Molecular Cloning and Characterization of the Intermediate-Conductance Ca2+-Activated K+ Channel in Vascular Smooth Muscle
Relationship Between KCa Channel Diversity and Smooth Muscle Cell Function
Abstract—Recent evidence suggests that functional diversity of vascular smooth muscle is produced in part by a differential expression of ion channels. The aim of the present study was to examine the role of Ca2+-activated K+ channels (KCa channels) in the expression of smooth muscle cell functional phenotype. We found that smooth muscle cells exhibiting a contractile function express predominantly large-conductance (≈200 pS) KCa (BK) channels. In contrast, proliferative smooth muscle cells express predominantly KCa channels exhibiting a much smaller conductance (≈32 pS). These channels are blocked by low concentrations of charybdotoxin (10 nmol/L) but, unlike BK channels, are insensitive to iberiotoxin (100 nmol/L). To determine the molecular identity of this K+ channel, we cloned a 1.9-kb cDNA from an immature-phenotype smooth muscle cell cDNA library. The cDNA contains an open reading frame for a 425 amino acid protein exhibiting sequence homology to other KCa channels, in particular with mIK1 and hIK1. Expression in oocytes gives rise to a K+-selective channel exhibiting intermediate-conductance (37 pS at −60 mV) and potent activation by Ca2+ (Kd 120 nmol/L). Thus, we have cloned and characterized the vascular smooth muscle intermediate-conductance KCa channel (SMIK), which is markedly upregulated in proliferating smooth muscle cells. The differential expression of these KCa channels in functionally distinct smooth muscle cell types suggests that KCa channels play a role in defining the physiological properties of vascular smooth muscle. The full text of this article is available at http://www.circresaha.org.
Functional plasticity is a fundamental property of vascular smooth muscle, important for regulating blood pressure and controlling vascular growth and repair during development, in response to disease states, and after mechanical injury.1 2 3 Plasticity is dependent on extensive diversity in the properties, distribution, and function of individual smooth muscle cell populations, arising from both phenotypic modulation between contractile and de-differentiated states and the establishment of smooth muscle cell lineages early in embryonic life, which have the potential to become major cell populations in pathological conditions.1 2 3 4
Recent studies suggest that differential expression of ion channels contributes to smooth muscle plasticity.5 6 7 8 Morphologically distinct smooth muscle cell types exhibit marked differences in ion channel expression, giving rise to differences in electrical properties and responsiveness to vasoactive substances.5 Much of the variation in contractile reactivity between different arterial segments, vessels, and physiological and pathophysiological states can be explained, at least in part, by differences in the expression and/or activity of ion channels on functionally distinct smooth muscle cell types.5 7 9 Because calcium ions (Ca2+) play a pivotal role in the control of many smooth muscle cell functions, ion channels that regulate Ca2+ are clearly of functional importance. A class of ion channels known to modulate intracellular Ca2+ are the Ca2+-activated K+ (KCa) channels.10 Activation of these channels produces membrane hyperpolarization which reduces vascular contractility by limiting Ca2+ influx through voltage-gated Ca2+ channels.11 12 Recently, we reported a marked contrast in the magnitude of a KCa current between smooth muscle cell types that differ in their morphology and proliferative capacity.5 6 This finding indicated that the functional properties of smooth muscle may be dependent in part on the expression and activity of KCa channels.
To investigate the relationship between KCa channels and smooth muscle cell function, we examined the expression of KCa channels on functionally distinct smooth muscle cell types. Three types of KCa channels, large (BK),11 12 small (SK),13 and intermediate (IK)14 -conductance are known to exist in vascular smooth muscle. IK channels, blocked by charybdotoxin but not iberiotoxin or apamin,15 have recently been cloned from human16 17 18 19 and mouse20 tissues; however, their molecular identity in vascular smooth muscle is not known. We describe the first cloning and functional characterization of a smooth muscle IK channel (which we refer to as SMIK). We also show that SMIK and BK are associated with different smooth muscle functions, indicating that these 2 KCa channels help to define the physiological properties of the vessel wall.
