Introduction

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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  subunits contain six membrane-spanning domains comprise the largest family. Molecular cloning of  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  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 K_ATP, 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 (I_AC) 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, I_AC channels 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, I_AC 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, I_AC 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  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  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 by l-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 I_AC channels, with respect to gating and permeation, would be reflected in their sensitivity to the different ion channel antagonists described above. Surprisingly, although I_AC K⁺ 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

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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.

	Results

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Inhibition of I_AC 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 (I_AC) 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, I_AC K⁺ current did not "rundown" in recordings lasting 1 h or longer. The absence of time-dependent inactivation of the I_AC 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 (I_A) as well as the noninactivating I_AC K⁺ current (Fig. 1, left traces). When voltage steps of 300-ms duration were applied, I_AC 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, I_AC 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).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Time- and concentration-dependent inhibition of K+ currents by 4-AP, TEA, and BaCl2. Whole-cell K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of 80 mV with or without 10-s prepulses to 20 mV that inactivated A-type K+ current. After IAC reached a stable amplitude, cells were superfused with 4-AP (A), TEA (B), or BaCl2 (C) at various concentrations. IAC amplitudes recorded with () or without () depolarizing prepulses are plotted against time (right). Numbers on current traces correspond to current amplitudes recorded at times indicated on the graph.

We determined the potency of five well known antagonists of voltage-gated K⁺ channels with respect to their ability to inhibit I_AC K⁺ current. These included the four organic antagonists: 4-AP, TEA, quinidine, bupivacaine, and the divalent cation Ba²⁺ (Figs. 1 and 2). 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 I_AC, 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 × 10⁵ M) > bupivacaine (1.13 × 10⁴ M) > BaCl₂ (1.02 × 10³ M) > 4-AP (2.75 × 10³ M) > TEA (2.43 × 10² M) (Fig. 2B).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Time- and concentration-dependent inhibition of IAC by quinidine and bupivacaine. A, whole-cell K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of 80 mV. After IAC reached a stable amplitude, cells were superfused with quinidine or bupivacaine at various concentrations as indicated. IAC amplitudes are plotted against time (right). Numbers on current traces correspond to current amplitudes recorded on the graph. B, inhibition curves for the five antagonists as indicated. Fraction of unblocked IAC current is plotted against antagonist concentration. Data were fit with an equation of the form I/Imax = 1/[1 + (B/IC50)]x, where B is the antagonist concentration, IC50 is the concentration that produces half-maximal inhibition, and x is the Hill slope. IC50 values have been estimated from the fits, and these are quinidine, 2.41 × 105 m; bupivacaine, 1.13 × 104 m; BaCl2, 1.02 × 103 m; 4-AP, 2.75 × 103 m; and TEA, 2.43 × 102 m. For each drug, data are normalized mean values of four to seven measurements at each concentration.

None of these agents selectively or even preferentially blocked I_AC. Each also inhibited the rapidly inactivating I_A current at concentrations sufficient to inhibit I_AC (Figs. 1 and 2). Of the five drugs, 4-AP preferentially blocked the rapidly inactivating I_A current (Fig. 1, left). In a previous study of I_A current in these cells, we found that 4-AP inhibits I_A with an IC₅₀ value of 630 µM (Mlinar and Enyeart, 1993), a concentration approximately 4-fold lower than the IC₅₀ value for I_AC inhibition by this agent.

