Abstract
Bovine adrenal zona fasciculata cells express a novel K+current (IAC) that sets the resting potential while it couples adrenocorticotropin and angiotensin II receptors to membrane depolarization and cortisol secretion. IAC is distinctive among K+ channels both in its activation by ATP and its inhibition by cyclic AMP. Whole-cell and single-channel patch-clamp recording was used to establish a pharmacological profile of IAC K+ channels. IAC was blocked by antagonists of cyclic nucleotide-gated channels, including the diphenylbutylpiperidine (DPBP) antipsychotic pimozide andl-cis-diltiazem. Other DPBPs, including penfluridol and fluspirilene, also potently inhibited this channel. The inhibition of IAC by DPBPs was selective because 200-fold higher concentrations of penfluridol were required to inhibit voltage-gated IA K+ channels in adrenal zona fasciculata cells. Standard K+ channel antagonists blocked IAC at concentrations 100- to 100,000-fold higher than the DPBPs. IAC channels were also inhibited by the sulfonylureas glyburide and tolbutamide but at concentrations higher than those that typically block ATP-sensitive inward rectifier K+ channels. Overall, the relative order of potency and associated IC50 values for IAC antagonists were as follows: penfluridol (0.187 μM) > fluspirilene (0.232 μM) > pimozide (0.354 μM) ≫l-cis-diltiazem (24.9 μM) ≈ quinidine (24.1 μM) > bupivacaine (113.2 μM) > tolbutamide (784.4 μM) > BaCl2 (1027 μM) > 4-aminopyridine (2750 μM) > tetraethylammonium (24,270 μM). IAC channels are unique in combining the pharmacological properties of K+-selective channels with those of cyclic nucleotide-gated cation channels. The potent block of IACchannels identifies DPBPs as a new class of K+ channel antagonists and suggests additional targets for these neuroleptics in the central nervous system.
Eucaryotic cells express a large number of K+ selective ion channels that have been identified and characterized with electrophysiological and molecular techniques. These K+ channels have been separated into two large families based on differences in their structure, pharmacology, and gating (Chandy and Gutman, 1995; Jan and Jan, 1997). The voltage-gated K+ channels whose α subunits contain six membrane-spanning domains comprise the largest family. Molecular cloning of α subunits from the voltage-gated channels revealed the presence of multiple subfamilies that contain a conserved core region and a variable flanking region. Within this subfamily are included the large conductance Ca2+-activated K+ channels and a group of cyclic nucleotide-gated (CNG) K+-selective channels (eag channels).
Inward rectifiers form the second major group of K+-selective ion channels. Unlike the voltage-gated ion channels, the inward rectifier α subunits include two rather than six hydrophobic segments (Jan and Jan, 1997). Many inward rectifiers are inhibited by the nonhydrolytic binding of ATP. These ATP-sensitive, or KATP, channels act as metabolic sensors in a variety of cell types (Ashcroft, 1988; Takano and Noma, 1993; Jan and Jan, 1997). Although inward rectifiers are structurally quite different from voltage-gated K+ channels, homologous pore regions account for their similar ionic selectivity (Ho et al., 1993; Jan and Jan, 1997).
With the molecular cloning of many types of ion channels, a striking similarity between the voltage-activated K+channels and the CNG nonselective cation channels from retinal and olfactory neurons was discovered (Guy and Durell, 1991; Heginbotham et al., 1992). In particular, eag K+channels are more closely related to polypeptides of CNG channels than to other voltage-gated K+ channels (Guy and Durell, 1991). The eag K+ channels contain a cyclic AMP (cAMP)-binding region in the carboxyl-terminal region, and they are modulated by cAMP (Bruggemann et al., 1993). Other K+ channels have recently been identified that may be directly modulated by cAMP (Mlinar et al., 1993; Enyeart et al., 1996). Taken together, these results suggest that interposed between the voltage-gated K+ channels and the CNG cation channels, a continuum of intermediate forms exists that includes K+-selective channels whose gating is directly controlled by cAMP and perhaps other nucleotides.