Materials and Methods
Smooth Muscle Cell Types
All smooth muscle cell types examined in this study were derived from enzymatic digestion of aortic smooth muscle media of adult (12-week-old) WKY rats.5 The smooth muscle cell types examined are (1) the fully differentiated contractile phenotype, (2) the de-differentiated phenotype, and (3) the immature smooth muscle cell type. Contractile smooth muscle cells4 are isolated from the smooth muscle medial layer and used within 24 hours. De-differentiated smooth muscle cells have undergone phenotypic modulation in culture (2 to 4 weeks).4 Immature smooth muscle cells are epithelioid-shaped cells derived from multipassaged cultures.5
Determination of Membrane Potential
Changes in membrane potential were determined using the potential-sensitive fluorescent dye, bis-oxonol, as described previously.5 Cells on glass coverslips were incubated with bis-oxonol in a cuvette mounted within the thermostatically controlled (37°C) chamber of a SPEX 1681 fluorolog spectrometer (540-nm and 340-nm excitation, 580-nm emission wavelengths; 1 s integration time, and readings collected at 3-s intervals). Fluorescence responses to ET-1 were calibrated against standard KCl-induced depolarizations in the presence of gramicidin (2 μg/mL).5
Single-channel and macropatch experiments were performed at room temperature (22°C to 25°C) using the gigohm seal patch-clamp method in the excised inside-out configuration, as described previously.21 Patch pipettes were fabricated from Corning 7056 glass capillary tubing (OD 1.5 mm) (Warner) on a programmable micropipette puller (Model P-87, Sutter Instruments), and their tips were fire-polished to resistances of 2 to 4 MΩ using a Narishige MF-83 microforge. For single-channel recordings, electrode shanks were coated with Sylgard (Dow Corning) to reduce capacitive current noise. Single-channel and macropatch currents were amplified using an Axopatch 200 amplifier (Axon Instruments), low-pass filtered at 2 kHz using an 8-pole Bessel filter (Frequency Devices), digitized by an Axon AD/DA converter (Digidata 1200 DMA interface), sampled at >10 kHz, and stored on hard disk. No series resistance compensation was used, and leak currents were not subtracted from macropatch currents. Capacitance compensation was performed using the built-in amplifier circuitry. Pipette potentials were nulled immediately before seal formation. Grounding was achieved via an agar bridge to avoid junction potential shifts caused by solution changes.
High K+ solution for electrophysiological recordings of smooth muscle cell currents had the following composition (in mmol/L): KCl 130, NaHEPES 10, MgCl2 1, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) 0.3, and CaCl2 (free Ca2+ concentration, 0.1 μmol/L). Recording solutions for oocyte expression studies contained gluconate as a nonpermeant anion to prevent activation of Ca2+-activated Cl− channels. The composition of the high K+ solution was (in mmol/L) K+-gluconate 145, EGTA 1, KCl 2, KOH 5, and MOPS 10, pH 7.4. Free Ca2+ ion concentrations (0 to 1 μmol/L) at pH 7.4 were adjusted as described elsewhere.21 In experiments using different K+ concentration gradients, K+ was replaced by Na+ in the appropriate proportions in the pipette solutions. Osmolarity of pipette and bath solutions were measured using an osmometer and adjusted to 270 and 280 mOs, respectively. All solutions were equilibrated to room temperature (22°C to 25°C) before use.
