A human intermediate conductance calcium-activated potassium channel

Abstract

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is ≈50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K_0.5 = 0.3 μM). Although the K_0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3.5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (K_i = 2.5 nM) and clotrimazole (K_i = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 μM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Three distinct classes of calcium-activated K⁺ channels (K_Ca channels) have been described. Large conductance calcium-activated K⁺ (BK) channels are gated by the concerted actions of internal calcium ions and membrane potential and have a unit conductance of between 100 and 220 pS. Small (SK) and intermediate (IK) conductance calcium-activated K⁺ channels are gated solely by internal calcium ions, with a unit conductance of 2–20 and 20–85 pS, respectively, and are more sensitive to calcium than are BK channels (for review, see ref. 1). In addition, each type of K_Ca channel shows a distinct pharmacology, and the activity of each hyperpolarizes the membrane potential. Members of the BK (2–4) and SK (5) subfamilies have been cloned and expressed in heterologous cell types, where they recapitulate the fundamental properties of their native counterparts.

The first demonstration that internal calcium ions regulate potassium flux was provided by Gardos from red blood cells (6). A rise in intracellular Ca²⁺ in red blood cells opens the Gardos channel, causing potassium loss and cell dehydration, a condition that is exacerbated in sickle cell anemia. Promising therapeutic approaches for sickle cell anemia involve specifically blocking the Gardos channel (7, 8). Like IK channels, the Gardos channel is opened by submicromolar concentrations of internal calcium and has a rectifying unit conductance, ranging from 50 pS at −120 mV to 13 pS at 120 mV (symmetrical 130 mM K⁺; ref. 9). It is blocked by charybdotoxin (CTX) but not the structurally related peptide iberiotoxin (IBX), both of which block BK channels (10). Apamin, a potent blocker of certain native (11, 12) and cloned (5) SK channels, does not block IK channels (13). The Gardos channel is also blocked by some imidazole compounds, such as clotrimazole, but not ketoconazole (ref. 7; S. Alper, personal communication). The electrophysiological and pharmacological properties of the Gardos channel show that it belongs to the IK subfamily.

IK channels have been described in a variety of other cell types. IK channels have also been implicated in the microvasculature of the kidney, where they may be responsible for the vasodilatory effects of bradykinin (14). In brain capillary endothelial cells, IK channels, activated by endothelin that is produced by neurons and glia, shunt excess K⁺ into the blood (15). Neutrophil granulocytes, mobile phagocytic cells that defend against microbial invaders, undergo a large depolarization subsequent to agonist stimulation, and IK channels have been implicated in repolarizing the stimulated granulocyte (16).

IK channels are distinguished from other K_Ca channel types by their biophysical and pharmacological profiles. Here, we describe the isolation and heterologous expression of the first member of this class of calcium-activated potassium channels. hIK1 is structurally related to the SK subfamily, and expression in Xenopus oocytes results in intermediate conductance calcium-activated K⁺ channels with a pharmacology that is consistent with the Gardos channel from red blood cells and IK channels from other tissues.

MATERIALS AND METHODS

Molecular Biology.

A blast search of the Expressed Sequence Tag database using the nucleotides encoding the C-terminal 180 aa of rSK2 (5) retrieved AA076337. Oligonucleotides corresponding to nucleotides 1–36 were synthesized (Genosys, The Woodlands, TX), radiolabeled using polynucleotide kinase (BRL) and [³²P]ATP (NEN), and used to screen ≈10⁶ recombinant phage from the human pancreatic cDNA library [Stratagene; 40% formamide; 1 M NaCl, 1% SDS, 37°C; washed at 1× SSC (150 mM NaCl, 15 mM sodium citrate, pH 7.2), 50°C]. Double positively hybridizing phage were purified by rescreening at reduced densities. cDNA inserts were subcloned into M13, and the nucleotide sequences were determined using T7 DNA polymerase (Sequenase, Upstate Biotechnology). Computer analyses were performed using the gcg software suite (Genetics Computer Group; version 8.1). Northern blots, prepared with 2 μg of poly(A)⁺ mRNA isolated from the indicated human tissues, were purchased from CLONTECH and probed in Expresshyb solution (CLONTECH) at 68°C with a radiolabeled DNA fragment derived from the coding region of hIK1 (corresponding to amino acid residues 319–428 and including ≈100 bp of 3′ untranslated sequence) washed with 0.1× SSC, 0.1% SDS at 65°C, and exposed to x-ray film at −80°C with an intensifying screen for 48 hr. The probe sequence used for Northern blot analysis does not cross-hybridize with the related SK channel clones. 5′ rapid amplification of cDNA ends (RACE) reactions were performed as previously described (17).

