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(Circulation Research. 2001;89:1030.)
© 2001 American Heart Association, Inc.

Cellular Biology

Molecular Composition of 4-Aminopyridine-Sensitive Voltage-Gated K⁺ Channels of Vascular Smooth Muscle

Kevin S. Thorneloe, Tim T. Chen, Paul M. Kerr, Elizabeth F. Grier, Burton Horowitz, William C. Cole, Michael P. Walsh

From the Smooth Muscle Research Group and Canadian Institutes of Health Research (CIHR) Group in Regulation of Vascular Contractility (K.S.T., T.T.C., P.M.K., E.F.G., W.C.C., M.P.W.), University of Calgary, Alberta, Canada, and the Department of Physiology, University of Nevada School of Medicine (B.H.), Reno, Nev.

Correspondence to Dr William C. Cole, Smooth Muscle Research Group, University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta T2N 4N1, Canada. E-mail wcole{at}ucalgary.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Voltage-gated K⁺ channels (Kv) play a critical role in regulating arterial tone by modulating the membrane potential of vascular smooth muscle cells. Our previous work demonstrated that the dominant 4-aminopyridine (4-AP)-sensitive, delayed rectifier Kv current of rabbit portal vein (RPV) myocytes demonstrates similar 4-AP sensitivity and biophysical properties to Kv1-containing channels. To identify the molecular constituents underlying the 4-AP-sensitive Kv current of vascular myocytes, we characterized the expression pattern of Kv1 subunits and their modulatory Kvß subunits in RPV. The mRNAs encoding pore-forming subunits Kv1.2, Kv1.4, and Kv1.5 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas Kv1.1, Kv1.3, and Kv1.6 transcripts were undetectable. Kvß1.1, ß1.2, ß1.3, ß2.1, and ß2.2 messages were expressed, whereas Kvß3.1 and ß4 mRNAs were undetected by RT-PCR. Kv1.2, Kv1.4, Kv1.5, Kvß1.2, ß1.3, and ß2.1 proteins were detected in RPV by Western blotting and/or immunocytochemistry of freshly isolated myocytes. We provide the first evidence, from coimmunoprecipitation studies, for the formation of heteromultimeric Kv channel complexes composed of Kv1.2, Kv1.5, and Kvß1.2 subunits in vascular smooth muscle.

Key Words: vascular smooth muscle • Kv1.5 • Kv1.2 • Kvß subunits • voltage-gated K⁺ channel

	Introduction

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Control of arterial diameter and peripheral vascular resistance is dependent on the development of tone by vascular smooth muscle cells. Membrane potential of vascular myocytes is a major determinant of tone development, as it controls the magnitude of steady-state Ca²⁺ influx across the membrane through voltage-gated Ca²⁺ channels.¹ The level of membrane potential is the result of a dynamic balance between opposing inward (depolarizing) and outward (hyperpolarizing) ionic currents.¹ Hyperpolarizing K⁺ current in vascular myocytes is primarily due to the activity of four classes of K⁺ channels: inward rectifier, ATP-sensitive, large-conductance Ca²⁺-activated, and voltage-gated K⁺ (Kv) channels.^1,2 Alterations in K⁺ channel activity modulate the level of membrane potential and, thereby, cytosolic free Ca²⁺ concentration and contractile filament activation.^1,2

Voltage-dependent outward current of vascular myocytes is due to at least three components: a slowly inactivating 4-aminopyridine (4-AP)-sensitive delayed rectifier (K_DR) current^1–6; a 4-AP-insensitive outward current⁵; and a rapidly inactivating, 4-AP-sensitive transient A-type current (K_TO). ⁶ 4-AP-sensitive K_DR channels are thought to contribute to the control of membrane potential in vascular myocytes.^1–3,7 This view is supported by (1) electrophysiological evidence that K_DR channels are active at voltages consistent with the membrane potential of vascular myocytes^1–5 and (2) pharmacological evidence indicating that submillimolar 4-AP inhibits K_DR currents of vascular myocytes^1–5 and elicits depolarization and contraction of intact blood vessels.^1,2,7 Moreover, the activity of vascular 4-AP-sensitive K_DR channels is modulated by agonists that signal through protein kinases A and C^4,5,8,9 and may contribute to the physiological regulation of arterial diameter by locally released or circulating vasoactive factors.

