|Kv1.5 is a major component underlying the A-type potassium current in retinal arteriolar smooth muscle.|
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|PMID: 17040965 Owner: NLM Status: MEDLINE|
|Little is known about the molecular characteristics of the voltage-activated K(+) (K(v)) channels that underlie the A-type K(+) current in vascular smooth muscle cells of the systemic circulation. We investigated the molecular identity of the A-type K(+) current in retinal arteriolar myocytes using patch-clamp techniques, RT-PCR, immunohistochemistry, and neutralizing antibody studies. The A-type K(+) current was resistant to the actions of specific inhibitors for K(v)3 and K(v)4 channels but was blocked by the K(v)1 antagonist correolide. No effects were observed with pharmacological agents against K(v)1.1/2/3/6 and 7 channels, but the current was partially blocked by riluzole, a K(v)1.4 and K(v)1.5 inhibitor. The current was not altered by the removal of extracellular K(+) but was abolished by flecainide, indicative of K(v)1.5 rather than K(v)1.4 channels. Transcripts encoding K(v)1.5 and not K(v)1.4 were identified in freshly isolated retinal arterioles. Immunofluorescence labeling confirmed a lack of K(v)1.4 expression and revealed K(v)1.5 to be localized to the plasma membrane of the arteriolar smooth muscle cells. Anti-K(v)1.5 antibody applied intracellularly inhibited the A-type K(+) current, whereas anti-K(v)1.4 antibody had no effect. Co-expression of K(v)1.5 with K(v)beta1 or K(v)beta3 accessory subunits is known to transform K(v)1.5 currents from delayed rectifers into A-type currents. K(v)beta1 mRNA expression was detected in retinal arterioles, but K(v)beta3 was not observed. K(v)beta1 immunofluorescence was detected on the plasma membrane of retinal arteriolar myocytes. The findings of this study suggest that K(v)1.5, most likely co-assembled with K(v)beta1 subunits, comprises a major component underlying the A-type K(+) current in retinal arteriolar smooth muscle cells.|
|Mary K McGahon; Jennine M Dawicki; Aruna Arora; D A Simpson; T A Gardiner; A W Stitt; C Norman Scholfield; J Graham McGeown; Tim M Curtis|
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|Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2006-10-13|
|Title: American journal of physiology. Heart and circulatory physiology Volume: 292 ISSN: 0363-6135 ISO Abbreviation: Am. J. Physiol. Heart Circ. Physiol. Publication Date: 2007 Feb|
|Created Date: 2007-02-08 Completed Date: 2007-03-20 Revised Date: 2013-06-07|
Medline Journal Info:
|Nlm Unique ID: 100901228 Medline TA: Am J Physiol Heart Circ Physiol Country: United States|
|Languages: eng Pagination: H1001-8 Citation Subset: IM|
|Centre of Vision Sciences, The Queen's University of Belfast, Institute of Clinical Sciences, The Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BA. UK.|
|APA/MLA Format Download EndNote Download BibTex|
Arterioles / metabolism
Kv1.5 Potassium Channel / analysis, drug effects, metabolism*
Membrane Potentials / drug effects
Muscle, Smooth, Vascular / chemistry, drug effects, metabolism*
Myocytes, Smooth Muscle / metabolism
Potassium / metabolism
Potassium Channel Blockers / pharmacology
Protein Subunits / metabolism
RNA, Messenger / analysis
Retinal Vessels / chemistry, drug effects, metabolism*
Reverse Transcriptase Polymerase Chain Reaction
|0/Kv1.5 Potassium Channel; 0/Potassium Channel Blockers; 0/Protein Subunits; 0/RNA, Messenger; 7440-09-7/Potassium|
Journal ID (nlm-ta): Am J Physiol Heart Circ Physiol
Journal ID (publisher-id): ajpheart
Publisher: American Physiological Society
Copyright ? 2007, American Physiological Society
open-access: This document may be redistributed and reused, subject to www.the-aps.org/publications/journals/funding_addendum_policy.htm.
Received Day: 13 Month: 9 Year: 2006
Accepted Day: 6 Month: 10 Year: 2006
Print publication date: Month: 2 Year: 2007
Electronic publication date: Day: 13 Month: 10 Year: 2006
pmc-release publication date: Day: 1 Month: 2 Year: 2007
Volume: 292 Issue: 2
First Page: H1001 Last Page: H1008
Publisher Id: H-01003-2006
PubMed Id: 17040965
|Kv1.5 is a major component underlying the A-type potassium current in retinal arteriolar smooth muscle|
|Mary K. McGahon1|
|Jennine M. Dawicki1|
|D. A. Simpson1|
|T. A. Gardiner1|
|A. W. Stitt1|
|C. Norman Scholfield2|
|J. Graham McGeown2|
|Tim M. Curtis1|
1Centre for Vision Sciences, School of Biomedical Sciences, The Queen's University of Belfast, Institute of Clinical Sciences, The Royal Victoria Hospital; 2School of Medicine, The Queen's University of Belfast, Medical Biology Centre, Belfast, United Kingdom
|Address for reprint requests and other correspondence: T. M. Curtis, Centre of Vision Sciences, The Queen's Univ. of Belfast, Institute of Clinical Sciences, The Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BA. UK (E-mail: email@example.com)
SMOOTH MUSCLE TONE in resistance arteries and arterioles is an important determinant of peripheral vascular resistance and blood pressure (29). By controlling membrane potential and the level of free intracellular Ca2+ available to the contractile apparatus, voltage-activated K+ channels (Kv) are crucial in regulating arterial smooth muscle tone (38). Based on electrophysiological studies, two major Kv current components have been identified in vascular smooth muscle cells; namely, the delayed rectifier K+ current and the transient A-type K+ current. However, this broad classification fails to reveal the highly diverse nature of the Kv channels that underlie these currents, and in fact, a host of delayed rectifier and A-type K+ currents have been reported in arterial smooth muscle with distinct biophysical and pharmacological properties (9). This diversity stems in part from the large number of gene families that encode the pore-forming Kv? subunits (Kv1?11) but also from other processes such as alternative splicing, heterotetrameric assembly of members within the same Kv? subfamily, and interaction of the channels with accessory subunits (8). Despite this complexity, recent efforts have been directed toward identifying the molecular composition of the Kv channels that underlie functional Kv currents in arterial smooth muscle, and evidence has begun to emerge suggesting that some delayed rectifier currents may arise as a consequence of heterotetrameric assembly of distinct Kv1? subunits (2, 4?7, 15, 22, 26, 27, 31, 39). The molecular composition of A-type currents in myocytes of the systemic circulation is not clear at this time (9); however, Kv3.4 and Kv4 channels appear to be the most likely determinants of these currents in pulmonary arterial smooth muscle (20).