Electrophysiology.

All channel subunits were subcloned into the oocyte expression vector pBF (graciously provided by B. Fakler), which provides 5′ and 3′ untranslated regions from the Xenopus β-globin gene flanking a polylinker that contains multiple restriction sites. In vitro mRNAs were generated using SP6 polymerase (GIBCO/BRL). After synthesis, mRNAs were evaluated spectrophotometrically and by ethidium bromide staining after agarose gel electrophoresis. Xenopus care and handling were in accordance with the highest standards of institutional guidelines. Frogs underwent no more than two surgeries, separated by at least 3 weeks, and surgeries were performed using well established techniques. Frogs were anesthetized with an aerated solution of 3-aminobenzoic acid ethyl ester. Oocytes were studied 2–14 days after injection with 0.5–5 ng of mRNA. Inside-out macropatches were excised into an intracellular solution containing 116 mM K-gluconate, 4 mM KCl, and 10 mM Hepes (pH 7.2, adjusted with KOH) supplemented with CaCl₂ to give free calcium concentration of 5 μM; the proportion of calcium binding to gluconate was determined by a computer program (cabuf) assuming a stability constant for Ca²⁺ gluconate of 15.9 M⁻¹ (18). To obtain Ca²⁺ concentrations below 1 μM, 1 mM EGTA was added to the bath solution and CaCl₂ was added as calculated using the cabuf program and published stability constants (19). Electrodes were pulled from thin-walled, filamented borosilicate glass (World Precision Instruments, Sarasota, FL) and filled with 116 mM K-gluconate, 4 mM KCl, and 10 mM Hepes (pH 7.2). Electrode resistance was typically 2–5 MΩ. For outside-out macropatches, the solutions were reversed. Membrane patches were voltage-clamped using an Axopatch 1B amplifier (Axon Instruments, Foster City, CA). The data were low-pass filtered (8-pole Bessel filter; Frequency Devices, Haverhill, MA) at 1 kHz and acquired using pulse software (HEKA Electronics, Lambrecht, Pfalz, Germany). Analysis was performed using pulse, kaleidagraph (Abelbeck Software, Reading, PA), or igor (Wavemetrics, Lake Oswego, OR) software. All experiments were performed at room temperature from a holding potential of 0 mV. Voltage ramps (2.5 s) from −100 to either 60 or 100 mV were acquired at a sampling frequency of 500 Hz. Values are expressed as mean ± SD. Statistical differences were determined using an unpaired t test; P values <0.05 were considered significant. Clotrimazole was from Sigma, ketoconazole and IBX were from Research Biochemicals, apamin was from Calbiochem, and CTX was the generous gift of Chris Miller.

For single channel recordings, oocytes were bathed in 116 mM k-gluconate, 4 mM KCl, 10 mM Hepes, 5 mM EGTA, pH 7.2, adjusted with CaCl₂ to yield the reported concentration of free Ca²⁺. All recordings were performed in the inside-out patch configuration using thick-walled quartz electrodes (13–15 MΩ) containing 116 mM k-gluconate, 4 mM KCl, and 10 mM Hepes, pH 7.2. Membrane patches were voltage-clamped with an Axopatch 200 amplifier (Axon Instruments). Continuous recordings were low-pass filtered (8-pole Bessel filter, Frequency Devices) at 1 kHz, acquired at 10 kHz using pulse software (Heka Electronics) and stored directly on a Macintosh Quadra 650. Single channel recordings were analyzed with mactac (Skalar Instruments, Seattle, WA) using the “50% threshold” technique to estimate event amplitudes, and each transition was visually inspected before being accepted. Event histograms were constructed using mactacfit (Skalar Instruments). Only events lasting at least 1 msec were included, and amplitude histograms were fitted by single Gaussian distributions. All experiments were performed at room temperature.

RESULTS

Isolation and Characterization of hIK1 cDNA.