Bovine adrenal zona fasciculata (AZF) cells express an interesting example of these hybrid K+ channels. Noninactivating potassium current in bovine AZF cells (IAC) channels set the resting potential of AZF cells while they couple adrenocorticotropin receptor activation to depolarization-dependent Ca2+ entry and cortisol secretion (Enyeart et al., 1993, 1996; Mlinar et al., 1993). Aside from their central role in steroidogenesis, IACchannels are unique among K+-selective channels in their modulation by nucleotides. Specifically, these channels are among the first channels described that are inhibited by cAMP through an A-kinase-independent mechanism (Enyeart et al., 1996). Furthermore, IAC channels are distinctive because they are activated by the nonhydrolytic binding of ATP and other nucleotides, including ADP, GTP, UTP, and AMP-PNP (Enyeart et al., 1997). Overall, IAC channels incorporate features of voltage-gated and ATP-sensitive K+ channels, as well as CNG nonselective cation channels.
Voltage-gated K+ channels, ATP-sensitive inward rectifier K+ channels, and CNG nonselective cation channels have separate pharmacological profiles. Homology among the pores of all K+-selective channels confers sensitivity to a group of inorganic and organic blockers that originally were identified as antagonists of voltage-gated K+ channels (Cook and Quast, 1990; Lancaster, 1991; Hille, 1992). The β subunit of ATP-sensitive K+ channels is a member of the ABC transporter family of membrane proteins (Aguilar-Bryan et al., 1995; Inagaki et al., 1995). This β subunit or sulfonylurea receptor is responsible for the unique pharmacology of ATP-sensitive K+channels conferring sensitivity to antagonists such as glyburide and tolbutamide (Edwards and Weston, 1993; Inagaki et al., 1996).
CNG nonselective cation channels are much less sensitive to any of the K+ channel antagonists mentioned above, but they are blocked relatively potently byl-cis-diltiazem and the diphenylbutylpiperidine antipsychotic pimozide (Koch and Kaupp, 1985; Stern et al., 1986;Haynes, 1992; Nicol, 1993).
In establishing a pharmacological profile of this new class of metabolically regulated K+ channels, we wanted to determine whether the hybrid properties of IACchannels, with respect to gating and permeation, would be reflected in their sensitivity to the different ion channel antagonists described above. Surprisingly, although IACK+ channels were inhibited by standard K+ channel antagonists, they were far more sensitive to antagonists of CNG cation channels, particularly the DPBPs.
Materials and Methods
Tissue culture media, antibiotics, fibronectin, and FBS were obtained from GIBCO (Grand Island, NY). Coverslips were from Bellco Glass, Inc. (Vineland, NJ). Enzymes, MgATP, NaGTP, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 4-aminopyridine (4-AP), tetraethylammonium (TEA), quinidine, bupivacaine, BaCl2, and pimozide were obtained from Sigma Chemical Co. (St. Louis, MO). Glyburide and tolbutamide were obtained from BIOMOL (Plymouth Meeting, PA). Fluspirilene was obtained from Research Biochemicals Inc. (Natick, MA). Penfluridol was obtained from Janssen Pharmaceutical (Beerse, Belgium).l-cis-Diltiazem was a generous gift from Tanabe Seiyaku, Ltd. (Saitama, Japan).
Isolation and Culture of AZF Cells.
Bovine adrenal glands were obtained from steers (age range, 1–3 years) within 30 min of slaughter at a local slaughterhouse. Fatty tissue was removed immediately, and the glands were transported to the laboratory in ice-cold PBS containing 0.2% dextrose. Isolated AZF cells were prepared as previously described (Enyeart et al., 1997). Cells were plated onto 35-mm dishes containing 9-mm2 glass coverslips that had been treated with fibronectin (10 μg/ml) at 37°C for 30 min and then rinsed with warm, sterile PBS immediately before addition of the cells. Dishes were maintained at 37°C in a humidified atmosphere of 95% air/5% CO2.
Patch-Clamp Experiments.