K+ channel ?-subunits with A-type properties are found in several Kv channel subfamilies, including Shaker (Kv1.3, Kv1.4, and Kv1.7), Shaw (Kv3.3 and Kv3.4), and Shal (Kv4.1, Kv4.2, and Kv4.3), and transcripts for all of these subunits have been detected in vascular smooth muscle (9). In addition, co-expression of Kv?1 or K?3 accessory subunits with certain Kv1? subunits confers rapid inactivation on otherwise noninactivating delayed rectifier channels (18, 25, 33). Kv? subunit expression has been identified in several types of arterial smooth muscle (9). Rapid inactivation of A-type Kv channels is thought to be conferred by ?ball? domains in the NH2 termini of the relevant Kv? subunits and Kv?1 and Kv?3 subunits (N-type inactivation) (32). Upon depolarization, these domains occlude the channel pore by binding to a receptor near or at the cytoplasmic side of the pore (21).
We have recently described a rapidly inactivating A-type current in retinal arteriolar smooth muscle cells, which appears to play a physiological role in suppressing cell excitability and contractility (28). In the present study we have used this preparation as a model system to begin to resolve the molecular nature of the A-type current in myocytes of the peripheral systemic circulation. By combining a range of pharmacological, molecular, immunohistochemical, and neutralizing antibody approaches, we show that Kv1.5, most likely co-assembled with Kv?1, comprises a principal component underlying the A-type K+ current in this tissue.
Male Sprague-Dawley rats (200?300 g) were anesthetized with CO2 and killed by cervical dislocation. Retinas were rapidly removed, and arterioles, devoid of surrounding neuropile, were isolated as previously described (37). In brief, retinas were lightly triturated using a fire-polished Pasteur pipette (internal tip diameter 0.3 mm) in a low Ca2+ Hanks' solution. The resulting homogenates were centrifuged at 2,800 rpm (952 g) for 1 min, the supernatant was aspirated off, and the tissue was washed again with low Ca2+ medium. The suspension was then stored at 21?C until needed. Arteriolar segments remained useable for up to 10 h under these conditions.
Voltage-clamp experiments were performed using the whole cell-perforated patch-clamp technique as previously described (28). One milliliter of homogenate was pipetted into a rectangular glass-bottomed recording bath on the stage of an inverted microscope (Nikon Eclipse, TE2000). Arterioles were anchored down with tungsten wire slips (50 ?m diameter, 2 mm length) and continuously superfused with normal Hanks' solution at 37?C to remove extraneous retinal tissue from the bathing medium. Before the electrophysiological recording, vessels were digested for 20-min with an enzyme cocktail of collagenase 1A (0.1 mg/ml) and protease type XIV (0.01 mg/ml) to remove surface basal lamina. Enzyme and drug solutions were delivered via a seven-way micromanifold with an exchange time of 1 s as measured by switching over to dye solution. The flow from the manifold into the bath was through a single tube (350 ?m in diameter, 6 mm in length, 0.2-?l volume) long enough to allow the temperature to equilibrate with the solution flowing through the bath. Gigaseals were formed directly on arteriolar smooth muscle cells still embedded within their native arterioles. Electrodes (1?2 M? in free bathing solution) were pulled from filamented borosilicate glass capillaries (1.5 mm od w 1.17 mm id, Clark Electromedical Instruments, UK), and the patch-clamp amplifier used was an Axopatch-1D (Axon Instruments, Foster City, CA). The pipette solution was K+ based with amphotericin B as the perforating agent (see Drugs and solutions). Contaminating currents through Ca2+-activated K+ and Cl? channels were minimized in all experiments by including 100 nM Penitrem A and 1 mM 9-anthracene carboxylic acid in the external bathing medium. Recordings were delayed until full perforation of the membrane patch had been achieved, as judged from the development of repeatable currents in response to step depolarizations: this usually took 3?5 min. Recordings were low pass filtered at 0.5 kHz and sampled at 2 kHz by a National Instruments PC1200 interface using software provided by John Dempster (University of Strathclyde, UK). Leakage currents were subtracted off-line from the active currents with the use of the standard leak subtraction protocol contained within the Patch software suite. Liquid junction potentials (<2 mV) were compensated electronically. Series resistance (?30 M?) was routinely compensated by >70%. For determination of whole cell current densities, cell membrane capacitance was determined from the time constant of a capacitance transient elicited by a 20-mV depolarization from ?80 mV with a sampling frequency of 20 kHz.