The C-terminal domain of rSK2, an SK channel (5), was used in a blast search of the National Center for Biotechnology Information database. One of the retrieved sequences (AA076337), cloned from human pancreas, showed significant homology with rSK2 but varied in regions conserved among the SK subfamily. Based on this sequence, an oligonucleotide was synthesized and used to screen a human pancreas cDNA library. The most 5′ methionine codon in the longest positively hybridizing clone initiated an ORF with homology to the SK subfamily (Fig. 1A). The sequence predicts a protein of 428 aa with 6 transmembrane domains and is 42–44% identical and 50–55% conserved compared with SK channels (Fig. 1A; ref 5). The pore region contains the canonical GYG K⁺ selectivity sequence (20) and is more related to the SK pore regions than to those from other K⁺ channel subfamilies. However, the determinants for apamin and tubocurarine block of SK channels are not present (27), and the N-terminal domain of the pore region shows several differences compared with SK channels.

Figure 1.

(Upper) Amino acid sequence of hIK1 and comparison with SK channel subunits. Alignments were generated by eye; dots represent gaps introduced to optimize the alignment. The six predicted transmembrane domains and the pore region are overlined. Residues that are conserved between hIK1 and any of the SK sequences are boxed. Amino acid numbers for the full-length coding sequences are given on the right. The hIK1 sequence has been deposited in GenBank (accession number AF022150). The asterisks indicate stop codons. (Lower) Northern blot analyses of hIK1 mRNA distribution. Poly(A)⁺ mRNA (2 μg), isolated from the indicated tissue sources, was loaded in each lane. Sizes are indicated to the left. hIK1 mRNA was detected in many peripheral tissues, particularly smooth muscle tissues, but not in brain.

The predicted N-terminal domain of hIK1 was notably shorter than that of any of the SK channel clones (ref. 5; unpublished work), having only 24 aa prior to the predicted first transmembrane domain. To determine whether additional coding sequences were missing, Northern blot and 5′ RACE (21) analyses were performed. Northern blots showed that the hIK1 mRNA is expressed in many peripheral tissues, particularly smooth muscle tissues. All tissues containing hIK1 mRNA showed a band of ≈2.1 kb (Fig. 1B), very close to the length of the hIK1 cDNA clone (1.95 kb), although several mRNA species, presumably partially processed transcripts, are detected in some tissues. In addition, RACE reactions were performed using cDNA from human pancreas or placenta as substrate. Analysis of >20 clones from each tissue source, using several independent RACE reactions, yielded no additional 5′ nucleotides (not shown). These results suggested that the entire hIK1 coding sequence was present on the pancreatic cDNA clone.

Expression of hIK1 in Xenopus Oocytes.

hIK1 was expressed in Xenopus oocytes. Voltage ramp commands delivered to inside-out patches excised into 5 μM Ca²⁺ evoked robust, inwardly rectifying macroscopic current responses, which were not present in patches from uninjected oocytes (not shown) or inside-out patches bathed in Ca²⁺-free media (Fig. 2A). Voltage step commands evoked large, time-independent currents only when Ca²⁺ was included in the (bath) internal solution (Fig. 2 B and C). The selectivity of hIK1 was determined in oocytes using the two-electrode voltage-clamp technique, in which IK1 channels were activated by bathing the oocyte in 0.25 mM Ca²⁺ ND96 containing 1 μM ionomycin. Using 2, 6, 20, and 60 mM K⁺, which was substituted for Na⁺ in the ionomycin-containing ND96, in response to voltage ramps (0.28 V/s) from a holding potential of −90 mV, the reversal potential shifted in accord with the Nernst potential for a K⁺-selective channel (57.6 ± 1.4 mV/10-fold change in K⁺, n = 4; not shown). Similar to rSK2, hIK1 currents recorded from voltage ramp commands were dependent upon the concentration of Ca²⁺ applied to the internal face of the membrane (Fig. 2D). Currents normalized by the maximum response evoked by 10 μM and 3 μM Ca²⁺ for hIK1 and rSK2, respectively, were plotted as a function of Ca²⁺ concentration, and the data points were fitted with the Hill equation. Both channels showed a similar K_0.5 [concentration for half-maximal activation, 0.32 ± 0.03 μM (n = 7) for hIK1 and 0.31 ± 0.05 μM (n = 4) for rSK2; P = 0.68] but differed in the steepness of the Ca²⁺-dependence; rSK2 had a Hill coefficient of 3.5 ± 0.4 (n = 4), whereas hIK1 had a Hill coefficient of 1.7 ± 0.3 (n = 7, P < 0.001; Fig. 2E). These results demonstrate that hIK1 is a calcium-activated potassium channel.

