absence of cytosolic ATP, the Kd value being significantly different from the corresponding value (0.66 ± 0.13 µM, with a Hill coefficient of 3.28 ± 1.04) (n = 7, P < 0.05) in the presence of cytosolic ATP. Maximal response to cytosolic Ca2+ also appeared to be regulated by cytosolic ATP because the addition of ATP (2 mM) to the bath solution having free Ca2+ (10 µM) (i.e., a saturating concentration) significantly increased the currents (n = 13, P < 0.05) (Fig. 7D), although we could not perform further experiments with a [Ca2+]i >10 µM due to a strong activation of nonselective cation currents in the absence of ATP, which prevented evaluation of the KCa currents accurately.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Possible involvement of rSK4/IK1 in TEA-insensitive KCa currents in RSA cells. A previous whole cell patch-clamp study on RSA cells showed the presence of a TEA-insensitive KCa conductance, which was activated by physiological cytosolic Ca2+ levels (pCa = 6), highly permeable to Rb+, and blocked by external Ba2+ (17). The present study has now provided evidence that rSK4/IK1 contributes to a TEA-insensitive KCa conductance in RSA cells. First, electrophysiological properties of the native currents in the excised macropatches obtained from RSA cells include those of the whole cell currents mentioned above. The Ca2+-sensitivity observed in the present inside-out macropatches in an ATP-free environment is also in agreement with the finding that a TEA-insensitive whole cell KCa conductance was not activated by dialyzing with a similar ATP-free pipette solution containing a [Ca2+]i of 100 nM (17). Furthermore, we have found that a TEA-insensitive whole cell KCa conductance in RSA cells is inhibited by clotrimazole (1 µM) (C. Kunii and T. Ishikawa, unpublished observation). Second, RT-PCR analysis has confirmed the presence of transcripts of rSK4/IK1 in RSA cells (34). Finally, heterologous expression of rSK4/IK1 channels expressed from RSA cells induced KCa currents whose biophysical and pharmacological properties were similar to those of RSA cells: 1) time- and voltage-independent activation of the currents with a slightly inwardly rectifying current-voltage relationship in symmetrical K+ conditions, 2) inhibition by ChTX and clotrimazole, but not by apamin, and 3) activation by 1-EBIO.

The similarity of native and rSK4/IK1 channels was further strengthened by a voltage-dependent block of the currents by external Ba2+. As expected for an ion binding within the membrane electrical field, the Kd (0 mV) for Ba2+ and the slope ({delta}) of the block were estimated to be 311 and 95 mM and 0.61 and 0.49 mV for native and the cloned currents, respectively. Although the Kd (0 mV) values for the native and expressed channels appeared to be slightly different, it should be noted that the two values were determined under different conditions. The TEA-insensitive KCa channels naturally expressed in RSA cells were stimulated with 1 µM Ca2+ in the pipette solution, whereas the SK4/IK1 channels expressed heterologously in HEK-293 cells were stimulated with 10 µM Ca2+. In particular, the different level of [Ca2+]i may explain the discrepancy in the Kd (0 mV) values given that Ba2+ is probably an open channel blocker and that channels will undergo a greater degree of block when they are more likely to open.

The pharmacological properties of the native currents differed from those of SK4/IK1 currents heterologously expressed in HEK-293 cells in one respect. Calmodulin antagonists such as trifluoperazine, calmidazolium, and W-7 did not affect native currents, whereas expressed rSK4/IK1 currents were significantly inhibited by external trifluoperazine and W-7, the latter findings being consistent with a previous study on rSK4/IK1 cloned from rat colon expressed in Xenopus oocytes (36). The reason for these apparent discrepancies is unclear at present. However, it should be mentioned that the absence of inhibitory effects of calmodulin antagonists on SK4/IK1 channels is not without precedent. Fanger et al. (6) have shown that native hIK1-like currents in human T lymphocytes and hIK currents in COS-7 cells stably transfected with hIK1 are not affected by W-7, trifluoperazine, or calmidazolium and indicated the presence of a novel Ca2+-independent binding of calmodulin to hIK1. Del Carlo et al. (3) have shown that native intermediate Ca2+-activated K+ channels (IKCa or Gardos channels) of human erythrocytes are insensitive to the antagonists and interpreted the data as indicating a constitutive association of calmodulin with the native Gardos channels, thereby causing the unavailability of hydrophobic sites to which calmodulin antagonists could bind to prevent the interactions between calmodulin and the channel (3). Furthermore, Gerlach et al. (9) have suggested that Ca2+/calmodulin-dependent regulation of SK4/IK1 channels may involve an additional unidentified regulatory protein, whose expression might be cell or tissue specific.