The molecular identity of vascular 4-AP-sensitive K_DR channels is not established, but recent reports describe the expression of several transcripts encoding Kv channel subunits in vascular tissues.^10–16 Kv channels are composed of pore-forming Kv and modulatory Kvß subunits. Kv proteins with six transmembrane segments are encoded by nine related families, Kv1 to Kv9, within the superfamily of Kv channel genes. Kv1 to Kv4 proteins assemble to form homotetrameric channels, or coassemble with members of the same family to produce heterotetrameric channel complexes.¹⁷ Subunit proteins of the Kv5 to Kv9 families do not form functional channels, but they coassemble with Kv2 or Kv3 subunits into functional complexes with unique properties.^17–19 Additional functional diversity and appropriate trafficking to the membrane are obtained by association of the Kv1 subunits with Kvß subunits.^20–23 Four genes encoding Kvß subunits, Kvß1 to 4, as well as splice variants of the Kvß1 and ß2 genes, have been identified.^20–23 The assembly pattern of Kv and ß subunits provides a mechanism for obtaining diverse populations of channels with unique properties for appropriate cellular function.

Approximately 70% of the K_DR current, as well as the K_TO current, of rabbit portal vein (RPV) myocytes is inhibited by 4-AP with an IC₅₀ in the submillimolar range.^{4,5,8,9,11,24} This level of 4-AP sensitivity and the biophysical properties of the currents are consistent with those previously identified for Kv1-containing,^{11,17,22–26} but not Kv2- to 4-containing,^26–31 Kv channels. The properties of whole-cell and unitary currents due to expression of Kv1.5 cloned from RPV closely mimicked those of native RPV K_DR channels.¹¹ However, homomultimeric Kv1.5 channels do not share functional identity with vascular K_DR because (1) differences in rate and voltage dependence of inactivation are apparent¹¹; (2) RPV Kv1.5 channels in inside-out membrane patches of mouse L cells or human embryonic kidney 293 (HEK) cells do not exhibit modulation by purified protein kinase A in the presence of ATP³²; and (3) 4-AP treatment is associated with a positive shift in the voltage dependence of activation of native K_DR, but not Kv1.5 channels, as described in another study published in this issue of Circulation Research by Kerr et al.²⁴ This lack of functional identity indicates that Kv1.5 cannot be the only Kv1 subunit contributing to K_DR channels of vascular myocytes.

In this study, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry were performed to identify the expression profile of Kv1 and Kvß subunits in RPV. Furthermore, the pattern of subunit coassembly was assessed by coimmunoprecipitation. We demonstrate expression in RPV of Kv1.2, Kv1.4, and Kv1.5 pore-forming subunits, as well as Kvß1 and ß2 modulatory subunits. Additionally, we present the first direct evidence that Kv1.2 and Kv1.5 coassemble to form heteromultimeric Kv channels in vascular myocytes.

	Materials and Methods

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Standard methods for RT-PCR, antibody production, immunocytochemistry, Western blotting, and immunoprecipitation were used. Full-length clones were prepared for Kv1.2, Kvß1.1, ß1.2, ß1.3, ß2.1, and ß2.2 of RPV via RT-PCR for expression in HEK cells. GenBank accession numbers are the following: for Kv1.2, AF284420; for Kvß1.1, AF131934; for ß1.2, AF131935; for ß1.3, AF131936; for ß2.1, AF247701; and for ß2.2, AF247702. Western blotting, immunocytochemistry, and immunoprecipitation were performed using commercial antibodies against Kv1.2, Kv1.4, and Kv1.6 (Upstate Biotechnology), as well as Kv1.5 (Alomone Laboratories). Novel peptide-directed rabbit polyclonal antibodies against RPV ß1.1, ß1.2, ß1.3, ß2.1, and ß2.2 raised in conjunction with Macromolecular Resources (Colorado State University, Fort Collins, Colo) were also used after affinity purification. The rabbit Kv1.2- and Kvß-subunit amino acid sequences and details of all methods used are included in the online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
RT-PCR for Kv1 and Kvß Subunits of RPV
Specific primers for Kv1 subunits (Kv1.1 to 1.6) were used for RT-PCR of mRNA isolated from RPV. Rat or mouse brain mRNA was utilized as a positive control to confirm the function of all primers and integrity of each RT-PCR reaction performed. All PCR products were sequenced to confirm their identity. RT-PCR products of appropriate size for Kv1.2 (1500, 1418, 449, and 367 bp), Kv1.4 (188 bp), and Kv1.5 (923 and 591 bp) were obtained from RPV mRNA after 35 cycles of PCR (Figure 1). The RT-PCR product representing the full-length coding sequence of RPV Kv1.2 was cloned into pcDNA3 for use in subsequent heterologous expression experiments. The amino acid sequence of rabbit Kv1.2 exhibits a very high level of identity with Kv1.2 expressed in other species (online Figure 1, available at http://www.circresaha.org). Products for Kv1.1 (710 bp), Kv1.3 (653 bp), and Kv1.6 (1590 bp) were detected utilizing brain mRNA after 35 cycles, but were consistently not detected in RPV, even with a second round of PCR (total 70 cycles) (Figure 1).