Figure 2.

(A) Current traces elicited by 2.5-s voltage ramps from −100 to 100 mV from inside-out macropatches excised from oocytes expressing hIK1. The traces were obtained in the presence (+Ca²⁺) or absence (−Ca²⁺) of 5 μM internal Ca²⁺. (B) Currents evoked by voltage steps from inside-out macropatches excised from an oocyte expressing hIK1, in the presence (Upper) or absence (Lower) of 5 μM internal Ca²⁺; voltage protocol is shown below the current traces. The membrane was stepped from a holding potential of 0 mV to test potentials between −100 and 100 mV. Currents activated instantaneously within the resolution of the recording configuration and showed no inactivation during the 900-ms test pulses. (C) Current–voltage relationship for the traces shown in B. Squares are in the absence of Ca²⁺; circles are in the presence of 5 μM Ca²⁺. (D) Current traces elicited by 2.5-s voltage ramps from −100 to 60 mV from inside-out macropatches excised from oocytes expressing hIK1 or rSK2. The traces were obtained in the presence of the indicated concentrations of intracellular Ca²⁺; current amplitudes increased as the Ca²⁺ concentration was raised. (E) Calcium concentration response for hIK1 and rSK2. For hIK1 (solid squares; n = 7) and rSK2 (solid circles; n = 4), the current measured at −100 mV normalized by the response in saturating Ca²⁺ (10 μM for hIK1 and 3 μM for rSK2) was plotted as a function of the calcium concentration. The data were fit with the Hill equation, yielding a K_0.5 of 0.3 μM for both channels and a Hill coefficient of 1.7 for hIK1 and 3.5 for rSK2. (Error bars are ±SD.)

The single channel conductance of hIK1 was determined. Stationary recordings from inside-out patches excised into a bathing solution containing 0.2–1.0 μM free calcium showed short-duration openings not seen in the absence of calcium. Fig. 3A shows representative traces recorded at −60 mV. The degree of channel activity depended on the concentration of internal calcium. Reducing intracellular calcium reduced channel activity, and removing internal calcium abolished channel activity, which returned after reapplication of Ca²⁺. For the patch in Fig. 3B, the amplitudes of openings recorded at different voltages were measured, assembled into histograms, and fit by Gaussian distributions. The resulting mean amplitudes were used to construct the current–voltage relationship shown in Fig. 3C. Like the macroscopic current–voltage relationship, the single channel current–voltage relationship showed inward rectification. For this patch, linear regression analysis of the current–voltage relationship between −60 and −100 mV yielded a single channel conductance of 42 pS; results from four patches gave a unit conductance of 39 ± 4 pS. Measurements of the outward conductance were more variable, ranging from 5 to 12 pS.

Figure 3.

(A) Continuous recording at different internal calcium concentrations from a representative inside-out patch containing several hIK1 channels. Decreasing the calcium concentration from 0.8 to 0.6 μM decreased channel activity. Channel activity ceased when calcium was removed and returned following reapplication of 0.6 μM calcium. Gaps represent breaks in the continuously acquired recording. (B) Channel activity from a representative patch recorded in the presence of 0.4 μM calcium at −80, −60, −40, and 60 mV. The patch contained more than one channel, and double openings are apparent. (C) Single channel current–voltage relationship for the patch presented in B. Data points were derived from fitting amplitude histograms at each membrane potential; a representative histogram (−40 mV) and fit are shown as an Inset. Linear regression fitting of the current–voltage relationship between −60 and −100 mV yielded a single channel conductance of 42 pS.

Pharmacology of hIK1.

The functional characteristics of hIK1 are reminiscent of intermediate conductance calcium-activated K⁺ channels described from red blood cells (the Gardos channel; ref. 6) and other tissues. Native IK channels present a distinguishing pharmacology; they are blocked by CTX but, different from large conductance voltage- and BK channels, are not blocked by IBX. Also, IK channels are not sensitive to the bee venom peptide toxin apamin, a blocker of certain native and cloned SK channels. In addition, some IK channels, notably the Gardos channel, are sensitive to several imidazole derivatives, such as clotrimazole, but are not sensitive to others such as ketoconazole (refs. 7, 8, 13, and 22; S. Alper, personal communication). hIK1 currents were potently blocked by CTX, with a K_i of 2.5 nM (n = 4) (Fig. 4A), whereas 50 nM IBX blocked only 15 ± 3% (n = 4; Fig. 4C). hIK1 was sensitive to clotrimazole with a K_i of 24.8 nM (n = 4; Fig. 4B) but was only 24 ± 6% (n = 4) blocked by 10 μM ketoconazole (Fig. 4C). Apamin (100 nM) reduced hIK1 currents by only 12 ± 5% (n = 5; Fig. 4C).