Detection of KCa Channel mRNAs by Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from vessels and smooth muscle cells by the guanidinium thiocyanate method, the concentration determined by spectroscopic analysis at 260 nm, and purity checked on ethidium bromide-stained formaldehyde/agarose gels. RT-PCR assays were performed using the GeneAmp RNA PCR Kit (Perkin Elmer). Reverse transcription was started using 0.1 μg RNA in a total volume of 5.25 μL containing (in mmol/L) Tris-HCl 10, KCl 50, MgCl2 5, pH 8.3, and 1 mmol/L each of dATP, dTTP, dGTP, and dCTP, 5 U of ribonuclease inhibitor, 2.5 μmol/L random hexamers, 12.5 U of MuLV reverse transcriptase in a Perkin Elmer thermal cycler using 1 cycle of 15 minutes at 42°C, and 5 minutes at 99°C before cooling to room temperature. This mixture was then used for amplification of specific cDNAs by PCR. The primers used for amplification of the BK reverse transcript (5′-AGC TCA AGT TGG GTT TCA TAG CCC-3′, sense; 5′-GAC AGT CAT GGC AGC TTC ACT TCG-3′, antisense) were designed from the mouse Slo1 sequence22 , and those used to amplify IK (5′-TGC ACG CTG AGA TGT TGT GG-3′, sense; 5′-GTG TCT GTG AGG TGC CCC GT-3′, antisense) were designed from the mIK1 sequence.20 PCR buffer contained (in mmol/L) Tris-HCl 10 (pH 8.3), KCl 50, MgCl2 1, and 0.2 mmol/L each of dATP, dTTP, dGTP, and dCTP, 0.8 μmol/L oligonucleotide PCR primers, 0.625 U of AmpliTaq DNA Polymerase, and a 1:200 dilution of anti-Taq DNA polymerase monoclonal antibody. Samples were incubated at 94°C for 5 minutes, followed by 1 minute at 60°C and 3 minutes at 72°C, followed by 35 cycles of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes. A final extension step was performed at 72°C for 8 minutes. PCR products were examined on 2% agarose gels.
Isolation and Cloning of SMIK
To clone SMIK, a cDNA library was constructed from immature smooth muscle cells, which express high activity of the IK channel.5 PolyA+ RNA was extracted from multipassaged (>20 times) rat aortic smooth muscle cells.5 First strand cDNA was synthesised using MuLV reverse transcriptase and a primer containing an 18-base poly(dT) sequence (Stratagene). Double-stranded cDNAs were size-fractionated (>400 bp), ligated into the Uni-ZAP XR vector using the ZAP-cDNA Synthesis Kit, and in vitro packaged using the Gigapack III Gold Cloning Kit (Stratagene). The resultant primary library contained approximately 1.0×106 independent clones, and following amplification of the library to 6.0×109 pfu/mL, pBluescript phagemids containing the cloned DNA inserts were excised and used for subsequent library screening.
To generate a cDNA probe for cDNA library screening, a 588-bp RT-PCR fragment was amplified from immature smooth muscle cell polyA+ RNA using mIK1 PCR primers (see above).20 The resultant fragment was excised from the gel, purified, and cloned into the pCR 2.1 vector by TA cloning (Invitrogen). DNA sequencing showed that this PCR product corresponded to a partial sequence of the rat SMIK. Internal nested primers were designed (5′-CAT GAC TGA CAA CGG GCT CCG GGA-3′, sense; and 5′-TCA GCC ACA GAC AGC ACC CAA GCT-3′, antisense) and used to amplify a 415-bp PCR fragment from <1 ng of pCR 2.1/DNA template. This fragment was then used to screen the smooth muscle cell cDNA library.
XL1-blue cells infected with the pBluescript phagemid library were plated onto 137-mm Duralose-UV filter membranes at a density of 25 000 cfu/plate. The PCR product corresponding to SMIK was purified on NucTrap purification columns (Stratagene) and radiolabeled with [α-32P]dCTP (specific activity, 3000 Ci/mmol; Amersham Pharmacia Biotech) using the Prime-It RmT Random Primer Labeling Kit (Stratagene). [α-32P]-labeled probe was purified from unincorporated radioactivity and boiled with herring sperm DNA before addition to hybridization solution. Hybridization was performed on replica filters at 42°C in 50% formamide, 2× PIPES buffer, 0.5% SDS, and herring sperm DNA (100 g/mL) for 14 hours on an orbital shaker. Filters were washed 6 times (15 minutes each) at 50°C in wash buffer (0.1×SSC, 0.1% SDS) and exposed to x-ray film for 1 to 2 days. The 15 positive regions (3 to 5 mm) of the master filter were scraped, replated, and screening was repeated to obtain single positive colonies. These colonies were placed into liquid culture overnight and plasmid DNA isolated using Qiagen (Chatsworth) Miniprep columns. Plasmid DNA was sequenced using T7 and T3 primers by the Duke DNA Sequencing Facility. One of the clones was found to contain the full-length SMIK sequence including both 5′ and 3′ untranslated regions. Use of internal sequencing primers allowed the complete bidirectional sequencing of the SMIK cDNA.