View larger version (96K): [in this window] [in a new window]   Figure 1. Kv1 transcript expression by RPV. A, 710-bp product of Kv1.1 was detected by RT-PCR in rat brain (RB), but not in RPV, mRNA. B, 1500-, 1418-, 449-, and 367-bp products of Kv1.2 were detected in rat brain and RPV mRNA. C, 653-bp product of Kv1.3 was detected in mouse brain (MB), but not in RPV, mRNA. D, 188-bp product of Kv1.4 was detected in rat brain and RPV mRNA. E, 923- and 591-bp products of Kv1.5 were detected in RPV mRNA. F, 1590-bp product of Kv1.6 was detected in rat brain, but not in RPV, mRNA.

RT-PCR was performed to identify the Kvß subunits expressed in RPV. Products for Kvß1.1 (1206 bp), ß1.2 (1227 bp), ß1.3 (1260 bp), and ß2 (1102 and 1062 bp for Kvß2.1 and ß2.2, respectively) were obtained using RPV mRNA (Figure 2). RT-PCR products representing full-length coding sequences of each of these Kvß subunits were obtained for (1) sequence comparison with Kvß subunits expressed by other species, (2) generation of peptide-directed antibodies, and (3) heterologous expression experiments. Rabbit Kvß subunit amino acid sequences demonstrated a high level of identity, except within the differentially spliced N-termini (online Figure 2), as previously described for Kvß subunits cloned from other species.^20,23 A novel ß2.2 splice variant that lacked 14 amino acids within the N-terminus of ß2.1 was identified in RPV, and was subsequently reported to be expressed in glioma and astrocytes.³³ We consistently noted the additional presence of a 435-bp product when using the ß2 primers (Figure 2A, arrow). This product was sequenced and found to correspond to the 3' end of the Kvß1 coding sequence. RT-PCR for ß3.1 and ß4 subunits yielded 1213-bp and 750-bp products, respectively, from brain mRNA, but not from RPV (Figure 2), even after 70 cycles of PCR.

View larger version (55K): [in this window] [in a new window]   Figure 2. Kvß transcript expression by RPV. A, 1206-, 1277-, and 1260-bp products for Kvß1.1, ß1.2, and ß1.3, as well as 1102- and 1062-bp products of Kvß2, were detected by RT-PCR in RPV mRNA. Arrow indicates 435-bp product of Kvß1.2 detected using Kvß2 primers. B, 1213-bp product of Kvß3.1 was detected in rat brain (RB), but not in RPV, mRNA. C, 750-bp product of Kvß4 was detected in mouse brain (MB), but not in RPV, mRNA.

Western Blotting for Kv and Kvß Subunits
Commercial antibodies were used to confirm the expression in RPV of Kv1.2, Kv1.4, and Kv1.5 subunit proteins. Western blotting experiments were conducted using HEK cells transfected with Kv1.2, Kv1.4, or Kv1.5 cDNA to confirm the specificity of the antibodies (online Figure 3, available at http://www.circresaha.org). Immunoreactive bands of appropriate molecular weight were apparent in transfected, but not untransfected, HEK cells. Multiple bands were observed for Kv1.4 and Kv1.5 and may represent products with different posttranslational modification.³⁴ Blotting of RPV protein with anti-Kv1.5 revealed an immunoreactive band in RPV with a molecular weight similar to that of the Kv1.5 products expressed in HEK cells, and the immunoreactivity was eliminated by competition with antigenic peptide (Figure 3). Blotting of RPV protein for Kv1.2 and Kv1.4 failed to detect specific immunoreactivity to these two subunits (data not shown). However, the presence of Kv1.2 subunit in RPV protein was subsequently detected by immunoprecipitation followed by Western blotting (see below).

View larger version (32K): [in this window] [in a new window]   Figure 3. Western blots for RPV Kv1.5, Kvß1.2, and ß2.1. A, Anti-Kv1.5 detected Kv1.5 (arrow) in RPV. Immunoreactive bands were absent after antigenic peptide preabsorption (+peptide). B, Kvß1.2 was detected in RPV. Immunoreactive bands were absent after preabsorption with respective antigenic peptide (+peptide). C, Kvß2.1 and ß2.2 were detected in RPV. Immunoreactive bands were absent after preabsorption with antigenic peptide (+peptide).