Figure 4.

(A and B) Dose response for block by external CTX (A) or clotrimazole (B). Block was determined from outside-out patches exposed to increasing concentrations of blocker. Each data point represents the fractional current (drug/control) at −100 mV. Currents were elicited by voltage ramps; representative traces with the concentrations of blockers (nM) are shown as Insets. Nonlinear least squares fit to a Langmuir isotherm (continuous line) yielded a K_i of 2.5 nM for CTX and 24.8 nM for clotrimazole. An offset was included to account for residual, unblocked current (0.17 for CTX and 0.13 for clotrimazole). (Error bars are ±SD.) Insets show onset and reversal of block. (C) Bar graph showing normalized current in the presence of IBX (50 nM; n = 4), ketoconazole (10 μM; n = 4), and apamin (100 nM; n = 5). Normalized current was determined by comparing the current amplitude at −100 mV in the presence or absence of drug. Currents were elicited by voltage ramp commands, and representative traces are shown above each column. Reduced current traces are in the presence of drug. (Error bars are ±SD.)

DISCUSSION

The first description of a calcium-activated potassium flux was provided by Gardos (6), and further studies identified the erythrocyte Gardos channel as an IK channel (9). IK and SK channels require intracellular calcium for activation and may be distinguished by their unit conductance values and pharmacological profiles. The cloned potassium channel hIK1 presents the defining characteristics of the Gardos channel, an IK channel. In addition, Northern blot analysis shows that the mRNA for hIK1 is expressed in tissues from which IK channels have been recorded.

Structurally, hIK1 is closest to the SK channel subfamily, showing ≈50% similarity (identical and conservative amino acid substitutions). As for SK channels, there are no E–F hand motifs (23, 24) in the hIK1 sequence suggesting where calcium may interact with the channel. The sensitivity of hIK1 for calcium is the same as that of SK channels; however, the slope of the concentration–response curve is more shallow. This difference has significant physiological implications. At low concentrations of calcium, in the range of resting internal calcium (≈0.1 μM), SK channels would be silent, whereas IK channels would show some activity. Indeed, SK channels expressed in Xenopus oocytes are not active in the intact cell without elevating internal calcium, whereas IK channels show some basal activity (unpublished work).

Macroscopic and unitary hIK1 currents show inward rectification, which is similar to the Gardos channel (9). The reduction of single channel current amplitudes suggests that the rectification may be due to a fast, unresolved block. Unit current reduction was seen in the absence of internal Mg²⁺ or polyamines, and it has been suggested that the rectification of the Gardos channel may be due to a voltage-dependent block by potassium ions at a site close to the external mouth of the pore (9).

hIK1, but not SK, channels are blocked by CTX with an affinity similar to that of Shaker or Kv1.3 potassium channels. Structure-activity studies of these channels have identified several residues that mediate toxin binding (25, 26). One of these residues, D240 in hIK1, is conserved, suggesting that this residue is responsible for blocking by CTX. IK channels are distinguished from SK channels by their lack of sensitivity to the bee venom peptide toxin apamin or the plant alkaloid d-tubocurarine, and the amino acids mediating block by apamin and d-tubocurarine in SK channels are not present in the hIK1 pore region (27).

IK channels from different tissues have been reported to possess a wide range of unit conductance values. This may reflect measurements of the same channel type, and the variability may be due to the use of different concentrations of K⁺ and the particular region of the rectifying current–voltage relationship chosen for conductance determinations. Alternatively, hIK1 may represent the first described member of the IK subfamily, in which several members possess different unit conductances. In either case, hIK1 shows a biophysical and pharmacological profile consistent with the erythrocyte Gardos channel, in particular, the unit conductance, single channel rectification, and block by CTX and clotrimazole but not by IBX or ketoconazole. Selective block of the Gardos channel provides a promising therapy for sickle cell anemia (8), and the availability of hIK1 may help identify novel compounds for the treatment of this disorder.