To evaluate the effects of pharmacological agents, outward A-type K+ current was first elicited by voltage steps from ?80 mV to +20 mV (500 ms, 10-s intervals) in drug-free medium and then in bath solution containing the drug of interest. We have previously shown that the A-type K+ current in retinal arteriolar smooth muscle is closely approximated by the peak minus the sustained components of the net outward current in Penitrem A and 9-anthracene carboxylic acid (28). With the use of this method to isolate the A-type K+ current, the peak current densities for each vessel was averaged from five voltage steps before and then after drug application, and changes were expressed as a percentage for a minimum of four vessels per treatment group. For experiments using antibodies, conventional whole cell recordings were performed. Pipettes were dipped in an antibody-free intracellular solution and then back filled with the pipette solution containing the antibody of interest [anti-Kv1.4(589?655) or anti-Kv1.5(513?602); Alomone Labs, Jerusalem, Israel]. Aliquoted antibodies were defrosted daily to avoid degradation. Because the antibody experiments did not permit pairwise comparisons to be made within the same vessel, to improve resolution of changes across treatment groups, voltage steps were applied from ?80 mV to +80 mV, where average peak current densities in control vessels are twice those observed at +20 mV.
Each rat retina yielded approximately two to three first-order arterioles (35?50 ?m diameter, 50?500 ?m in length). The arterioles were uncontracted and easily distinguished from venules by their thick wall of circularly arranged smooth muscle cells (Fig. 1). As noted above, just before the patch-clamp recording, vessels were anchored down in the recording bath and digested for 20 min with an enzyme cocktail of collagenase and protease. In addition to removing the basal lamina to facilitate gigaseal formation, this treatment electrically uncouples the endothelial cells from the overlying arteriolar smooth muscle (28). In other types of arterioles, the smooth muscle cells are also known to be electrically coupled via gap junctions (10, 19, 41, 42). In our earlier work, we did not investigate whether the retinal arteriolar smooth muscle layer remains electrically coupled following enzyme treatment. An indirect method to evaluate this is to compare cell capacitance measurements with the estimated cell surface area for a single arteriolar myocyte. In the present study, cell capacitance was measured as 13.1 ? 0.48 pF (n = 31), indicating a total patch-clamped membrane surface area of ?1,310 ?m2. The dimensions of individual retinal arteriolar myocytes from first-order arterioles were estimated by confocal scanning laser microscopy in vessels loaded for 10 min with the membrane-tracking dye di-4-ANEPPs (10 ?M) (11); average dimensions for length (based on the vessel circumference), width, and height were 121.3, 5.7, and 2 ?m, respectively (21 cells; n = 4 vessels). When we used these values and assumed a scalene ellipsoid structure, the approximate surface area for a single retinal arteriolar myocyte was calculated according to the Knud Thomsen formula:
The approximated cell surface area using this method was calculated to be 1,220 ?m2, suggesting our electrophysiological recordings are most likely confined to individual myocytes. To further verify this, experiments were undertaken using the gap junction inhibitor 18?-glycyrrhetinic acid (18?-GA). In mesenteric arterioles, 40 ?M 18?-GA causes a rapid block of electrical communication within the smooth muscle layer, as denoted by a switch from predominantly slow to fast capacitative transients (41). In the present study, no changes in the capacitative currents were observed in enzyme-digested arterioles exposed to 100 ?M 18?-GA (capacitances were 12.53 ? 0.81 pF and 11.86 ? 0.97 pF, before and after 18?-GA, respectively; n = 9; P = 0.18). Taken together, the above results strongly suggest that following collagenase and protease treatment retinal arteriolar smooth muscle cells within intact vessel segments are electrically uncoupled from their neighboring cells.
Retinal homogenates were placed in a 2-ml recording chamber on the stage of an inverted microscope and between 5 and 13 vessels collected for each PCR experiment using single tungsten wire slips (50 ?m in diameter, 5 mm length). Total RNA was extracted using RNeasy minikit (Qiagen, Crawley, UK) according to the manufacturer's protocol. Total RNA was also extracted from brain pia. Samples were split into two aliquots, and first-strand cDNA was prepared from one aliquot using Sensiscript Reverse Transcription kit (Qiagen). The other aliquot was used in an equivalent reaction lacking enzyme to control for potential genomic or extraneous DNA contamination [no reverse transcriptase (RT)]. The cDNA RT products were amplified with Kv1.4-, Kv1.5-, Kv?1-, and Kv?3-specific primers by RT-PCR using Qiagen HotStar Taq reagents. The primer pairs, relevant Genbank entries, and expected product sizes are listed in Table 1. All products were resolved on 2.5% agarose gels and visualized by ethidium bromide fluorescence.
For immunofluorescence experiments, retinal arterioles were visualized while embedded within retinal flatmount preparations. The advantage of this technique over the use of isolated arterioles is that negative results within the vasculature can be cross-referenced with other cell types in the retina to gauge the degree of antibody reactivity. Freshly enucleated eyes were placed in low Ca2+ Hanks' solution, and the anterior segment lens complexes were removed. The posterior eye cup was fixed in 4% paraformaldehyde for 20 min and washed extensively in phosphate-buffered saline (PBS) throughout a 4-h period. Retinas were subsequently detached and soaked in PBS containing 0.5% Triton X-100 to permeabilize the tissue and 5% normal donkey serum (Chemicon International, Temecula, CA) to block nonspecific binding of the primary antibody. Kv1.4, Kv1.5, and Kv?1 were detected in separate experiments using rabbit polyclonal antibodies targeted to rat, mouse, and human sequences, respectively [anti-Kv1.4(589?655) and anti-Kv1.5(513?602); Alomone Labs, Jerusalem, Israel; anti-Kv?1(311?360); abcam, Cambridge, UK]. Tissue was incubated in primary antibody at a dilution of 1:100 (anti-Kv1.4 and anti-Kv1.5) or 1:200 (anti-Kv?1) in the permeabilization buffer overnight at 4?C and then extensively washed for 4 h at 21?C. A 1:200 dilution of a donkey anti-rabbit IgG labeled with Alexa-488 (Molecular Probes Europe BV, Leiden, The Netherlands) was used for secondary detection; the staining time and subsequent washing were the same as that used for the primary antibody. The specificity of the antibodies was investigated by parallel control experiments in the absence of primary antibody. To facilitate rapid identification of arterioles within the retinal neuropile, propidium iodide nuclear stain was used. Retinas were incubated in 5 nmol/l of propidium iodide (Invitrogen, Carlsbad, CA) in PBS for 30 min at 37?C. Retinas were flattened by placing four radial cuts from the retinal periphery to points within 1 mm from the optic disk and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were acquired using an Olympus BX60 fluorescence microscope (Olympus, London, UK) fitted with a MicroRadiance confocal-scanning laser microscope (Bio-Rad).