Cytosolic ATP-dependent regulation of native SK4/IK1-like channels in RSA cells. The following results obtained in the present outside-out macropatch experiments are consistent with the interpretation that cytosolic ATP may regulate native SK4/IK1-like currents via a protein phosphorylation in RSA cells: 1) ChTX-sensitive, TEA-insensitive currents attributable to the SK4/IK1-like channel activity ran down unless both ATP and Mg2+ were present on the cytosolic surface of the membrane; 2) the rundown was not prevented when the pipette solution contained the nonhydrolyzable ATP analogue, AMPPNP with Mg2+; 3) inclusion of a nonspecific protein kinase inhibitor, staurosporine (1 µM), into the pipette solution having ATP/Mg2+ caused a marked rundown of the SK4/IK1-like channel activity. Three lines of other indirect evidence further support the view that a possible mechanism of the ATP-dependent regulation may involve, at least in part, endogenous PKA. First, inclusion of a PKA inhibitor Rp-cAMPS into the pipette solution caused a marked rundown even in the presence of ATP/Mg2+. Second, cAMP had a stimulatory effect on the currents in the presence of ATP. Because cAMP itself was not able to prevent the rundown observed in the absence of ATP, a direct effect of cAMP on the channel is unlikely to be involved. Finally, forskolin increased the currents induced by a lower concentration of free Ca2+ (0.1 µM). Although these experiments were performed in excised outside-out membrane patches, they do not allow us to determine whether PKA is membrane bound or is present in the submembrane region. It should be also stressed that the present study cannot exclude a role of other protein kinases in the ATP-dependent regulation of the native currents in RSA cells.

It remains unclear whether a PKA-dependent activation of native SK4/IK1-like channels is mediated by a direct phosphorylation of the channel itself or a closely associated protein in RSA cells, either. ATP-dependent activation, which involves a protein phosphorylation, has been described for both artificially expressed SK4/IK1 (8, 36, 40) and native SK4/IK1-like channels (8, 28, 31). Pellegrino and Pellegrini (31) demonstrated that in inside-out patches obtained from human erythrocytes a PKA inhibitor, PKI5–24 reversibly blocked the native human IK1 (hIK1)-like channels activated by the addition of a cocktail of cAMP, ATP/Mg2+, and theophylline to the cytosolic surface of the membrane. Gerlach et al. (8) also showed that the PKA inhibitor reversibly inhibited hIK1 and hIK1-like currents in inside-out patches excised from Xenopus oocytes heterologously expressing hIK1 and from T84 cells, but not from HEK-293 cells expressing hIK1. Interestingly, these authors have demonstrated that mutation of the single PKA consensus phosphorylation site at serine 334 to alanine in hIK1 failed to affect the PKA-dependent activation of the currents in Xenopus oocytes expression systems. In contrast to these studies, rSK4/IK1 and hIK1 currents expressed in Xenopus oocytes and HEK-293 cells, respectively, are not affected by PKA itself, PKA inhibitors or mutation of the PKA phosphorylation site at serine 332 to alanine in rSK4/IK1 and at serine 334 to glycine in hIK1 (32, 36). Given these contradictory results obtained in different experimental systems, it is tempting to speculate that PKA-dependent activation of SK4/IK1 like channels in RSA cells might also occur via phosphorylation of a closely associated protein rather than being a direct effect on the channel. Future studies are indeed required to examine whether such an interacting protein is present in RSA cells.