Patch-clamp recordings of K+ channel currents were made in the whole-cell and outside-out patch configurations. For both recording configurations, the standard pipette solution was 115 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 20 mM HEPES, 11 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, and 200 μM GTP, with pH buffered to 7.2 using KOH. For whole-cell and patch recordings, pipette solutions contained 5 and 2 mM MgATP, respectively. Pipette [Ca2+] was 22 nM as determined using the “Bound and Determined” program (Brooks and Storey, 1992). The external solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM CaCl, 2 mM MgCl2, 10 mM HEPES, and 5 mM glucose, pH 7.4 using NaOH. All solutions were filtered through 0.22-μm cellulose acetate filters.
AZF cells were used for patch-clamp experiments 2 to 12 h after plating. Typically, cells with diameters of less than 15 μm and capacitances of 8 to 12 pF were selected. Coverslips were transferred from 35-mm culture dishes to the recording chamber (volume, 1.5 ml), which was continuously perfused by gravity at a rate of 3 to 5 ml/min. For whole-cell recordings, patch electrodes with resistances of 1.0 to 2.0 MΩ were fabricated from Corning 0010 glass (Garner Glass Co., Claremont, CA); these routinely yielded access resistances of 1.5 to 4 MΩ and voltage-clamp time constants of less than 100 μs. For single-channel recordings, patch electrodes with higher resistances of 3 to 5 MΩ were used. K+ currents were recorded at room temperature (22–25°C) according to the procedure of Hamill et al. (1981) using an Axopatch 1-D (Axon Instruments, Inc., Burlingame, CA) patch-clamp amplifier.
Pulse generation and data acquisition were done using a personal computer and pCLAMP software with a TL-1 interface (Axon Instruments). Currents were digitized at 1 to 20 kHz after filtering with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA). Linear leak and capacity currents were subtracted from current records using scaled hyperpolarizing steps of one-third to one-fourth amplitude. Data were analyzed and plotted using pCLAMP Versions 5.5 and 6.02 (CLAMPAN, CLAMPFIT, FETCHAN, and PSTAT) and SigmaPlot 4.0. Drugs were applied by bath perfusion, controlled manually by a six-way rotary valve. Series resistance compensation was not used in experiments where the IAC currents were less than 1 nA. A current of this size in combination with a 4 MΩ access resistance produces a voltage error of 4 mV that was not corrected.
Results
Inhibition of IAC K+ Current by K+ Channel Blockers
Bovine AZF cells express two separate K+currents that are easily distinguished in whole-cell patch-clamp recordings; these include a voltage-gated, rapidly inactivating A-type K+ current, and a noninactivating K+ current (IAC) that grows to a stable maximum amplitude over a period of many minutes, provided that the pipette contains ATP or other nucleotides at millimolar concentrations (Mlinar et al., 1993; Mlinar and Enyeart, 1993; Enyeart et al., 1997). Under these conditions, IACK+ current did not “rundown” in recordings lasting 1 h or longer. The absence of time-dependent inactivation of the IAC K+ current allow it to be easily isolated for measurement in whole-cell recordings. Using either of two voltage-clamp protocols, voltage steps to +20 mV applied from a holding potential of −80 mV activated the rapidly inactivating A-type K+ current (IA) as well as the noninactivating IAC K+ current (Fig.1, left traces). When voltage steps of 300-ms duration were applied, IAC could be selectively measured near the end of a step at a time when the A-type K+ current had inactivated entirely. Using a second protocol, IAC was selectively activated using an identical voltage step after a 10-s prepulse to −20 mV had fully inactivated the A-type current (Fig. 1, right traces).
We determined the potency of five well known antagonists of voltage-gated K+ channels with respect to their ability to inhibit IAC K+current. These included the four organic antagonists: 4-AP, TEA, quinidine, bupivacaine, and the divalent cation Ba2+ (Figs. 1 and2). These agents were superfused at increasing concentrations, and voltage steps were applied at 30-s intervals. Antagonist concentration was increased only when steady-state block was achieved as determined by three consecutive current traces of nearly constant amplitude. These agents all reversibly inhibited IAC, with IC50 values that varied over a 1000-fold range from 24.1 μM for quinidine to 24.3 mM for TEA. The order of potency and corresponding IC50 values were quinidine (2.41 × 10−5 M) > bupivacaine (1.13 × 10−4 M) > BaCl2 (1.02 × 10−3M) > 4-AP (2.75 × 10−3 M) > TEA (2.43 × 10−2 M) (Fig. 2B).