Hanks' solution contained (in mM) 140 NaCl, 5 KCl, 5 D-glucose, 2 CaCl2, 1.3 MgCl2, 10 HEPES; pH 7.4 with NaOH. Low Ca2+ medium differed only in that it contained 0.1 mM CaCl2. K+-free medium was the same as normal Hanks' solution without 5 mM KCl. For patch-clamp recordings the pipette solution contained (in mM) 138 KCl, 1 MgCl2, 0.5 EGTA, 10 HEPES (pH adjusted to 7.2 using NaOH) to which 300 ?g/ml amphotericin B was added in perforated patch mode.
Amphotericin B, collagenase 1A, penitrem A, protease type XIV, 9-anthracene carboxylic acid, 18?-glycyrrhetinic acid, flecainide, and riluzole were purchased from Sigma-Aldrich (Poole, UK). Correolide was a kind gift from Dr. Maria Garcia and Dr. Jianming Bao of Merck Research Laboratories, Rahway, NJ. Phrixotoxin-2, rHeteropodatoxin-2, rHongotoxin-1, rNoxiustoxin, and BDS-I were obtained from Alomone Labs.
Data are reported as means ? SE; n refers to the number of vessels tested. Significant differences between control and experimental treatments were determined using the paired t-test. Antibody experiments were analyzed using one-way ANOVA. P values <0.05 were considered significant.
Over recent years there has been a substantial increase in the number of toxins available that inhibit Kv channels. Taking impetus from this, we tested a range of pharmacological blockers as a first step in resolving likely Kv channel components underlying the A-type K+ current in retinal arteriolar myocytes. Initially, we screened agents that selectively block A-type Kv? subunits within the main Kv channel subfamilies. Cells were held at ?80 mV, and command voltage steps to +20 mV were applied. Phrixotoxin-2 and heteropodatoxin-2 are peptides from spider venoms that specifically inhibit Kv4 channels (12, 36). Neither of these toxins applied at concentrations higher than reported IC50 values affected the A-type K+ current in retinal arteriolar myocytes nor did BDS-I, a Kv3.4 channel antagonist (13) (see Fig. 2 and Table 2). Correolide is a novel nortriterpine from the Costa Rican tree Spachea correa that selectively binds to and blocks members of the Kv1 channel subfamily (14, 17). In four retinal arterioles, 10 ?M correolide inhibited the peak A-type K+ current by >70% (Fig. 2 and Table 2). In accord with its slow-onset kinetics (14), and consistent with previous reports in cerebral arteriolar myocytes (2), the blocking effects of correolide on the Kv current developed slowly taking 5?10 min to reach maximal levels.
The above data support the idea that Kv1 channels may underlie the A-type K+ current in retinal arteriolar myocytes. The Kv1 channel subfamily consists of multiple members (Kv1.1?1.7), and several Kv1? subunits are known to display A-type properties, including Kv1.3, Kv1.4, and Kv1.7 (3). Moreover, the accessory subunit Kv?1.1 can induce rapid inactivation of delayed rectifier type Kv1 channels, including Kv1.1, Kv1.2, and Kv1.5, but not Kv1.6 (18). Since most Kv1 channels could potentially represent molecular candidates of the A-type K+ current in retinal arteriolar myocytes, we extended our pharmacological approach using known inhibitors that specifically target various members of this subfamily. Hongotoxin is a peptide from venom of the scorpion Centruroides limbatus, which is known to block homotetrameric Kv1.1, Kv1.2, and Kv1.3 channels (IC50s in 31?170 pM range) and also Kv1.6 with low affinity (IC50 = 6 nM) (23). In retinal arteriolar smooth muscle, 100 nM recombinant hongotoxin did not suppress the A-type K+ current (Fig. 3, Table 3). To further eliminate a possible contribution by Kv1.2?1.3 homotetramers, we tested the effects of noxiustoxin, another scorpion-derived toxin that binds to and blocks these channels at low concentrations (IC50 values of 1 and 2 nM, respectively) (16). Kv1.7 is also potently blocked by noxiustoxin at nanomolar levels (IC50 of 18 nM) (8). Recombinant noxiustoxin (20 nM) had no effect on the A-type K+ current in retinal arterioles (Fig. 3, Table 3). Riluzole is a neuroprotective drug that modulates Kv1.4 channels via the oxidation of a cysteine residue in the NH2-terminal inactivation ball (IC50 of 70 ?M) (40). More recently, it has also been shown that this drug inhibits cloned Kv1.5 channels by preferentially binding to the inactivated and to the closed states of the channel (IC50 40 ?M) (1). Riluzole (100 ?M) inhibited the A-type K+ current by 32 ? 7% at +20 mV (Fig. 3, Table 3). To begin to isolate the possible contributions of Kv1.4 and Kv1.5 channels to the A-type K+ current, we investigated the effects of K+-free bathing medium and the anti-arrhythmic agent flecainide (Fig. 3, Table 3). Removal of extracellular K+ is known to suppress current through Kv1.4 channels but not Kv1.5 (30), and in retinal arteriolar myocytes, exposure to K+-free medium elicited no change in the peak A-type K+ current. Classically, flecainide has been used to discriminate Kv4-based A-type K+ currents (flecainide sensitive; IC50 ? 10 ?M) from those mediated by Kv1.4 channels (flecainide insensitive; IC50 = 700 ?M) (43). Similar to Kv4 channels, Kv1.5 is also known to be sensitive to flecainide at low micromolar concentrations (16), and application of 20 ?M flecainide inhibited the A-type K+ current in retinal arterioles by >80%.