Physiological significance of a PKA-dependent regulation of SK4/IK1-like channel. A critical question concerns the physiological significance of a PKA-dependent regulation of SK4/IK1-like currents in RSA cells. An attractive possibility is that the regulation may be involved in synergistic effects of Ca2+-and cAMP-mediated agonists, such as ACh and VIP, respectively, both of which are shown to be coreleased from parasympathetic nerves innervating salivary glands (24), on salivary fluid secretion (5, 22, 24). Given the currently accepted model for the secretion in terms of the role of K+ channels, a cAMP-dependent modulation (probably via PKA activation) of SK4/IK1 may be a mechanism underlying the synergistic effects. In fact, the present study has demonstrated that a maneuver, which increases cytosolic cAMP, further enhances native SK4/IK1-like channel currents induced by a fixed [Ca2+]i in the presence of ATP/Mg2+, being also consistent with a previous report demonstrating that Ca2+-dependent fluid secretion, which is enhanced by an increase in cytosolic cAMP in rat submandibular gland is associated with an increase in a TEA-insensitive KCa conductance in RSA cells (16). Furthermore, we have shown that ATP-dependent activation of native SK4/IK1-like currents is likely associated with an increase in both the maximum current [proportional to the maximum channel activity, which is defined as the product of overall open probability (Po) of active channel and the number of active channel (NPo)] and Ca2+ sensitivity of the currents, giving a clue for the mechanism of a cAMP-dependent potentiation of a TEA-insensitive KCa conductance. However, we cannot say at this stage as to whether PKA-mediated phosphorylation increases the maximum Po or it recruits latent channels to open. Further single-channel experiments are required to address this isssue.

Potentiation of a Ca2+-activated fluid secretion involving SK4/IK1-like channels may also occur in cooperation with other types of KCa conductance. A recent study on perfused rabbit submandibular glands showed that a high concentration (5 mM) of a nonspecific K+ channel inhibitor, Ba2+ caused a partial inhibition of an ACh-induced transient fluid secretion for 1 min, but clotrimazole, an inhibitor of SK4/IK1 failed to block the secretion (33). These results may be interpreted as indicating a minor role of SK4/IK1 channels in an initial transient phase of the secretion induced by ACh in this gland. Although contribution of SK4/IK1-like channels to ACh-induced fluid secretion has not been determined in rat submandibular gland yet, even assuming that an unidentified KCa channel other than SK4/IK1 might support the initial phase of secretion, PKA-dependent upregulation of SK4/IK1-like channel activity would contribute to the increase in the basolateral K+ conductance.

The other possible role of a PKA-dependent regulation of SK4/IK1 might be involved in cell volume regulation because the cloned hIK1 channel is shown to be stimulated by cell swelling (11, 35, 37), which triggers regulatory volume decrease (RVD) via a loss of cellular KCl and water in various cell types (30). Although cell shrinkage rather occurs in salivary acinar cells during fluid secretion (7), an increase in cell volume, if it could happen under a pathophysiological condition, might be limited by an activation of SK4/IK1 channel. From this point of view, it is interesting to note that a RVD response to a hypotonic extracellular solution is largely reduced by clotrimazole (1 µM) in mouse parotid acinar cells (26). However, it remains to be examined whether PKA activation would modulate such a RVD in salivary acinar cells. Further experiments are necessary to elucidate the role of PKA-dependent regulation of SK4/IK1-like channels in native salivary acinar cells under physiological and pathophysiological conditions.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by a Grant-in-Aid for the Japan Society for the Promotion of Science (JSPS), and in part by a grant from the Northern Advancement Center for Science and Technology Foundation. M. Hayashi was supported by a JSPS Research Fellowship for Young Scientists.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. Ishikawa, Laboratory of Physiology, Dept. of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido Univ., Sapporo 060-0818, Japan (E-mail: torui{at}vetmed.hokudai.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Barry PH and Lynch JW. Liquid junction potentials and small cell effects in patch-clamp analysis. J Membr Biol 121: 101-117, 1991.[ISI][Medline]

2. Cook DI, Van Lennep EW, Roberts ML, and Young JA. Secretion by the major salivary glands. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR, Alpers DH, Christensen J, Jacobson ED, and Walsh JH. New York: Raven, 1994, p. 1061-1117.