None of these agents selectively or even preferentially blocked IAC. Each also inhibited the rapidly inactivating IA current at concentrations sufficient to inhibit IAC (Figs. 1 and 2). Of the five drugs, 4-AP preferentially blocked the rapidly inactivating IA current (Fig. 1, left). In a previous study of IA current in these cells, we found that 4-AP inhibits IA with an IC50value of 630 μM (Mlinar and Enyeart, 1993), a concentration approximately 4-fold lower than the IC50 value for IAC inhibition by this agent.
Inhibition of IAC by Sulfonylureas
Inwardly rectifying KATP channels are potently blocked by sulfonylureas including glyburide and tolbutamide (Inagaki et al., 1995, 1996). We found that ATP-activated IAC K+ channels were much less sensitive to inhibition by sulfonylureas. Glyburide is the most potent sulfonylurea, inhibiting ATP-sensitive K+channels in insulin-secreting cells at concentrations of less than 10 nM (Inagaki et al., 1995, 1996; Aguilar-Bryan et al., 1995). As illustrated in Fig. 3, glyburide inhibited IAC only at much higher concentrations (IC50 = 63.8 μM). Even though glyburide was a weak antagonist of IAC, it was relatively selective. Figure 3A shows that at the highest concentration used (100 μM), glyburide produced little inhibition of IAcurrent, whereas IAC was inhibited by more than 60%. A second sulfonylurea, tolbutamide, is much less potent than glyburide as an inhibitor of ATP-sensitive K+channels (Inagaki et al., 1995). Tolbutamide inhibited IAC with an IC50 value of 784 μM (Fig. 3B).
Inhibition of IAC by CNG Channel Antagonists
DPBPs.
IAC K+channels are one of a select group of ion channels that are modulated by cAMP through an A-kinase-independent mechanism. Sequence similarity between K+ channels and CNG nonselective cation channels has been mentioned above. CNG channels of the rod photoreceptor are blocked by the DPBP antipsychotic pimozide with an IC50 value of 0.8 μM (Nicol, 1993). We measured the inhibition of IAC K+channels by pimozide and two other DPBPs, fluspirilene and penfluridol, in whole-cell and single-channel patch-clamp recordings.
Each of these agents potently and preferentially inhibited IAC. Figure 4illustrated the concentration-dependent inhibition of IAC by fluspirilene. K+currents were recorded with (Fig. 4A, bottom) or without (Fig. 4A, top) depolarizing prepulses. When IAC reached a stable amplitude, the cell was sequentially superfused with increasing concentrations of fluspirilene (Fig. 4B). At each concentration, currents were recorded until inhibition reached a clear steady-state value. Inhibition by this lipid soluble agent was partially reversible with prolonged washing. Fluspirilene inhibited IAC with an IC50 value of 2.32 × 10−7 M (Fig. 4C). As shown in Fig.4A, inhibition of IAC by fluspirilene appeared to be relatively selective. Even the highest concentration (10 μM), which completely blocked IAC, produced less than 15% inhibition of the rapidly inactivating A-type current.
The other two DPBPs, penfluridol and pimozide, inhibited IAC with IC50 values of 1.84 × 10−7 and 3.54 × 10−7 M, respectively (Fig.5, A and C). To quantify the selective inhibition of IAC compared with IA K+ current by DPBPs, we measured the concentration-dependent inhibition of IA by penfluridol in cells where IAC current had been specifically and completely eliminated by first superfusing AZF cells with adrenocorticotropin (200 pM) (Mlinar et al., 1993; Enyeart et al., 1996). Penfluridol inhibited IA with an IC50value of 4.18 × 10−5 m, a concentration more than 200-fold higher than that required to produce 50% inhibition of IAC (Fig. 5, B and C). Because of this large difference in potency, penfluridol can be used at concentrations near 1 μM to produce complete and selective inhibition of IAC.