cRNA Synthesis and Expression in Xenopus Oocytes
Plasmid DNA was linearized using the restriction endonuclease, Sal1, and transcribed using a modified T3 RNA polymerase in the presence of the cap analog m7G(5′)ppp(5′)G (Ambion MEGAscript kit, Austin, Tex). Template DNA was removed using RNase-free DNase 1, and the RNA was extracted using phenol-chloroform, ethanol precipitated, resuspended in RNase-free water at a concentration of 750 ng/μL, and stored in 5-μL aliquots at −80°C. RNA was examined on ethidium bromide-stained denaturing agarose minigels to assure the presence of an undegraded transcript.
Oocytes were obtained from female Xenopus laevis as previously described.21 Ovarian segments containing Stage V-VI oocytes were surgically removed from anesthetized frogs and, following brief collagenase digestion, the oocytes were manually defolliculated, washed extensively, and incubated in ND-92 medium (in mmol/L: NaCl 92, KCl 1.5, CaCl2 1.2, MgCl2 2, HEPES 10, and 50 mg/mL Gentamycin, pH 7.6) for 24 hours at 17°C before RNA injection. Each oocyte was injected with 46 nL of in vitro transcribed, capped cRNA (diluted to 250 ng/μL for macroscopic currents and 150 ng/μL for single channels) using a Drummond Nanojector (Broomall). Functional channel expression was observed within 2 days, and abundant current levels observed for up to 14 days after injection. Immediately before patch clamp experiments, the vitelline membrane was removed with fine forceps. Oocytes were maintained at 17°C in sterile ND-92 medium that was changed daily.
Quantitative data are expressed as mean±SE (n=number of patches). Differences between means were tested for significance (P<0.05) using the Student’s unpaired t test. Computer software packages, pClamp 6.0 (Axon Instruments) and Origin 5.0 (MicroCal), aided acquisition and analysis of single-channel and macropatch data. DNA and protein sequence editing and alignments (pileup) were performed using GCG software (Genetics Computer Group).
Data deposition: The sequence reported in this study has been deposited in the GenBank database (accession No. AF190458).
Characterization of the KCa Channel Responsible for Differences in Electrical Properties of Smooth Muscle Cell Types
Figure 1A⇓ shows the difference in electrical properties of de-differentiated and immature smooth muscle cell populations. Exposure of de-differentiated smooth muscle cells to endothelin-1 (ET-1, 0.1 μmol/L) induces membrane depolarization (top trace). In contrast, exposure of immature smooth muscle cells to ET-1 produces hyperpolarization (second trace), an effect which is abolished by charybdotoxin (20 nmol/L) (third trace). These changes in membrane potential were assessed using the membrane potential-sensitive probe, bis-oxonol.5 To determine whether a BK channel is involved, we examined the effect of the selective BK inhibitor, iberiotoxin.15 Iberiotoxin, used at concentrations known to produce effective blockade of BK in other tissues (10 to 100 nmol/L), had no effect on ET-1–induced hyperpolarization of immature smooth muscle cells (fourth trace); ET-1-induced hyperpolarization was 15.1±1.5 and 15.1±3.3 mV in control and iberiotoxin-treated cells (100 nmol/L, 10 minutes), respectively (n=3). Thus, the sensitivity to charybdotoxin, but not iberiotoxin (present study) or apamin (shown previously,5 ) indicates that the KCa current responsible for the difference in electrical responses between the 2 smooth muscle cell populations is mediated by a novel type of KCa channel.