3. Del Carlo B, Pellegrini M, and Pellegrino M. Calmodulin antagonists do not inhibit IKCa channels of human erythrocytes. Biochim Biophys Acta 1558: 133-141, 2002.[ISI][Medline]

4. Devor DC, Singh AK, Frizzell RA, and Bridges RJ. Modulation of Cl- secretion by benzimidazolones. I. Direct activation of a Ca2+-dependent K+ channel. Am J Physiol Lung Cell Mol Physiol 271: L775-L784, 1996.[Abstract/Free Full Text]

5. Ekström J and Olgart L. Complementary action of substance P and vasoactive intestinal peptide on the rat parotid secretion. Acta Physiol Scand 126: 25-31, 1986.[ISI][Medline]

6. Fanger CM, Ghanshani S, Logsdon NJ, Rauer H, Kalman K, Zhou J, Beckingham K, Chandy KG, Cahalan MD, and Aiyar J. Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1. J Biol Chem 274: 5746-5754, 1999.[Abstract/Free Full Text]

7. Foskett JK and Melvin JE. Activation of salivary secretion: coupling of cell volume and [Ca2+]i in single cells. Science 244: 1582-1585, 1989.[ISI][Medline]

8. Gerlach AC, Gangopadhyay NN, and Devor DC. Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. J Biol Chem 275: 585-598, 2000.[Abstract/Free Full Text]

9. Gerlach AC, Syme CA, Giltinan L, Adelman JP, and Devor DC. ATP-dependent activation of the intermediate conductance, Ca2+-activated K+ channel, hIK1, is conferred by a C-terminal domain. J Biol Chem 276: 10963-10970, 2001.[Abstract/Free Full Text]

10. Grissmer S, Nguyen AN, and Cahalan MD. Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology. J Gen Physiol 102: 601-630, 1993.[Abstract]

11. Grunnet M, MacAulay N, Jorgensen NK, Jensen S, Olesen SP, and Klaerke DA. Regulation of cloned, Ca2+-activated K+ channels by cell volume changes. Pflügers Arch 444: 167-177, 2002.[CrossRef][ISI][Medline]

12. Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85-100, 1981.[ISI][Medline]

13. Hille B. Ionic Channels of Excitable Membranes (2nd ed.). Sunderland, MA: Sinauer Associates, 1992.

14. Hoffman JF, Joiner W, Nehrke K, Potapova O, Foye K, and Wickrema A. The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells. Proc Natl Acad Sci USA 100: 7366-7371, 2003.[Abstract/Free Full Text]

15. Ishii TM, Silvia C, Hirschberg B, Bond CT, Adelman JP, and Maylie J. A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci USA 94: 11651-11656, 1997.[Abstract/Free Full Text]

16. Ishikawa T. cAMP modulation of a Ca2+-dependent K+ conductance in rat submandibular acinar cells. Am J Physiol Gastrointest Liver Physiol 272: G454-G462, 1997.[Abstract/Free Full Text]

17. Ishikawa T and Murakami M. Tetraethylammonium-insensitive, Ca2+-activated whole-cell K+ currents in rat submandibular acinar cells. Pflügers Arch 429: 748-750, 1995.[ISI][Medline]

18. Jensen BS, Strøbæk D, Christophersen P, Jørgensen TD, Hansen C, Silahtaroglu A, Olesen SP, and Ahring PK. Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel. Am J Physiol Cell Physiol 275: C848-C856, 1998.[Abstract]

19. Joiner WJ, Wang LY, Tang MD, and Kaczmarek LK. hSK4, a member of a novel subfamily of calcium-activated potassium channels. Proc Natl Acad Sci USA 94: 11013-11018, 1997.[Abstract/Free Full Text]

20. Khanna R, Chang MC, Joiner WJ, Kaczmarek LK, and Schlichter LC. hSK4/hIK1, a calmodulin-binding KCa channel in human T lymphocytes. Roles in proliferation and volume regulation. J Biol Chem 274: 14838-14849, 1999.[Abstract/Free Full Text]

21. Larsson O, Detsch T, and Fredholm BB. VIP and forskolin enhance carbachol-induced K+ efflux from rat salivary gland fragments by a Ca2+-sensitive mechanism. Am J Physiol Cell Physiol 259: C904-C910, 1990.[Abstract/Free Full Text]

22. Larsson O and Olgart L. The enhancement of carbachol-induced salivary secretion by VIP and CGRP in rat parotid gland is mimicked by forskolin. Acta Physiol Scand 137: 231-236, 1989.[ISI][Medline]

23. Logsdon NJ, Kang J, Togo JA, Christian EP, and Aiyar J. A novel gene, hKCa4, encodes the calcium-activated potassium channel in human T lymphocytes. J Biol Chem 272: 32723-32726, 1997.[Abstract/Free Full Text]

