This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Neylon, C. B. Articles by Reinhart, P. H. Search for Related Content PubMed PubMed Citation Articles by Neylon, C. B. Articles by Reinhart, P. H. Related Collections Genomics Ion channels/membrane transport Smooth muscle proliferation and differentiation

(Circulation Research. 1999;85:e33-e43.)
© 1999 American Heart Association, Inc.

UltraRapid Communication

Molecular Cloning and Characterization of the Intermediate-Conductance Ca²⁺-Activated K⁺ Channel in Vascular Smooth Muscle

Relationship Between K_Ca Channel Diversity and Smooth Muscle Cell Function

Craig B. Neylon, Richard J. Lang, Ying Fu, Alex Bobik, Peter H. Reinhart

From the Department of Neurobiology (C.N., P.H.), Duke University Medical Center, Durham, NC; Baker Medical Research Institute (C.N., Y.F., A.B.), Victoria; and Department of Physiology (R.L.), Monash University, Victoria, Australia.

Correspondence to Dr Craig B. Neylon, Department of Neurobiology, Duke University Medical Center, Box 3209, Durham, NC 27710. E-mail neylon{at}neuro.duke.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 (K_Ca 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) K_Ca (BK) channels. In contrast, proliferative smooth muscle cells express predominantly K_Ca 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 K_Ca 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⁺ (K_d 120 nmol/L). Thus, we have cloned and characterized the vascular smooth muscle intermediate-conductance K_Ca channel (SMIK), which is markedly upregulated in proliferating smooth muscle cells. The differential expression of these K_Ca channels in functionally distinct smooth muscle cell types suggests that K_Ca 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.

Key Words: molecular cloning • K⁺ channel • vascular smooth muscle • [Ca²⁺]_i • charybdotoxin

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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⁺ (K_Ca) 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 K_Ca 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 K_Ca channels.

To investigate the relationship between K_Ca channels and smooth muscle cell function, we examined the expression of K_Ca channels on functionally distinct smooth muscle cell types. Three types of K_Ca 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 human^{16 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 K_Ca channels help to define the physiological properties of the vessel wall.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
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

Electrophysiology
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 M 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.

Electrophysiological Solutions
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(ß-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 K_Ca 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 1.0x10⁶ independent clones, and following amplification of the library to 6.0x10⁹ 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 [-³²P]dCTP (specific activity, 3000 Ci/mmol; Amersham Pharmacia Biotech) using the Prime-It RmT Random Primer Labeling Kit (Stratagene). [-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, 2x 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 (0.1xSSC, 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.

Data Analysis
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).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of the K_Ca 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 K_Ca current responsible for the difference in electrical responses between the 2 smooth muscle cell populations is mediated by a novel type of K_Ca channel.

View larger version (43K): [in this window] [in a new window]   Figure 1. Involvement of IK channels in the altered electrical properties of vascular smooth muscle cell types. A, Toxin sensitivity of the agonist-induced electrical response to vasoactive agonists in immature vascular smooth muscle cells. Electrical responses in confluent smooth muscle cell populations were measured using the membrane potential-sensitive fluorescent probe, bis-oxonol. Top 2 traces show the effect of ET-1 (0.1 µmol/L) on membrane potential in de-differentiated and immature vascular smooth muscle cells. Lower 2 traces show the effect of charybdotoxin (20 nmol/L) and iberiotoxin (100 nmol/L) on the hyperpolarization of immature vascular smooth muscle cells. Traces are representative of at least 3 similar experiments. B, Diversity of KCa channel mRNA expression in vascular smooth muscle cell types. RT-PCR assays were used to determine expression of BK and IK mRNA in aortic, de-differentiated, and immature smooth muscle cells. DNA size standards (kb) are shown on left. C, Electrophysiological characterization of the K+ channels expressed in vascular smooth muscle cell types. Top, Traces show typical single-channel recordings from inside-out patches of contractile, de-differentiated, and immature smooth muscle cell types. Closed states are represented by "C"; multiple open-state levels are denoted by dashed lines. BK channels occasionally opened to a subconductance level. Middle, Single-channel current amplitudes recorded at a range of voltages; different symbols on each plot are taken from different membrane patches. A comparison of the single-channel current amplitudes from the 2 cell types is shown in the histogram where amplitudes were measured from recordings taken at 40 mV. Bottom, Traces show the effect of charybdotoxin (10 nmol/L) on channel activity in de-differentiated vascular smooth muscle cells. Control channel activity (left) was recorded before diffusion of charybdotoxin onto the extracellular surface of the patch (right). All points histograms show the absence of open transitions after exposure to charybdotoxin.