Ramp voltage-clamp protocols showed that IACcurrent was effectively blocked by penfluridol over a wide range of test potentials. Linear voltage ramps applied between +60 and −140 mV, with a slope of 100 mV/s from a holding potential of 0 mV, selectively recorded the outwardly rectifying IAC current (Fig. 6). Penfluridol (2.5 μM) effectively blocked this current by more than 90% at potentials between −50 and +60 mV, where IAC could be accurately measured.
Effect of Penfluridol on Unitary IAC Currents.
Penfluridol inhibited single-channel IAC activity in excised outside-out patches without reducing the amplitude of the unitary current. Figure 7 shows unitary IAC currents in an outside-out patch, recorded in response to depolarizing steps to +30 mV from a holding potential of −40 mV, a potential where IA channels are inactivated (Mlinar and Enyeart, 1993). Under these conditions, a single type of K+ channel was typically present in the membrane patch. In control saline, histogram analysis of unitary current amplitudes showed a major peak with a mean of 3.96 ± 0.67 pA. Two smaller peaks with mean values approximately twice and three times that of the major peak (7.87 ± 0.55 and 11.57 ± 0.72 pA) were also present, indicating that at least three active IAC channels were present in this patch (Fig.7A).
IAC channel activity was dramatically reduced on superfusion of the membrane patch with 200 nM penfluridol (Fig. 7B). Although the amplitude of the unitary IAC current remained essentially constant (3.77 ± 0.32 pA), the open probability (Po) was reduced by 95%.
Inhibition by penfluridol was reversible in outside-out recordings (Fig. 7C). IAC channel activity increases spontaneously with time in excised-patch recordings. However, in contrast to whole-cell recordings, IAC channel activity does not typically reach a stable maximum value. Accordingly, in the experiment illustrated in Fig. 7C, IACchannel activity increased compared with the control value on washing. Histogram analysis showed that four distinct peaks, each a multiple of the unitary current amplitude, were present after washing with control saline. At this time Po was increased by 86% compared with the control value.
Block of IAC by l-cis-Diltiazem.
Along with pimozide, l-cis-diltiazem is the most potent known antagonist of CNG cation channels, having reported IC50 values of less than 10 μM (Koch and Kaupp, 1985; Stern et al., 1986; Haynes, 1992). l-cis-Diltiazem blocked IAC K+ current at slightly higher concentrations (IC50 = 24.9 μM) (Fig. 8). Compared with the DPBPs, this drug was less selective for IAC. IA current was significantly reduced by diltiazem at concentrations that maximally inhibited IAC(Fig. 8A). In contrast to inhibition by penfluridol, inhibition of IAC by l-cis-diltiazem was rapidly reversible (data not shown).
Discussion
We discovered that IACK+ channels possess a unique pharmacological profile combining sensitivity to standard K+channel blockers and antagonists of CNG cation channels. Standard K+ channel blockers inhibited IAC at concentrations typical of those that inhibit a wide range of K+ channels. By comparison, antagonists of CNG-gated channels, particularly the DPBPs, were much more potent inhibitors of IAC channels. Block of IAC K+ channels by the DPBPs was selective because 200-fold higher concentrations were required to inhibit the rapidly inactivating IAK+ current.
Block of IAC by DPBPs andl-cis-Diltiazem.
These results demonstrate that the distinctive hybrid properties of IACK+ channels extend to their pharmacology. Potent inhibition of K+ channels by DPBPs is rare but appears to be a common characteristic of CNG cation channels (Nicol, 1993; Broillet and Firestein, 1997; Wible et al., 1997). The DPBP pimozide blocks CNG channels of rod photoreceptors with an IC50 value of 0.8 μM (Nicol, 1993), a concentration only 2-fold higher than that blocking IAC half-maximally.
In addition to IAC K+channels and CNG channels, DPBPs also inhibit several subtypes of voltage-gated Ca2+ channels in endocrine and neurally derived cells (Enyeart et al., 1990, 1993). Why DPBPs block ion channels as diverse as these with similar potency is not clear. However, the recent finding that the β subunit of CNG channels is itself a Ca2+-selective channel that is inhibited by pimozide may identify a common link among these channels (Chen et al., 1993; Broillet and Firestein, 1997).
l-cis-Diltiazem blocked IACK+ channels at concentrations only slightly higher than those that inhibit CNG channels (Koch and Kaupp, 1985;Stern et al., 1986; Haynes, 1992). The β subunit of the human rod CNG channel dramatically increases the sensitivity of this channel tol-cis-diltiazem by 100-fold (Chen et al., 1993). When expressed along with the α subunit, the heteroligomeric channel is inhibited by micromolar l-cis-diltiazem, much like the native channel.