24. Lundberg JM, Änggård A, and Fahrenkrug J. Complementary role of vasoactive intestinal polypeptide (VIP) and acetylcholine for cat submandibular gland blood flow and secretion. I. VIP release. Acta Physiol Scand 113: 317-327, 1981.[ISI][Medline]

25. Neher E. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207: 123-131, 1992.[ISI][Medline]

26. Nehrke K, Arreola J, Nguyen HV, Pilato J, Richardson L, Okunade G, Baggs R, Shull GE, and Melvin JE. Loss of hyperpolarization-activated Cl- current in salivary acinar cells from Clcn2 knockout mice. J Biol Chem 277: 23604-23611, 2002.[Abstract/Free Full Text]

27. Nehrke K, Quinn CC, and Begenisich T. Molecular identification of Ca2+-activated K+ channels in parotid acinar cells. Am J Physiol Cell Physiol 284: C535-C546, 2003.[Abstract/Free Full Text]

28. Nielsen MS, Warth R, Bleich M, Weyand B, and Greger R. The basolateral Ca2+-dependent K+ channel in rat colonic crypt cells. Pflügers Arch 435: 267-272, 1998.[CrossRef][ISI][Medline]

29. Oiki S and Okada Y. Ca-EGTA buffer in physiological solutions. Seitai-no-kagaku 38: 79-83, 1987.

30. Okada Y. Volume expansion-sensing outward-rectifier Cl- channel: fresh start to the molecular identity and volume sensor. Am J Physiol Cell Physiol 273: C755-C789, 1997.[Abstract/Free Full Text]

31. Pellegrino M and Pellegrini M. Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase. Pflügers Arch 436: 749-756, 1998.[CrossRef][ISI][Medline]

32. Schrøder RL, Jensen BS, Strøbæk D, Olesen SP, and Christophersen P. Activation of the human, intermediate-conductance, Ca2+-activated K+ channel by methylxanthines. Pflügers Arch 440: 809-818, 2000.[CrossRef][ISI][Medline]

33. Stummann TC, Poulsen JH, Hay-Schmidt A, Grunnet M, Klaerke DA, Rasmussen HB, Olesen SP, and Jorgensen NK. Pharmacological investigation of the role of ion channels in salivary secretion. Pflügers Arch 446: 78-87, 2003.[ISI][Medline]

34. Takahata T, Hayashi M, and Ishikawa T. SK4/IK1-like channels mediate TEA-insensitive, Ca2+-activated K+ currents in bovine parotid acinar cells. Am J Physiol Cell Physiol 284: C127-C144, 2003.[Abstract/Free Full Text]

35. Vandorpe DH, Shmukler BE, Jiang L, Lim B, Maylie J, Adelman JP, de Franceschi L, Cappellini MD, Brugnara C, and Alper SL. cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation. J Biol Chem 273: 21542-21553, 1998.[Abstract/Free Full Text]

36. von Hahn T, Thiele I, Zingaro L, Hamm K, Garcia-Alzamora M, Köttgen M, Bleich M, and Warth R. Characterisation of the rat SK4/IK1 K+ channel. Cell Physiol Biochem 11: 219-230, 2001.[CrossRef][ISI][Medline]

37. Wang J, Morishima S, and Okada Y. IK channels are involved in the regulatory volume decrease in human epithelial cells. Am J Physiol Cell Physiol 284: C77-C84, 2003.[Abstract/Free Full Text]

38. Warth R, Hamm K, Bleich M, Kunzelmann K, von Hahn T, Schreiber R, Ullrich E, Mengel M, Trautmann N, Kindle P, Schwab A, and Greger R. Molecular and functional characterization of the small Ca2+-regulated K+ channel (rSK4) of colonic crypts. Pflügers Arch 438: 437-444, 1999.[CrossRef][ISI][Medline]

39. Woodhull AM. Ionic blockage of sodium channels in nerve. J Gen Physiol 61: 687-708, 1973.[Abstract/Free Full Text]

40. Wulf A and Schwab A. Regulation of a calcium-sensitive K+ channel (cIK1) by protein kinase C. J Membr Biol 187: 71-79, 2002.[CrossRef][ISI][Medline]