To determine the types of K_Ca channels expressed in the different smooth muscle cell types, we examined mRNA expression levels of 2 candidate K_Ca 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 K_Ca channel mRNA expressed in immature smooth muscle cells.

The electrophysiological properties of the K_Ca 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, nP_o, increased from 0.022 in 30 nmol/L Ca²⁺ to 2.76 in 300 nmol/L Ca²⁺) and K⁺-selective (E_K 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 E_K 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 K_Ca 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 K_Ca 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 calmodulin^{23 24 25} (Figure 2C).

View larger version (128K): [in this window] [in a new window]   Figure 2. cDNA cloning of SMIK. A, Schematic of vascular SMIK sequence. Transmembrane domains (S1-S6) are shown as dark cylinders, the pore is between S5 and S6. Regions shown below the transmembrane domains are predicted to face the cytoplasmic side. A putative site for N-linked glycosylation is shown following S5. The aspartate residue important of charybdotoxin-sensitivity is indicated by small gray box. K+ selectivity filter GYG is bracketed. ERS indicates endoplasmic reticulum retention signal; CBD, putative calmodulin-binding domain. Consensus sites for phosphorylation by protein kinases are denoted by asterisks; numbers 1 to 4 are PKA, PKG, PKC, and tyrosine kinase, respectively. Buried C-helix is indicated by horizontal gray box, and the leucine zipper by triangles. B, Kyte-Doolittle hydropathy plot using a window of 9 residues. Vertical scale refers to free energy transfer to water, with upward peaks indicating hydrophobic regions. Transmembrane domains (S1-S6) and the pore (P) are indicated along top. C, Comparison of SMIK protein sequence with other cloned IK and SK channels. SK sequences have been truncated at both the N- and C-termini for clarity. Dots represent gaps introduced to optimize alignment. Transmembrane domains (S1-S6) are overlined, amino acid numbers for the full-length sequences are given on the right, and identical residues among all sequences are boxed. Phosphorylation sites are shown by asterisks, N-linked glycosylation by the hash, and the aspartate residue important for charybdotoxin-sensitivity is indicated by a cross.

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).

View larger version (22K): [in this window] [in a new window]   Figure 3. Single-channel properties of recombinant SMIK expressed in Xenopus oocytes. A, Ca2+ activation of SMIK. Oocytes were injected with capped SMIK cRNA (6 ng) and single channels were observed on the second day after injection. Representative traces show single-channel activity on inside-out patches exposed to 0, 50, 100, and 500 nmol/L free Ca2+ (patch contains at least 2 channels). Channel openings are shown as upward deflections. Voltage was -60 mV, and K+ concentration was symmetrical at 150 mmol/L. Data were filtered at 2 kHz. B, Single-channel activity from a representative patch at different voltages. Single-channel amplitudes show inward rectification and reversal at 0 mV. C, Single-channel current-voltage (I-V) curve obtained from an inside-out patch exposed to symmetrical (150 mmol/L) K+ solutions. Intracellular free Ca2+ concentration was 500 nmol/L. Membrane potential was stepped from -80 mV to 80 mV manually, and steady-state channel activity was recorded for 30 s at each 20-mV voltage step. Data were sampled at 10 kHz with 2 kHz filtering.

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 E_K 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).

View larger version (27K): [in this window] [in a new window]   Figure 4. Ca2+-sensitivity of SMIK expressed in Xenopus oocytes. Oocytes were injected with capped SMIK cRNA (12 ng) and macroscopic currents recorded 3 to 12 days after injection. Shown are currents evoked by 20-mV voltage steps (-100 to 100 mV) from a representative inside-out macropatch exposed to 1 µmol/L Ca2+ (top left), and nominally Ca2+-free (bottom left) bath solutions, in symmetrical (150 mmol/L) KCl. Traces from mock-injected oocytes, recorded under identical conditions, have been subtracted. Top right, Current-voltage relationship of macroscopic currents recorded from 3 separate oocytes. , Currents in the presence of Ca2+ (1 µmol/L) and •, currents in Ca2+-free solution. Bottom right, Ca2+-sensitivity of SMIK currents recorded from inside-out macropatches excised from SMIK-injected oocytes. Average current was measured at -60 mV from macropatches exposed to different Ca2+ concentrations (0 to 1000 nmol/L). Current at each Ca2+ concentration within a membrane patch was normalized against the current recorded in 1 µmol/L Ca2+ solution (n=7). Due to some degree of block at 1 µmol/L Ca2+, the normalized current at 500 nmol/L is >1.