It is interesting that β subunits of the CNG channels, which are themselves Ca2+-conducting channels, confer sensitivity to l-cis-diltiazem, which is an isomer of the Ca2+ channel blockerd-cis-diltiazem and to a second Ca2+ antagonist, pimozide. The similarities between IAC channels and CNG channels with respect to gating by cAMP and sensitivity to DPBPs and diltiazem are intriguing. Perhaps functional IACK+ channels include a β subunit with homology to that of the CNG channels, which confers sensitivity to DPBPs andl-cis-diltiazem.
Mechanism of IAC Block by DPBPs.
The molecular mechanism by which DPBPs inhibit IAC channels remains to be determined. Block of voltage-gated Ca2+ channels by DPBPs is enhanced by repeated or prolonged depolarizations, suggesting preferential binding of these drugs to open or inactivated channels (Enyeart et al., 1990, 1992). However, unlike Ca2+ channels, IAC channels are only weakly voltage dependent, are open at negative potentials, and do not inactivate (Mlinar et al., 1993; Enyeart et al., 1996, 1997). Thus, the characteristics of block would likely be quite different for the two types of channels. In ramp voltage protocols, penfluridol blocked IACcurrent effectively over a wide range of test voltages. Similarly, block of rod photoreceptor CNG channels by pimozide was reported to be independent of membrane voltage in contrast to its action in blocking voltage-gated Ca2+ channels (Nicol, 1993).
At the single-channel level, penfluridol did not reduce the size of the individual unitary IAC currents, as is often observed by blockers whose kinetics of binding and unbinding are very rapid. Penfluridol blocks unitary IAC currents with characteristics of a slow or intermediate blocker (Moczydlowski, 1992).
K+ Channel Blockers.
Standard K+ channel blockers including TEA, 4-AP, quinidine, and Ba2+ inhibit K+-selective channels with varying potencies. Each of these agents inhibited IACK+ channels at concentrations typical of those that effectively block other K+-selective channels. TEA inhibited IAC with an IC50 of 24.3 mM, a value slightly higher than that reported for many delayed rectifier K+channels but lower than the concentration required to produce half-maximal inhibition of some other K+ currents (Cook and Quast, 1990; Bokvist et al., 1990; Lancaster, 1991; Edwards and Weston, 1993).
4-AP blocked IAC K+channels with an IC50 value of 2.75 mM. This drug blocks voltage-gated K+ channels, including some delayed rectifiers and A-type K+ currents with similar potency, whereas other K+ channels, including Ca2+-activated K+channels are far less sensitive (Cook and Quast, 1990; Edwards and Weston, 1993; Ludwig et al., 1994).
Compared with TEA and 4-AP, quinidine (IC50 = 24.1 μM) was approximately 100 and 1000 times more potent as an inhibitor of IAC K+channels. Quinidine is also a relatively more potent antagonist of voltage-gated K+ channels, including delayed rectifier and A-type channels with reported IC50values ranging from 20 to 120 μM (Cook and Quast, 1990; Lancaster, 1991). Quinidine also inhibits an ATP-sensitive K+ channel with an IC50value of less than 50 μM (Bokvist et al., 1990).
Ba2+ blocked IAC with an IC50 value of 1.03 mM, a value that is typical for block of a variety of K+ channels, including delayed rectifier, inward rectifiers, and ATP-sensitive K+ channels, all of which are blocked with IC50 between 1 and 5 mM (see Lancaster, 1991, for review). Overall, IAC channels display a sensitivity to standard K+ channel blockers that is typical of a wide range of K+ channels, and particularly those channels whose α subunits include six membrane-spanning segments.
Sulfonylureas.