Inhibition of I_AC by Sulfonylureas

Inwardly rectifying K_ATP channels are potently blocked by sulfonylureas including glyburide and tolbutamide (Inagaki et al., 1995, 1996). We found that ATP-activated I_AC 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 I_AC only at much higher concentrations (IC₅₀ = 63.8 µM). Even though glyburide was a weak antagonist of I_AC, it was relatively selective. Figure 3A shows that at the highest concentration used (100 µM), glyburide produced little inhibition of I_A current, whereas I_AC 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 I_AC with an IC₅₀ value of 784 µM (Fig. 3B).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Inhibition of IAC by sulfonylureas. Whole-cell K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of 80 mV with or without 10-s prepulses to 20 mV. After IAC reached a stable amplitude, cells were superfused with glyburide or tolbutamide at various concentrations as indicated. A and B, effect of glyburide. A, traces with (bottom) or without (top) depolarizing prepulses. Numbers on current traces correspond to current amplitudes measured at indicated times on the graph in B. B, IAC amplitudes recorded with () or without () depolarizing prepulses are plotted against time. C, inhibition curves for glyburide and tolbutamide. Inhibition curves for glyburide and tolbutamide were constructed from experiments as in A and B. Fraction of unblocked IAC current is plotted against antagonist concentration. Data are fit with an equation of the form: I/Imax = 1/[1 + (B/IC50)]x, where B is the antagonist concentration, IC50 is the concentration that produces half-maximal inhibition, and x is the Hill slope. IC50 values have been estimated from the fits; these are 63.8 and 784 µM for glyburide and tolbutamide, respectively. Data are normalized mean values of four or five measurements at each concentration for tolbutamide and 13 to 32 separate measurements for glyburide.

Inhibition of I_AC by CNG Channel Antagonists

DPBPs. I_AC 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 I_AC K⁺ channels by pimozide and two other DPBPs, fluspirilene and penfluridol, in whole-cell and single-channel patch-clamp recordings.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Inhibition of IAC K+ current by fluspirilene. Whole-cell K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of 80 mV with or without a 10-s prepulse to 20 mV. After IAC reached a stable amplitude, cells were superfused with fluspirilene at various concentrations, as indicated. A, current traces obtain with (bottom) or without (top) depolarizing prepulses. Numbers on current traces correspond to current amplitudes measured at indicated times on the graph in B (right). B, IAC amplitudes recorded with () or without () depolarizing pulses are plotted against time. C, inhibition curve for fluspirilene constructed from experiments as in A and B. Fraction of unblocked IAC current is plotted against fluspirilene. Data are fit with an equation of the form: I/Imax = 1/[1 + (B/IC50)]x, where B is the antagonist concentration, IC50 is the concentration that produces half-maximal inhibition, and x is the Hill slope. IC50 has been estimated from the fit as 2.32 × 107 m. Data are normalized mean values of six measurements at each concentration.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Preferential block of IAC over IA by DPBPs. A, whole-cell K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of 80 mV with or without 10-s prepulses to 20 mV. After IAC reached a stable amplitude, cell was superfused with penfluridol at the indicated concentrations. IAC amplitudes recorded with () or without () depolarizing prepulses are plotted against time (left). Numbers on current traces (right) correspond to current amplitudes measured at indicated times on corresponding graph. B, inhibition of IA K+ currents were recorded in isolation after completely eliminating IAC K+ current with 200 pM adrenocorticotropin. While recording currents at 30-s intervals, cell was superfused with penfluridol at various concentrations. Traces show IA current after steady-state block at indicated concentrations of penfluridol. C, inhibition curves for penfluridol on IA and IAC and for pimozide inhibition of IAC as indicated. Fraction of unblocked K+ current is plotted against penfluridol or pimozide as indicated. Inhibition curves were constructed from experiments as in A and B. Data are fit with an equation of the form: I/Imax = 1/[1 + (B/IC50)]x, where B is the antagonist concentration, IC50 is the concentration that produces half-maximal inhibition, and x is the Hill slope. IC50 values estimated from the fits are 1.87 × 107 m and 4.18 × 105 m for penfluridol inhibition of IAC and IA, respectively, and 3.54 × 107 m for pimozide inhibition of IAC. Data are normalized mean values for seven to nine separate measurements at each concentration.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Voltage-independent block of IAC by penfluridol. Voltage ramps of 100 mV/s were applied from a holding potential of 0 mV to potentials between +60 and 140 mV, before and 10 min after superfusing the cell with 2.5 µM penfluridol, as indicated. Similar results were obtained in a total of four experiments.

Effect of Penfluridol on Unitary I_AC Currents. Penfluridol inhibited single-channel I_AC activity in excised outside-out patches without reducing the amplitude of the unitary current. Figure 7 shows unitary I_AC 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 I_A 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 I_AC channels were present in this patch (Fig. 7A).