View larger version (19K): [in this window] [in a new window]   Figure 5. K+ ion selectivity of the smooth muscle cDNA clone. Current-voltage relationships were recorded in excised (inside-out) macropatches at 3 different extracellular/intracellular K+ concentration gradients: 5/150 mmol/L (), 27/150 mmol/L (•), and 150/150 mmol/L (). Voltage steps (-100 to 100 mV, 20-mV steps) were applied in intracellular bath solution containing 1 µmol/L Ca2+, and representative traces are shown. Inset is the dependence of K+ reversal potential (EK) on extracellular K+ concentration (mmol/L). Each point is the average from at least 5 different patches. A linear regression through these points results in a slope of 60.6 mV per 10-fold change in pipette K+ concentration.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 K_Ca channel types is likely to exert a profound influence on the functional properties of smooth muscle cells, modulation of K_Ca 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 K_Ca 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 K_Ca 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 K_Ca 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 K_Ca 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 K_Ca 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 K_Ca 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.

	Acknowledgments

 
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.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Owens G. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev. 1995;75:487–517.[Abstract/Free Full Text]

2. Sartore S, Chiavegato A, Franch R, Faggin E, Pauletto P. Myosin gene expression and cell phenotypes in vascular smooth muscle during development, in experimental models, and in vascular disease. Arterioscler Thromb Vasc Biol. 1997;17:1210–1215.[Abstract/Free Full Text]

3. Frid MG, Dempsey EC, Durmowicz AG, Stenmark KR. Smooth muscle cell heterogeneity in pulmonary and systemic vessels. Importance in vascular disease. Arterioscler Thromb Vasc Biol. 1997;17:1203–1209.[Abstract/Free Full Text]

4. Chamley-Campbell J, Campbell G, Ross R. The smooth muscle cell in culture. Physiol Rev. 1979;59:1–61.[Free Full Text]

5. Neylon C, Avdonin P, Dilley R, Larsen M, Tkachuk V, Bobik A. Different electrical responses to vasoactive agonists in morphologically distinct smooth muscle cell types. Circ Res. 1994;75:733–741.[Abstract/Free Full Text]

6. Neylon C, Avdonin P, Larsen M, Bobik A. Rat aortic smooth muscle cells expressing charybdotoxin-sensitive potassium channels exhibit enhanced proliferative responses. Clin Exp Pharmacol Physiol. 1994;21:117–120.

7. Archer S, Huang J, Reeve H, Hampl V, Tolarova S, Michelakis E, Weir E. Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ Res. 1996;78:431–442.[Abstract/Free Full Text]

8. Reeve HL, Weir EK, Archer SL, Cornfield DN. A maturational shift in pulmonary K⁺ channels, from Ca²⁺ sensitive to voltage dependent. Am J Physiol. 1998;275:L1019–1025.[Abstract/Free Full Text]

9. Archer S. Diversity of phenotype and function of vascular smooth muscle cells. J Lab Clin Med. 1996;127:524–529.[Medline] [Order article via Infotrieve]

10. Vergara C, Latorre R, Marrion NV, Adelman JP. Calcium-activated potassium channels. Curr Opin Neurobiol. 1998;8:321–329.[Medline] [Order article via Infotrieve]

11. Brayden J, Nelson M. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science. 1992;256:532–535.[Abstract/Free Full Text]

12. Nelson M, Cheng H, Rubart M, Santana L, Bonev A, Knot H, Lederer W. Relaxation of arterial smooth muscle by calcium sparks. Science. 1995;270:633–637.[Abstract/Free Full Text]

13. Feletou M, Vanhoutte PM. The alternative: EDHF. J Mol Cell Cardiol. 1999;31:15–22.[Medline] [Order article via Infotrieve]

14. Van Renterghem C, Lazdunski M. A small-conductance charybdotoxin-sensitive, apamin-resistant Ca(2+)-activated K⁺ channel in aortic smooth muscle cells (A7r5 line and primary culture). Pflugers Arch. 1992;420:417–423.[Medline] [Order article via Infotrieve]

15. Brugnara C, Armsby C, De Franceschi L, Crest M, Martin Euclaire M-F, Alper S. Ca²⁺-activated K⁺ channels of human and rabbit erythrocytes display distinctive patterns of inhibition by venom peptide toxins. J Membr Biol. 1995;147:71–82.[Medline] [Order article via Infotrieve]