Our pharmacological data suggest that the A-type K+ current in retinal arteriolar myocytes is mediated, at least in part, by Kv1.5-containing channels. To provide further evidence that Kv1.5 underlies the A-type K+ current, RT-PCR was initially used to screen for mRNAs encoding Kv1.4 and Kv1.5 in freshly isolated retinal arterioles. RNA samples were collected from 5 to 13 arterioles, and rat brain pial membrane was used as a positive control (6). Kv1.5 mRNA was consistently detected in retinal arterioles but Kv1.4 was not evident (Fig. 4A). Lack of Kv1.4 expression could not be attributed to primer design, since Kv1.4 transcript expression was verified in the brain using the same primer pairs. No products were detected in any of the minus-reverse transcriptase control experiments (no amplification controls). Immunofluorescence staining with commercially available polyclonal antibodies was also used to test for cell-specific expression of Kv1.4 and Kv1.5 in retinal arterioles embedded within retinal flatmount preparations. To ensure that the images collected originated from the arterioles, the retinas were counterstained with propidium iodide nuclear stain. First-order arterioles radiating from the optic disk were readily identified by their distinctive monolayer of smooth muscle cell nuclei (see, for example, Fig. 4B,i), which was absent on interdigitating venules. Endothelial cell nuclei could also be seen just below the focal plane of the arteriolar smooth muscle cells and orientated in a longitudinal direction (Fig. 4B,i). Consistent with the RT-PCR results, Kv1.4 could not be detected in retinal arterioles (Fig. 4B, i and ii). This was not due to a lack of antibody reactivity because intense staining was observed in the outer nuclear layer of the retina (Fig. 4B, iii and iv), which was not apparent in flatmounts exposed to only secondary antibodies (2?Control; Fig. 4B,v). In contrast to Kv1.4, strong immunoreactivity was detected for Kv1.5 in retinal arterioles localized to the plasma membrane of the arteriolar smooth muscle cells (Fig. 4B, vi and vii). Anti-Kv1.5 staining was not visible in endothelial cells (data not shown), and 2?Controls were negative (Fig. 4B, viii and ix).
Recently, neutralizing antibody approaches have been successfully used to demonstrate the contribution of specific Kv? subunits to observed macroscopic Kv currents in native vascular smooth muscle cells (44). Conventional whole cell recordings were used to dialyze anti-Kv1.4 and anti-Kv1.5 antibodies into the cytosol of individual retinal arteriolar myocytes. The anti-Kv1.5 antibody employed has previously been demonstrated to specifically block Kv1.5 currents when applied intracellularly to mesenteric artery smooth muscle cells (26). Since gene and protein expression for Kv1.4 could not be detected in retinal arterioles, anti-Kv1.4 antibody was chosen as a negative control. Without antibody in the pipette, the A-type current was stable for periods >20 min. In the presence of anti-Kv1.5 antibody in the pipette solution, the A-type current was inhibited within 2?5 min after establishment of the whole cell configuration by 64 ? 11% (Fig. 5). Application of anti-Kv1.4 antibody in the pipette solution did not have any effect on the A-type current even after 20 min of recording (Fig. 5). Anti-Kv1.5 antibody also inhibited the sustained component of the net outward current (Fig. 5A; current densities were 44.1, 52.2, and 22.3 pA/pF for controls, anti-Kv1.4 antibody, and anti-Kv1.5 antibody, respectively; P < 0.05 for anti-Kv1.5 vs. other groups), suggesting that some of the delayed rectifier channels in this tissue may also contain Kv1.5 subunits.
The above results strongly suggest that Kv1.5 constitutes a principal molecular component underlying the A-type current in retinal arteriolar smooth muscle. In heterologous expression systems, Kv1.5 classically gives rise to delayed rectifier, not A-type currents. However, co-expression of Kv1.5 with Kv?1 or Kv?3 accessory subunits can transform the current into a rapidly inactivating A-type current (18, 25). To examine whether this mechanism might be of relevance in retinal arteriolar myocytes, we initially sought evidence for mRNA transcript expression of Kv?1 and Kv?3 subunits in freshly isolated retinal arterioles. Kv?1 product was consistently detected; however, no definite expression of Kv?3 mRNA was observed (Fig. 6A). Kv?3 was detected in rat brain pial membrane (Fig. 6A). Kv?1 protein expression in retinal arterioles was confirmed by immunohistochemistry of retinal flatmount preparations (Fig. 6B). At low magnifications, Kv?1-associated fluorescence was most intense in retinal astrocytes, particularly in the end feet surrounding blood vessels (Fig. 6B,i). At higher magnifications, Kv?1 was also observed to be specifically localized to the plasma membrane of retinal arteriolar myocytes (Fig. 6B, ii and iii), but was absent in the endothelial cell layer (Fig. 6B,iv).