To determine the types of KCa channels expressed in the different smooth muscle cell types, we examined mRNA expression levels of 2 candidate KCa channels, BK and IK, by RT-PCR in the different smooth muscle cell types. Figure 1B⇑ shows that BK mRNA, detected using PCR primers based on mSlo1,22 is expressed in aortic smooth muscle and de-differentiated smooth muscle cells. Surprisingly, however, no expression of BK mRNA is observed in immature smooth muscle cells despite these cells exhibiting a robust charybdotoxin-sensitive, Ca2+-dependent hyperpolarization. Eight additional PCR primer pairs, designed to amplify widely separated regions of the BK cDNA, also did not yield an RT-PCR product from immature smooth muscle cells (results not shown). To investigate whether the recently characterized IK channel was responsible, we performed RT-PCR analysis using primers designed initially on the human IK1 sequence16 and later on the mouse IK1 sequence.20 Figure 1B⇑ (right) shows that an abundant RT-PCR product is found at the expected molecular size using a PCR primer set which spans mIK1 regions encoding S1 to S5, indicating that an IK mRNA is expressed in immature smooth muscle cells. Cloning of this PCR product into pCR 2.1 and DNA sequencing confirmed its identity as part of an IK sequence (results not shown). Another mIK1 primer set, which spans S4 to the C terminus, and 2 primer sets based on the human IK1 sequence also indicated abundant expression (results not shown). These results demonstrate that an IK, rather than a BK channel, is the major KCa channel mRNA expressed in immature smooth muscle cells.
The electrophysiological properties of the KCa channels expressed on different smooth muscle cell types were examined by single-channel recordings from inside-out membrane patches. In contractile cells, most single-channel openings are mediated by BK channels, as characterized by their large amplitude and voltage-dependent open probability (Figure 1C⇑, top left). These single-channel openings, readily observed in every excised patch, have an amplitude of 7.8±0.3pA (n=6) at 40 mV and a calculated single-channel conductance of 196±8 pS in symmetrical K+ solution (Figure 1C⇑, middle left). They are Ca2+-activated (open probability, nPo, increased from 0.022 in 30 nmol/L Ca2+ to 2.76 in 300 nmol/L Ca2+) and K+-selective (EK decreased from 0 mV in symmetrical high K+ to −70 mV in physiological K+ gradients). These properties are consistent with an involvement of BK channels in contractile smooth muscle cells.
In contrast, BK currents are rarely observed in patches excised from de-differentiated smooth muscle cells. BK-like activity was observed in 1 of 6 patches excised from de-differentiated cells at 3 weeks in culture, and never in membrane patches excised from smooth muscle cells cultured for ≥6 weeks. The predominant K+ current in these cells has a smaller unitary conductance (Figure 1C⇑, top middle), and 2 to 6 of these channels were recorded in every patch excised. Single-channel openings are K+-selective (shifts in EK in different K+ gradients are similar to BK), and steady-state levels of channel activity were observed at all potentials ranging from −60 to 60 mV (data not shown). The slope-conductance is ≈32 pS (n=4) (Figure 1C⇑, middle); however, accurate recording of single-channel openings is problematic because of the presence of numerous channels in the membrane patches. Transmembrane currents recorded from immature smooth muscle cell patches (n=5) were large with an irregular and noisy appearance (Figure 1C⇑, top right). This macro current was evident at all potentials tested (−80 to 80 mV). When single transitions could be discerned, their amplitudes appeared similar to the 32 pS channels observed in de-differentiated smooth muscle cells (Figure 1C⇑, top right). Average amplitudes of the BK and IK currents recorded from membrane patches of contractile and de-differentiated smooth muscle cells, respectively, are shown in Figure 1C⇑ (middle right).