16. Ishii T, Silvia C, Hirschberg B, Bond C, Adelman J, Maylie J. A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci U S A. 1997;94:11651–11656.[Abstract/Free Full Text]

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

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

19. Jensen BS, Strobaek D, Christophersen P, Jorgensen TD, Hansen C, Silahtaroglu A, Olesen SP, Ahring PK. Characterization of the cloned human intermediate-conductance Ca²⁺-activated K⁺ channel. Am J Physiol. 1998;275:C848–856.[Abstract/Free Full Text]

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

21. Tseng-Crank J, Foster C, Krause J, Mertz R, Godinot N, DiChiara T, Reinhart P. Cloning, expression, and distribution of functionally distinct Ca²⁺-activated K⁺ channel isoforms from human brain. Neuron. 1994;13:1315–1330.[Medline] [Order article via Infotrieve]

22. Butler A, Tsunoda S, McCobb D, Wei A, Salkoff L. mSlo, a complex mouse gene encoding "maxi" calcium-activated potassium channels. Science. 1993;261:221–224.[Abstract/Free Full Text]

23. Xia XM, Fakler B, Rivard A, Wayman G, Johnson-Pais T, Keen JE, Ishii T, Hirschberg B, Bond CT, Lutsenko S, Maylie J, Adelman JP. Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature. 1998;395:503–507.[Medline] [Order article via Infotrieve]

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

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

26. Zerangue N, Schwappach B, Jan YN, Jan LY. A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron. 1999;22:537–548.[Medline] [Order article via Infotrieve]

27. Lee A, Wong ST, Gallagher D, Li B, Storm DR, Scheuer T, Catterall WA. Ca²⁺/calmodulin binds to and modulates P/Q-type calcium channels. Nature. 1999;399:155–159.[Medline] [Order article via Infotrieve]

28. Levitan IB. Modulation of ion channels by protein phosphorylation. How the brain works. Adv Second Messenger Phosphoprotein Res. 1999;33:3–22.[Medline] [Order article via Infotrieve]

29. Huang Y, Rane SG. Potassium channel induction by the Ras/Raf signal transduction cascade. J Biol Chem. 1994;269:31183–31189.[Abstract/Free Full Text]

30. Lepple-Wienhues A, Berweck S, Bohmig M, Leo CP, Meyling B, Garbe C, Wiederholt M. K⁺ channels and the intracellular calcium signal in human melanoma cell proliferation. J Membr Biol. 1996;151:149–157.[Medline] [Order article via Infotrieve]

31. Verheugen JA, Le Deist F, Devignot V, Korn H. Enhancement of calcium signaling and proliferation responses in activated human T lymphocytes. Inhibitory effects of K⁺ channel block by charybdotoxin depend on the T cell activation state. Cell Calcium. 1997;21:1–17.[Medline] [Order article via Infotrieve]

32. Heistad DD, Breese K, Armstrong ML. Cerebral vasoconstrictor responses to serotonin after dietary treatment of atherosclerosis: implications for transient ischemic attacks. Stroke. 1987;18:1068–1073.[Abstract/Free Full Text]

33. Kuga T, Kadokami T, Kuwata K, Hata H, Ohara Y, Egashira K, Shimokawa H, Takeshita A. Central role of vascular smooth muscle hyperreactivity in coronary hyperconstriction after balloon injury in miniature pigs. Coron Artery Dis. 1997;8:69–75.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				M. C. Shepherd, S. M. Duffy, T. Harris, G. Cruse, M. Schuliga, C. E. Brightling, C. B. Neylon, P. Bradding, and A. G. Stewart KCa3.1 Ca2+Activated K+ Channels Regulate Human Airway Smooth Muscle Proliferation Am. J. Respir. Cell Mol. Biol., November 1, 2007; 37(5): 525 - 531. [Abstract] [Full Text] [PDF]

				E. T. Barfod, A. L. Moore, M. W. Roe, and S. D. Lidofsky Ca2+-activated IK1 Channels Associate with Lipid Rafts upon Cell Swelling and Mediate Volume Recovery J. Biol. Chem., March 23, 2007; 282(12): 8984 - 8993. [Abstract] [Full Text] [PDF]

				T. V. Nguyen, H. Matsuyama, J. Baell, B. Hunne, C. J. Fowler, J. E. Smith, K. Nurgali, and J. B. Furness Effects of Compounds That Influence IK (KCNN4) Channels on Afterhyperpolarizing Potentials, and Determination of IK Channel Sequence, in Guinea Pig Enteric Neurons J Neurophysiol, March 1, 2007; 97(3): 2024 - 2031. [Abstract] [Full Text] [PDF]