Although the gating of IAC K+ channels, like other ATP-sensitive K+ channels, appears to be controlled by the nonhydrolytic binding of ATP, several lines of evidence indicate that IAC channels are not members of the KATP family. First, IAC channels are activated rather than inhibited by ATP and other nucleotides (Enyeart et al., 1997). In addition, KATP channels of pancreatic β, cardiac, and skeletal muscle cells are inwardly rectifying, whereas IAC channels are not (Enyeart et al., 1996,1997). The very low sensitivity of IAC channels to inhibition by glyburide and tolbutamide indicates that these channels are pharmacologically distinct from KATPchannels.
Inwardly rectifying KATP channels are hetero-oligomers of α and β subunits (Inagaki et al., 1995;Aguilar-Bryan et al., 1995). The β subunits belong to a family of sulfonylurea receptors that confer sensitivity to drugs like glyburide and tolbutamide (Aguilar-Bryan et al., 1995). Separate high- and low-affinity sulfonylurea receptors (SUR1 and SUR2) have been cloned (Inagaki et al., 1996). High-affinity sulfonylurea receptors such as those associated with KATP channels of pancreatic β cells have Kd values for glyburide of 1 to 10 nM (Inagaki et al., 1995, 1996; Aguilar-Bryan et al., 1995). Lower-affinity sulfonylurea receptors associated with KATP channels of other cells bind glyburide with 50- to 100-fold lower affinity (Edwards and Weston, 1993; Inagaki et al., 1996). However, the Kd values for glyburide binding to these low-affinity receptors, and for inhibiting K+ channels in these cells, is still much lower than the IC50 value (63.8 μM) that we measured for glyburide inhibition of IAC.
The unduly high concentration of glyburide required to block IAC channels suggests that these channels do not include an SUR1 or SUR2 sulfonylurea receptor as a β subunit. In this regard, glyburide has been shown to inhibit voltage-gated K+ channels in the kidney and brain with a potency very similar (IC50 = 50–100 μM) to that we observed for inhibition of IAC (Crepel et al., 1993; Yao et al., 1996). Molecular cloning of the α subunit of one of these channels showed that it coded for a protein with six, rather than two, membrane-spanning domains (Yao et al., 1996). K+ channels of this type do not contain a sulfonylurea receptor as a β subunit. Presumably, these channels as well as IAC possess a distinct low-affinity glyburide-binding site. Thus, although gated by the nonhydrolytic binding of ATP, IAC channels do not appear to be a member of the family of inward rectifier KATPchannels.
Summary.
DPBP antipsychotics were identified as potent and relatively selective antagonists of IACK+ channels. Because penfluridol blocked IAC with an IC50 value more than 200-fold lower than that for IAK+ channels, it can be used as a completely selective IAC antagonist in AZF cells and in identifying related K+ channels in other tissues.
IAC K+ channels are unique in combining the pharmacological sensitivity of true K+-selective channels with that of CNG nonselective cation channels. They are the first K+ channels described that are blocked by DPBPs and l-cis-diltiazem. This hybrid pharmacology parallels other properties of IAC channels with respect to sensitivity to cyclic nucleotides and K+ selectivity. It remains to be seen whether these similarities are coincidental or indicative of structural homology between these channels.
Footnotes
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Send reprint requests to: Dr. John J. Enyeart, Department of Pharmacology, The Ohio State University, College of Medicine, 5188 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210-1239. E-mail:enyeart.1{at}osu.edu
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↵1 This work was supported by National Institute of Diabetes and Digestive and Kidney Grant DK47875 (to J.J.E.) and by American Heart Association, National Center, Grant-in-Aid 94011740 (to J.J.E.).
- Abbreviations:
- IAC
- noninactivating potassium current in bovine adrenal zona fasciculata cells
- IA
- rapidly inactivating voltage-dependent potassium current in bovine adrenal cells
- AZF
- adrenal zona fasciculata
- CNG
- cyclic nucleotide-gated
- DPBP
- diphenylbutylpiperidine
- 4-AP
- 4-aminopyridine
- TEA
- tetraethylammonium
- Received December 1, 1999.
- Accepted March 3, 1999.
- The American Society for Pharmacology and Experimental Therapeutics