This study is the first to examine the molecular composition of the Kv channels underlying the A-type K+ current in vascular smooth muscle cells of the peripheral systemic circulation. Similar studies have previously been undertaken to identify the molecular nature of the Kv channels underlying delayed rectifier currents in arterial myocytes. The general consensus from these studies is that slowly inactivating tetraethylammonium (TEA)-insensitive delayed rectifier currents arise through heterotetrameric complexes of Kv1.2 with Kv1.4 or Kv1.5 channels associated with a Kv?1 subunit, whereas TEA-sensitive delayed rectifier currents most likely contain at minimum Kv2.1 ?-subunits (9). From the present study it appears that the molecular identity of the A-type K+ current in retinal arteriolar smooth muscle closely resembles the TEA-insensitive delayed rectifier current observed in other types of arterial smooth muscle in that Kv1.5 appears to be a major component and is probably co-assembled with Kv?1 subunits.
Previous studies concerned with the molecular composition of Kv channels in arterial smooth muscle have begun by systematically screening Kv channel gene expression. Although this approach represents a logical starting point for identifying potential candidate ?-subunits, data interpretation is often confounded by the multitude of Kv channel isoforms expressed when compared with the number of Kv current components observed (9). For this reason we initially adopted a pharmacological strategy to narrow down likely Kv channel components underlying the A-type K+ current. This type of approach has only recently become feasible with the emergence of an increasing number of drugs and toxins that selectively target specific members of the Kv channel superfamily. A caveat to this kind of approach, however, is that some of the drugs and toxins are only active against homotetrameric Kv channels (35), and hence, a lack of effect does not necessarily exclude the possibility that the subunit(s) of interest forms part of heterotetrameric Kv complex. Thus, although we can confidently exclude Kv1.1/2/3/4/6 and 7 homotetrameric channels as contributing to the A-type K+ current in retinal arteriolar smooth muscle, these subunits could still play a crucial role in the observed currents by forming heterotetrameric complexes with Kv1.5. In fact, we have previously shown that the A-type K+ current in retinal arterioles is partially suppressed by low levels of TEA (10 mM) (28), yet it is established that Kv1.5 homotetrameric channels are resistant to the actions TEA (16). This strongly suggests that the channels underlying the A-type K+ current in retinal arteriolar smooth muscle cells are heterotetrameric complexes of Kv1.5 with TEA-sensitive Kv1? subunits such as Kv1.1 and Kv1.6 (8, 16).
A distinctive feature of A-type K+ currents bestowed by Kv1.5 channels assembled with Kv?1 subunits is the voltage for half-inactivation, which at ?31.5 mV (18) is around 20?30 mV more positive than A-type K+ currents mediated by the majority of ?-subunits in other Kv subfamilies (8). We have previously reported a half-inactivation voltage for the A-type K+ current in the retinal arteriolar smooth muscle of ?28.3 mV (28), and this adds further weight to the notion that Kv1.5-containing channels co-assembled with Kv?1 subunits form the basis of this current. There is, nonetheless, a discrepancy in the relation to the time constant for recovery from inactivation. A-type K+ currents mediated by the Kv1 family of channels recover relatively slowly from inactivation (3), and for Kv1.5/Kv?1 channels, the time constant is 2.4 s (18). The time constant for recovery from inactivation of the A-type K+ current in retinal arteriolar myocytes is 118 ms (28), which is more reminiscent of Kv4-derived currents. Few studies have specifically addressed those factors involved in modulating recovery from inactivation for Kv1 channels, although it is notable that Ca2+/calmodulin-dependent kinase (CAMKII) phosphorylation of an NH2 terminal residue of Kv1.4 leads to an accelerated recovery from N-type inactivated states (34). Retinal arteriolar smooth muscle cells exhibit a very high level of spontaneous subcellular Ca2+ signaling activity (11), and this would be consistent with the idea that CAMKII-dependent regulation of Kv1.5/Kv?1 may be more relevant in this tissue than in heterologous expression systems where the biophysical properties these channels have been previously determined. Although it remains unclear if the phosphorylation status of Kv? subunits modifies recovery from N-type inactivation of Kv1 channels, protein kinase A phosphorylation of serine-24 in the NH2 terminus of Kv?1.3 is known to alter the kinetics of inactivation of Kv1.5 (24).
By using a multi-faceted approach, we have identified Kv1.5 as being a principal component underlying the A-type K+ current in retinal arteriolar smooth muscle cells. It seems likely that Kv1.5 forms a heterotetrameric complex with other TEA-sensitive Kv1? subunits and that the transient nature of the current arises through association of the channels with Kv?1 subunits. Intriguingly, the molecular components underlying the A-type K+ current in retinal arterioles appear similar to those that mediate delayed rectifier K+ currents in other types of vascular smooth muscle. Although the physiological significance of these observations warrants further investigation, it is evident that we should not necessarily consider A-type and delayed rectifier K+ currents in vascular smooth muscle as separate Kv current components. Instead, these currents may be derived from Kv channels with similar ?-subunit compositions but reflect opposing ends of a spectrum of kinetically distinct currents that vary according to their degree of regulation by Kv? subunits.