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Effect of penfluridol on unitary IAC currents. Unitary IAC currents were recorded in the outside-out configuration, using pipettes containing standard pipette solution supplemented with 2 mM MgATP. Voltage steps to +30 mV were applied at 10-s intervals from a holding potential of 40 mV. Each amplitude histogram was constructed from unitary currents recorded in response to 48 to 64 separate voltage steps of 300-ms duration. Unitary current amplitudes were distributed into bins of 0.2 pA width. A-C, unitary currents and corresponding amplitude histograms recorded in control solution, after 5 min in the presence of 200 nM penfluridol and 7 min after return to control (wash) as indicated. Currents were sampled at 5 kHz and filtered at a cutoff frequency of 2 kHz. Mean peak amplitudes were (A) 3.96 ± 0.67, 7.87 ± 0.55, and 11.57 ± 0.72 pA; (B) 3.77 ± 0.32 pA; and (C) 4.17 ± 0.71, 7.95 ± 1.11, 12.13 ± 0.74, and 15.68 ± 0.79 pA. Similar results were obtained in each of three outside-out patches when penfluridol was applied at 200 nM.

Block of I_AC 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 I_AC K⁺ current at slightly higher concentrations (IC₅₀ = 24.9 µM) (Fig. 8). Compared with the DPBPs, this drug was less selective for I_AC. I_A current was significantly reduced by diltiazem at concentrations that maximally inhibited I_AC (Fig. 8A). In contrast to inhibition by penfluridol, inhibition of I_AC by l-cis-diltiazem was rapidly reversible (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Effect of l-cis-diltiazem on IAC currents. Whole-cell K+ currents were recorded at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of 80 mV with or without a 10-s prepulse to 20 mV. After IAC reached a stable amplitude, cells were superfused with l-cis-diltiazem at various concentrations as indicated. A, current traces recorded with (bottom) or without (top) depolarizing prepulses in control saline and after steady-state block with indicated concentration of l-cis-diltiazem. B, inhibition curve was constructed from four separate experiments as in A. Fraction of unblocked current is plotted against l-cis-diltiazem concentration. Data were fit with an equation of the form: I/Imax = 1/[1 + (B/IC50)]x, where B is the l-cis-diltiazem concentration, IC50 is the concentration that produces half-maximal inhibition, and x is the Hill slope.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We discovered that I_AC K⁺ channels possess a unique pharmacological profile combining sensitivity to standard K⁺ channel blockers and antagonists of CNG cation channels. Standard K⁺ channel blockers inhibited I_AC 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 I_AC channels. Block of I_AC K⁺ channels by the DPBPs was selective because 200-fold higher concentrations were required to inhibit the rapidly inactivating I_A K⁺ current.

Block of I_AC by DPBPs and l-cis-Diltiazem. These results demonstrate that the distinctive hybrid properties of I_AC K⁺ 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 I_AC half-maximally.

Mechanism of I_AC Block by DPBPs. The molecular mechanism by which DPBPs inhibit I_AC 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, I_AC 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 I_AC current 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).

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 I_AC K⁺ channels at concentrations typical of those that effectively block other K⁺-selective channels. TEA inhibited I_AC 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).

Sulfonylureas. Although the gating of I_AC K⁺ channels, like other ATP-sensitive K⁺ channels, appears to be controlled by the nonhydrolytic binding of ATP, several lines of evidence indicate that I_AC channels are not members of the K_ATP family. First, I_AC channels are activated rather than inhibited by ATP and other nucleotides (Enyeart et al., 1997). In addition, K_ATP channels of pancreatic , cardiac, and skeletal muscle cells are inwardly rectifying, whereas I_AC channels are not (Enyeart et al., 1996, 1997). The very low sensitivity of I_AC channels to inhibition by glyburide and tolbutamide indicates that these channels are pharmacologically distinct from K_ATP channels.

Summary. DPBP antipsychotics were identified as potent and relatively selective antagonists of I_AC K⁺ channels. Because penfluridol blocked I_AC with an IC₅₀ value more than 200-fold lower than that for I_A K⁺ channels, it can be used as a completely selective I_AC antagonist in AZF cells and in identifying related K⁺ channels in other tissues.