The intermediate-conductance KCa channels observed in de-differentiated smooth muscle cells are blocked by charybdotoxin. Single-channel openings were recorded from inside-out excised membrane patches in which the pipette was back-filled with charybdotoxin (10 nmol/L). Initially, K+ channel openings of intermediate-conductance were recorded immediately after formation of the high-resistance seal (n=3) (Figure 1C⇑, bottom left); however, channel activity disappeared within 2 to 3 minutes because of diffusion of the toxin to the membrane patch (bottom right). In control patches (no charybdotoxin included in pipette), the small K+ currents were recorded for >20 minutes at potentials both positive and negative of 0 mV, as described above (n=4).
The differential sensitivity to scorpion toxins, relative expression of KCa channel mRNAs, and unique single-channel properties are all consistent with the involvement of an IK in regulating the electrical properties of immature smooth muscle cells. Together, these results prompted the molecular cloning of the IK from immature smooth muscle cells.
Molecular Cloning of SMIK
SMIK was cloned by PCR homology screening of a cDNA library constructed from immature vascular smooth muscle cells. The hybridization probe was generated from a PCR fragment amplified from immature smooth muscle cell poly(A+) mRNA using PCR primers based on the mIK1 sequence.20 One of the several positive clones identified contained a 1.9-kb sequence consisting of a 112-bp 5′-UTR, 1.3 kb of coding sequence, and 512 bp of 3′-UTR containing a polyadenylation signal and polyA+ tail. The GC-rich (67%) 5′-UTR contains a single strong Kozak sequence generating an open reading frame for a 425 amino acid protein.
The translated protein conforms to the common K+ channel topographic model of 6 transmembrane domains (S1-S6) and a pore region (Figure 2A⇓ and 2B⇓). Amino acid alignment reveals close homology with other cloned members of the IK and SK families (Figure 2C⇓). In particular, it shares 97.9% and 86.4% sequence identity with mIK1 and hIK1, respectively. Of the 9 amino acids that differ between SMIK and mIK1, 5 are located within the distal 10% of the protein at the C-terminus, 3 lie within the extracellular loop following S3, and another extracellular substitution occurs before S6. The close homology of SMIK with the mouse and human IK channels suggests that SMIK is likely to be encoded by the IK1 gene. Overall, SMIK is only 46% to 48% homologous with rSKs; however, there are several regions of identity between members of the IK and SK families including, most notably, the pore and the region following S6 reported to bind calmodulin23 24 25 (Figure 2C⇓).
The SMIK protein has several distinctive features, including the S5-S6 pore region containing the K+ selectivity sequence GYG and an aspartate residue (D237) thought to be important for charybdotoxin binding16 (Figure 2A⇑). A consensus site for N-linked glycosylation (N230) is identified near the pore. The N-terminus contains an endoplasmic reticulum retention signal motif shown to be important for intracellular trafficking of assembled channels.26 The S6 domain contains a series of 4 cysteine residues spaced to predict disulfide bridge formation possibly important for protomer oligomerization or association with other proteins, and similarly, the C-terminus contains a leucine zipper motif located partially along a helical domain. Consistent with its lack of voltage-dependence, the S4 domain lacks the series of arginine residues that comprise the voltage sensor in voltage-gated K+ channels. Multiple consensus sites for protein kinase phosphorylation (PKA: S312, S332; PKG: S312, T327, S332; and PKC: T327, T348) are concentrated in the region Y312-T348 on the C-terminus, and 2 additional PKC sites (T101, S176) are located on the S2-S3 and S4-S5 intracellular loops (Figure 2A⇑). The C-terminus also contains a strong consensus site (RLLQEAWMY) for tyrosine phosphorylation (Y323). Of the 7 potential phosphorylation sites, at least 3 (T101, S176, T327) appear to be conserved across IK and SK families (Figure 2C⇑).