We thank The Juvenile Diabetes Research Foundation (US), Fight for Sight (UK) and The Wellcome Trust for financial support.
|1.||Ahn, HS. , Choi JS, Choi BH, Kim MJ, Rhie DJ, Yoon SH, Jo YH, Kim MS, Sung KW, Hahn SJ. Inhibition of the cloned delayed rectifier K+ channels, Kv1.5 and Kv3.1, by riluzole. Neuroscience 133: 1007?1019, 2005. [pmid: 15964489]|
|2.||Albarwani, S. , Nemetz LT, Madden JA, Tobin AA, England SK, Pratt PF, Rusch NJ. Voltage-gated K+ channels in rat small cerebral arteries: molecular identity of the functional channels. J Physiol 551: 751?763, 2003. [pmid: 12815189]|
|3.||Amberg, GC. , Koh SD, Imaizumi Y, Ohya S, Sanders KM. A-type potassium currents in smooth muscle. Am J Physiol Cell Physiol 284: C583?C595, 2003. [pmid: 12556357]|
|4.||Chen, TT. , Luykenaar KD, Walsh EJ, Walsh MP, Cole WC. Key role of Kv1 channels in vasoregulation. Circ Res 99: 53?60, 2006. [pmid: 16741158]|
|5.||Cheong, A. , Dedman AM, Beech DJ. Expression and function of native potassium channel [K (V)?1] subunits in terminal arterioles of rabbit. J Physiol 534: 691?700, 2001. [pmid: 11483700]|
|6.||Cheong, A. , Dedman AM, Xu SZ, Beech DJ. KV?1 channels in murine arterioles: differential cellular expression and regulation of diameter. Am J Physiol Heart Circ Physiol 281: H1057?H1065, 2001. [pmid: 11514271]|
|7.||Clement-Chomienne, O. , Ishii K, Walsh MP, Cole WC. Identification, cloning and expression of rabbit vascular smooth muscle Kv1.5 and comparison with native delayed rectifier K+ current. J Physiol 515: 653?667, 1999. [pmid: 10066895]|
|8.||Coetzee, WA. , Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de ME, Rudy B. Molecular diversity of K+ channels. Ann NY Acad Sci 868: 233?285, 1999. [pmid: 10414301]|
|9.||Cox, RH. Molecular determinants of voltage-gated potassium currents in vascular smooth muscle. Cell Biochem Biophys 42: 167?195, 2005. [pmid: 15858231]|
|10.||Curtis, TM. , Scholfield CN. Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle. J Physiol 532: 609?623, 2001. [pmid: 11313433]|
|11.||Curtis, TM. , Tumelty J, Dawicki J, Scholfield CN, McGeown JG. Identification and spatiotemporal characterization of spontaneous Ca2+ sparks and global Ca2+ oscillations in retinal arteriolar smooth muscle cells. Invest Ophthalmol Vis Sci 45: 4409?4414, 2004. [pmid: 15557449]|
|12.||Diochot, S. , Drici MD, Moinier D, Fink M, Lazdunski M. Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis. Br J Pharmacol 126: 251?263, 1999. [pmid: 10051143]|
|13.||Diochot, S. , Schweitz H, Beress L, Lazdunski M. Sea anemone peptides with a specific blocking activity against the fast inactivating potassium channel Kv3.4. J Biol Chem 273: 6744?6749, 1998. [pmid: 9506974]|
|14.||Felix, JP. , Bugianesi RM, Schmalhofer WA, Borris R, Goetz MA, Hensens OD, Bao JM, Kayser F, Parsons WH, Rupprecht K, Garcia ML, Kaczorowski GJ, Slaughter RS. Identification and biochemical characterization of a novel nortriterpene inhibitor of the human lymphocyte voltage-gated potassium channel, Kv1.3. Biochemistry 38: 4922?4930, 1999. [pmid: 10213593]|
|15.||Fergus, DJ. , Martens JR, England SK. Kv channel subunits that contribute to voltage-gated K+ current in renal vascular smooth muscle. Pfl?gers Arch 445: 697?704, 2003.|
|16.||Grissmer, S. , Nguyen AN, Aiyar J, Hanson DC, Mather RJ, Gutman GA, Karmilowicz MJ, Auperin DD, Chandy KG. Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol Pharmacol 45: 1227?1234, 1994. [pmid: 7517498]|
|17.||Hanner, M. , Schmalhofer WA, Green B, Bordallo C, Liu J, Slaughter RS, Kaczorowski GJ, Garcia ML. Binding of correolide to K (v)1 family potassium channels. Mapping the domains of high affinity interaction. J Biol Chem 274: 25237?25244, 1999. [pmid: 10464244]|
|18.||Heinemann, SH. , Rettig J, Graack HR, Pongs O. Functional characterization of Kv channel ?-subunits from rat brain. J Physiol 493: 625?633, 1996. [pmid: 8799886]|
|19.||Hill, CE. , Eade J, Sandow SL. Mechanisms underlying spontaneous rhythmical contractions in irideal arterioles of the rat. J Physiol 521: 507?516, 1999. [pmid: 10581319]|
|20.||Iida, H. , Jo T, Iwasawa K, Morita T, Hikiji H, Takato T, Toyo-Oka T, Nagai R, Nakajima T. Molecular and pharmacological characteristics of transient voltage-dependent K+ currents in cultured human pulmonary arterial smooth muscle cells. Br J Pharmacol 146: 49?59, 2005. [pmid: 15937516]|
|21.||Isacoff, EY. , Jan YN, Jan LY. Putative receptor for the cytoplasmic inactivation gate in the Shaker K+ channel. Nature 353: 86?90, 1991. [pmid: 1881453]|
|22.||Kerr, PM. , Clement-Chomienne O, Thorneloe KS, Chen TT, Ishii K, Sontag DP, Walsh MP, Cole WC. Heteromultimeric Kv-Kv1.5 channels underlie 4-aminopyridine-sensitive delayed rectifier K+ current of rabbit vascular myocytes. Circ Res 89: 1038?1044, 2001. [pmid: 11717161]|