				D. L. Tharp, B. R. Wamhoff, J. R. Turk, and D. K. Bowles Upregulation of intermediate-conductance Ca2+-activated K+ channel (IKCa1) mediates phenotypic modulation of coronary smooth muscle Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2493 - H2503. [Abstract] [Full Text] [PDF]

				E. Qamirani, H. M. Razavi, X. Wu, M. J. Davis, L. Kuo, and T. W. Hein Sodium azide dilates coronary arterioles via activation of inward rectifier K+ channels and Na+-K+-ATPase Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1617 - H1623. [Abstract] [Full Text] [PDF]

				J. Ledoux, M. E. Werner, J. E. Brayden, and M. T. Nelson Calcium-Activated Potassium Channels and the Regulation of Vascular Tone Physiology, February 1, 2006; 21(1): 69 - 78. [Abstract] [Full Text] [PDF]

				J. Thompson and T. Begenisich Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel J. Gen. Physiol., January 30, 2006; 127(2): 159 - 169. [Abstract] [Full Text] [PDF]

				A. Sato, K. Terata, H. Miura, K. Toyama, F. R. Loberiza Jr., O. A. Hatoum, T. Saito, I. Sakuma, and D. D. Gutterman Mechanism of vasodilation to adenosine in coronary arterioles from patients with heart disease Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1633 - H1640. [Abstract] [Full Text] [PDF]

				O. Platoshyn, C. V. Remillard, I. Fantozzi, M. Mandegar, T. T. Sison, S. Zhang, E. Burg, and J. X.-J. Yuan Diversity of voltage-dependent K+ channels in human pulmonary artery smooth muscle cells Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L226 - L238. [Abstract] [Full Text] [PDF]

				R. Kohler, H. Wulff, I. Eichler, M. Kneifel, D. Neumann, A. Knorr, I. Grgic, D. Kampfe, H. Si, J. Wibawa, et al. Blockade of the Intermediate-Conductance Calcium-Activated Potassium Channel as a New Therapeutic Strategy for Restenosis Circulation, September 2, 2003; 108(9): 1119 - 1125. [Abstract] [Full Text] [PDF]

				W. J. Joiner, S. Basavappa, S. Vidyasagar, K. Nehrke, S. Krishnan, H. J. Binder, E. L. Boulpaep, and V. M. Rajendran Active K+ secretion through multiple KCa-type channels and regulation by IKCa channels in rat proximal colon Am J Physiol Gastrointest Liver Physiol, June 9, 2003; 285(1): G185 - G196. [Abstract] [Full Text] [PDF]

				T. Karkanis, S. Li, J. G. Pickering, and S. M. Sims Plasticity of KIR channels in human smooth muscle cells from internal thoracic artery Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2325 - H2334. [Abstract] [Full Text] [PDF]

				H. Koegel, S. Kaesler, R. Burgstahler, S. Werner, and C. Alzheimer Unexpected Down-regulation of the hIK1 Ca2+-activated K+ Channel by Its Opener 1-Ethyl-2-benzimidazolinone in HaCaT Keratinocytes. INVERSE EFFECTS ON CELL GROWTH AND PROLIFERATION J. Biol. Chem., January 24, 2003; 278(5): 3323 - 3330. [Abstract] [Full Text] [PDF]

				M. Simoes, L. Garneau, H. Klein, U. Banderali, F. Hobeila, B. Roux, L. Parent, and R. Sauve Cysteine Mutagenesis and Computer Modeling of the S6 Region of an Intermediate Conductance IKCa Channel J. Gen. Physiol., June 24, 2002; 120(1): 99 - 116. [Abstract] [Full Text] [PDF]

				C.-C. Shieh, M. Coghlan, J. P. Sullivan, and M. Gopalakrishnan Potassium Channels: Molecular Defects, Diseases, and Therapeutic Opportunities Pharmacol. Rev., December 1, 2000; 52(4): 557 - 594. [Abstract] [Full Text] [PDF]

				T. Nagayama, Y. Fukushima, M. Yoshida, M. Suzuki-Kusaba, H. Hisa, T. Kimura, and S. Satoh Role of potassium channels in catecholamine secretion in the rat adrenal gland Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2000; 279(2): R448 - R454. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Neylon, C. B. Articles by Reinhart, P. H. Search for Related Content PubMed PubMed Citation Articles by Neylon, C. B. Articles by Reinhart, P. H. Related Collections Genomics Ion channels/membrane transport Smooth muscle proliferation and differentiation