Functional Expression of Recombinant SMIK in Oocytes
Xenopus oocytes were injected with SMIK cRNA (6 to 12 ng) or with water (mock-injected) and examined 2 to 14 days later. Single-channel openings were recorded in SMIK-injected oocytes within 48 hours. To determine whether these were Ca2+-activated, the intracellular side of the membrane patches was perfused sequentially with solutions containing varying Ca2+ concentrations (0 to 1000 nmol/L). As illustrated in Figure 3A⇓ for a representative patch, the open probability of the channel is set by the Ca2+ concentration. Maximal channel activity was apparent by 500 nmol/L Ca2+, with half maximal activation occurring at ≈100 nmol/L Ca2+. Channel activity was reversible when the patch was again perfused with Ca2+-free solution, and such Ca2+-activation curves could typically be repeated 4 to 5 times before significant amounts of channel rundown were observed. Above 500 nmol/L Ca2+, some inhibition of outward current was observed. Figure 3B⇓ shows single-channel activity in a representative patch bathed in 500 nmol/L Ca2+ solution and held at varying voltages from −80 to 80 mV. Openings were observed at all membrane voltages, demonstrating a lack of voltage-dependent gating; however, single-channel amplitudes showed clear inward rectification (Figure 3C⇓).
Channel density on membrane patches increased by the third day after injection of SMIK cRNA, facilitating the recording of macroscopic currents. In the presence of 1 μmol/L Ca2+, a prominent macroscopic current was observed at all membrane voltages (Figure 4⇓, top left). This current was abolished by perfusion of Ca2+-free solution over the intracellular surface of the patch (Figure 4⇓, bottom left), and reappeared after reintroduction of the Ca2+ solution, demonstrating its Ca2+-dependence. No Ca2+-dependent current was observed in mock-injected oocytes. The current exhibited moderate inward rectification and slower activation time constants at positive membrane potentials (Figure 4⇓, top left). Perfusion of bathing solutions containing different Ca2+ concentrations (0 to 1000 nmol/L) over the intracellular surface of the patch confirmed the sharp Ca2+-dependence observed in single-channel recordings (Figure 4⇓, bottom right). Half-maximal activation occurred at 120±4 nmol/L free Ca2+ with a Hill coefficient of 2.9±0.3 (n=7) indicative of a highly cooperative Ca2+-mediated activation of the channel. The macroscopic current was highly selective for K+ ions (Figure 5⇓). In the presence of 1 μmol/L Ca2+ and 150 mmol/L K+ in the bath, the reversal potential for extracellular (pipette) solutions of 5, 27, and 150 mmol/L K+ was −89.6±7.3, −45.5±3.7, and −0.32±0.43 mV, respectively (n=5). This corresponds to a shift in EK of 60.6 mV per 10-fold change in extracellular K+ concentration, in close agreement with that predicted by the Nernst equation for a K+-selective channel (Figure 5⇓, inset).
In the present study, we have cloned and functionally characterized a novel vascular smooth muscle Ca2+-activated K+ channel that possesses intermediate conductance and potent activation by Ca2+. This smooth muscle IK channel, termed SMIK, plays a major role in the electrical response to vasoactive factors in proliferative vascular smooth muscle cell types. The gating properties of SMIK are distinct from those of the BK channel, whose expression is restricted to contractile-phenotype smooth muscle cells. Because the differential expression of these 2 KCa channel types is likely to exert a profound influence on the functional properties of smooth muscle cells, modulation of KCa channel expression may be an important mechanism by which specific physiological processes are carried out in the vessel wall.
SMIK is expressed in all smooth muscle cell types examined, however, its activity is markedly increased in proliferative immature-phenotype smooth muscle cells. It is the channel responsible for the hyperpolarization induced by vasoactive agonists in immature smooth muscle cells because (1) the clone was isolated from an immature smooth muscle cell cDNA library, (2) RT-PCR analysis shows that SMIK mRNA is expressed in the immature smooth muscle cell type, (3) intermediate-conductance channel activity is markedly increased in the immature cells which are devoid of BK mRNA and BK activity, (4) the electrophysiological properties of the cloned SMIK channel are essentially identical to the KCa channel expressed in immature smooth muscle cells in terms of single-channel conductance, K+ ion selectivity, and voltage-independent gating; and (5) the hyperpolarization is potently blocked by charybdotoxin, but not iberiotoxin or apamin,5 a characteristic feature of IK channels.