|23.||Koschak, A. , Bugianesi RM, Mitterdorfer J, Kaczorowski GJ, Garcia ML, Knaus HG. Subunit composition of brain voltage-gated potassium channels determined by hongotoxin-1, a novel peptide derived from Centruroides limbatus venom. J Biol Chem 273: 2639?2644, 1998. [pmid: 9446567]|
|24.||Kwak, YG. , Hu N, Wei J, George AL Jr, Grobaski TD, Tamkun MM, Murray KT. Protein kinase A phosphorylation alters Kv?1.3 subunit-mediated inactivation of the Kv1.5 potassium channel. J Biol Chem 274: 13928?13932, 1999. [pmid: 10318802]|
|25.||Leicher, T. , Bahring R, Isbrandt D, Pongs O. Coexpression of the KCNA3B gene product with Kv1.5 leads to a novel A-type potassium channel. J Biol Chem 273: 35095?35101, 1998. [pmid: 9857044]|
|26.||Lu, Y. , Hanna ST, Tang G, Wang R. Contributions of Kv1 R.2, Kv1.5 Kv2.1. subunits to the native delayed rectifier K+ current in rat mesenteric artery smooth muscle cells. Life Sci 71: 1465?1473, 2002. [pmid: 12127166]|
|27.||Lu, Y. , Zhang J, Tang G, Wang R. Modulation of voltage-dependent K+ channel current in vascular smooth muscle cells from rat mesenteric arteries. J Membr Biol 180: 163?175, 2001. [pmid: 11318099]|
|28.||McGahon, MK. , Dawicki JM, Scholfield CN, McGeown JG, Curtis TM. A-type potassium current in retinal arteriolar smooth muscle cells. Invest Ophthalmol Vis Sci 46: 3281?3287, 2005. [pmid: 16123430]|
|29.||Nelson, MT. , Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799?C822, 1995.|
|30.||Pardo, LA. , Heinemann SH, Terlau H, Ludewig U, Lorra C, Pongs O, Stuhmer W. Extracellular K+ specifically modulates a rat brain K+ channel. Proc Natl Acad Sci USA 89: 2466?2470, 1992. [pmid: 1549610]|
|31.||Plane, F. , Johnson R, Kerr P, Wiehler W, Thorneloe K, Ishii K, Chen T, Cole W. Heteromultimeric Kv1 channels contribute to myogenic control of arterial diameter. Circ Res 96: 216?224, 2005. [pmid: 15618540]|
|32.||Pongs, O. , Leicher T, Berger M, Roeper J, Bahring R, Wray D, Giese KP, Silva AJ, Storm JF. Functional and molecular aspects of voltage-gated K+ channel beta subunits. Ann NY Acad Sci 868: 344?355, 1999. [pmid: 10414304]|
|33.||Rettig, J. , Heinemann SH, Wunder F, Lorra C, Parcej DN, Dolly JO, Pongs O. Inactivation properties of voltage-gated K+ channels altered by presence of ?-subunit. Nature 369: 289?294, 1994. [pmid: 8183366]|
|34.||Roeper, J. , Lorra C, Pongs O. Frequency-dependent inactivation of mammalian A-type K+ channel KV1.4 regulated by Ca2+/calmodulin-dependent protein kinase. J Neurosci 17: 3379?3391, 1997. [pmid: 9133364]|
|35.||Russell, SN. , Overturf KE, Horowitz B. Heterotetramer formation and charybdotoxin sensitivity of two K+ channels cloned from smooth muscle. Am J Physiol Cell Physiol 267: C1729?C1733, 1994.|
|36.||Sanguinetti, MC. , Johnson JH, Hammerland LG, Kelbaugh PR, Volkmann RA, Saccomano NA, Mueller AL. Heteropodatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol 51: 491?498, 1997. [pmid: 9058605]|
|37.||Scholfield, CN. , Curtis TM. Heterogeneity in cytosolic calcium regulation among different microvascular smooth muscle cells of the rat retina. Microvasc Res 59: 233?242, 2000. [pmid: 10684729]|
|38.||Standen, NB. , Quayle JM. K+ channel modulation in arterial smooth muscle. Acta Physiol Scand 164: 549?557, 1998. [pmid: 9887977]|
|39.||Thorneloe, KS. , Chen TT, Kerr PM, Grier EF, Horowitz B, Cole WC, Walsh MP. Molecular composition of 4-aminopyridine-sensitive voltage-gated K+ channels of vascular smooth muscle. Circ Res 89: 1030?1037, 2001. [pmid: 11717160]|
|40.||Xu, L. , Enyeart JA, Enyeart JJ. Neuroprotective agent riluzole dramatically slows inactivation of Kv1.4 potassium channels by a voltage-dependent oxidative mechanism. J Pharmacol Exp Ther 299: 227?237, 2001. [pmid: 11561084]|
|41.||Yamamoto, Y. , Fukuta H, Nakahira Y, Suzuki H. Blockade by 18?-glycyrrhetinic acid of intercellular electrical coupling in guinea-pig arterioles. J Physiol 511: 501?508, 1998. [pmid: 9706026]|
|42.||Yamazaki, J. , Kitamura K. Intercellular electrical coupling in vascular cells present in rat intact cerebral arterioles. J Vasc Res 40: 11?27, 2003. [pmid: 12644722]|
|43.||Yeola, SW. , Snyders DJ. Electrophysiological and pharmacological correspondence between Kv4.2 current and rat cardiac transient outward current. Cardiovasc Res 33: 540?547, 1997. [pmid: 9093524]|
|44.||Zhou, BY. , Ma W, Huang XY. Specific antibodies to the external vestibule of voltage-gated potassium channels block current. J Gen Physiol 111: 555?563, 1998. [pmid: 9524138]|
Keywords: voltage-dependent potassium channels, vascular, Kv? subunits, pharmacology, microvessels.
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