SMIK is similar in overall topography to many types of voltage-gated and Ca2+-activated K+ channels10 having 6 predicted transmembrane domains (S1-S6) and a pore region. Like other members of the IK and SK families, SMIK exhibits pronounced Ca2+-dependent activation, and Ca2+-dependent openings occur with equivalent probability at both positive and negative membrane potentials. This property suggests that SMIK is regulated predominantly by changes in local Ca2+ concentration. The mechanism for Ca2+-dependent activation does not involve any of the known Ca2+-binding motifs such as a calcium bowl or EF hands.10 Recent studies have suggested that Ca2+ sensing in these channels is mediated by calmodulin, which binds to the proximal portion of the C-terminus.24 25 SMIK is highly homologous to other members of the IK and SK families in this region (A284- M342), indicating that SMIK may use a similar mechanism; however, the consensus IQ motif found in the human IK is not conserved in SMIK. This suggests that alternative residues may be important in SMIK for the interaction of calmodulin such as the MKES (309–312) stretch that forms part of the calmodulin-binding motif in P/Q type Ca2+ channels.27 Like many ion channels,28 SMIK may be regulated by protein kinase/phosphatase signaling pathways. Multiple consensus sites for phosphorylation are present, most of which are located close to the putative calmodulin-binding domain. Additionally, growth factors such as PDGF and EGF29 may modulate SMIK activity directly through the tyrosine phosphorylation consensus site (Y323) to induce changes in smooth muscle cell properties.
The present results are consistent with our hypothesis that changes in KCa channel expression play a role in defining vascular smooth muscle cell function. In mature healthy vessels, the majority of smooth muscle cells exist in a fully differentiated contractile state required for the physiological regulation of vascular tone. Our studies show that these cells express predominantly BK channels, the activation of which reduces vascular contractility due to an inhibitory effect on L-type Ca2+ channels.11 12 However, in response to vascular injury, in developing vessels or in disease states, a significant proportion of smooth muscle cells exhibit enhanced growth responsiveness and matrix production associated with phenotypic modulation. Our results suggest that under these conditions, smooth muscle cells no longer express BK channels, but rather a novel IK channel, SMIK, with distinct properties. This phenotypic shift in KCa channel expression pattern gives rise to dramatic alterations in the cell’s electrical properties which will have functional consequences, in part because of an effect on Ca2+ influx. BK channels inhibit Ca2+ influx, whereas IK channels are reported to enhance Ca2+ influx by increasing its transmembrane electrical gradient.30 31 This increase in Ca2+ influx, caused by activation of SMIK, may result in stimulation of distinct cellular mechanisms associated with smooth muscle growth.
If programmed changes in KCa channel expression are critical events in the development and maintenance of the vascular system, then aberrant expression of these channels may be responsible for growth and contractile abnormalities associated with vascular pathologies and disease. Changes in KCa channel expression could be responsible for hypersensitivity to vasoconstrictors.32 33 This highlights the potential use of SMIK channel inhibitors in the management of these conditions.
Our understanding of vascular function increased dramatically with the discovery that smooth muscle cells exist in a variety of functional phenotypes depending on the developmental, physiological, and pathophysiological state. Recent findings suggest that changes in the expression, distribution, and functional state of ion channels play pivotal roles in the regulation of these different smooth muscle cell types.5 8 9 The present results highlight the importance of multiple KCa channel gene families in regulating the functional properties of smooth muscle. The dynamic regulation of these channels allows smooth muscle cells to respond in specific ways to diverse physiological and pathological stimuli.
This work was supported in part by a National Institutes of Health grant (to P.H.R.), and the National Health and Medical Research Council of Australia.
- Received September 28, 1999.
- Accepted October 4, 1999.
- © 1999 American Heart Association, Inc.
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