Three peptide-directed rabbit polyclonal antibodies were raised to specifically recognize the splice variants of the rabbit Kvß1 gene based on the unique N-terminal domains of ß1.1, ß1.2, and ß1.3 (online Figure 2). Two additional antibodies were raised, one specific for Kvß2.1 based on the 14-amino acid sequence present in ß2.1, but not ß2.2, and a Kvß2 antibody raised against a peptide based on the common region of the N-termini of Kvß2.1 and ß2.2, but not present in the Kvß1 subunits (online Figure 2). The antibodies were first utilized for Western blotting of HEK cells expressing the RPV Kvß subunit clones (Figure 4). No cross-reactivity of the Kvß antibodies was apparent (data not shown). Kvß1.1, ß1.2, and ß1.3 antibodies recognized proteins of 36 kDa in transfected, but not in untransfected, HEK cells, and the immunoreactivity was eliminated by competition with antigenic peptide (Figures 3 and 4). Small differences in molecular weight were apparent, as expected on the basis of the distinct amino acid sequences of these subunits (online Figure 2). Anti-Kvß2 recognized a band in lysates of both Kvß2.1- and ß2.2-transfected, but not in untransfected, HEK cells (Figure 4). These bands were at a lower molecular weight than those identified with anti-Kvß1, consistent with the different amino acid sequences of the Kvß2 splice variants and Kvß1 subunits (online Figure 2). Western blotting with anti-Kvß2.1 detected a band of 33 kDa in HEK cells expressing Kvß2.1, consistent with the molecular weight observed with anti-Kvß2 (Figure 4). However, an identical band was also observed with anti-Kvß2.1 in HEK cells transfected with Kvß2.2, and in untransfected HEK cells (Figure 4) that were reported to lack endogenous Kvß expression.³⁵ Preabsorbing anti-Kvß2.1 with antigenic peptide eliminated the immunoreactive band in extracts of cells transfected with Kvß2.1, as well as the band in Kvß2.2-transfected and untransfected HEK cell extract (Figures 3 and 4). RT-PCR and PCR product sequencing confirmed the endogenous expression of both Kvß2 splice variants in untransfected HEK cells (data not shown). This Kvß2 subunit expression by HEK cells may reflect differences in passage number or culture conditions compared with those used previously.³⁵

View larger version (35K): [in this window] [in a new window]   Figure 4. Western blotting for Kvß subunits of HEK cells. Anti-Kvß1.1 (A), anti-ß1.2 (B), anti-ß1.3 (C), anti-ß2 (D), and anti-ß2.1 (E) immunoreactive bands (arrows) were detected in protein of transfected, but not untransfected, HEK cells, with the exception of the band detected in HEK cells with anti-Kvß2.1 (E). E, Right panel, Lack of anti-ß2.1 immunoreactive bands in HEK cells transfected with Kvß2.1 or ß2.2 and untransfected HEK cells after preabsorption with respective antigenic peptide.

Kvß1.2 and ß2.1 proteins were detected by Western blotting of RPV lysate with antibodies to Kvß1.2 and ß2.1, respectively. Immunoreactive bands corresponding to the proteins identified in transfected HEK cells were observed and eliminated by preabsorption with the respective antigenic peptides (Figure 3). Western blotting with antibodies to Kvß1.1, ß1.3, and ß2 did not demonstrate specific immunoreactivity in RPV protein extracts. This may be due to the relatively low titer of these three antibodies observed in transfected HEK cells (data not shown).

Immunocytochemistry of RPV Kv1 and Kvß Subunits
We previously detected expression of Kv1.5 in isolated RPV myocytes by immunocytochemistry.¹¹ In this study, we demonstrate immunolabeling of myocytes with antibodies to Kv1.2, Kv1.4, and the Kvß1.2 and ß1.3 subunits. The specificity of the monoclonal Kv1.2 and Kv1.4 antibodies was first determined: HEK cells were cotransfected with cDNAs encoding the subunits and green fluorescent protein (GFP), the latter a marker of successfully transfected cells. The Kv1.2 and Kv1.4 antibodies recognized the expression of the subunits, as indicated by the consistent correlation of Cy3 secondary antibody immunofluorescence with GFP fluorescence in the HEK cells and lack of cross-reactivity of anti-Kv1.2 and anti-Kv1.4 with Kv1.4- and Kv1.2-transfected cells, respectively (online Figure 4, available at http://www.circresaha.org). On the basis of these control experiments, the antibodies were then used to identify the expression of Kv1.2 and Kv1.4 protein in RPV myocytes (Figure 5). Immunofluorescence was not detected in cells exposed to secondary antibody alone (Figure 5).

View larger version (57K): [in this window] [in a new window]   Figure 5. Immunocytochemistry for Kv1 and Kvß of RPV myocytes. Isolated myocytes were exposed to monoclonal anti-Kv1.2 (A), monoclonal anti-Kv1.4 (B), secondary antibody alone (C), anti-ß1.2 (D), anti-ß1.2 plus antigenic peptide (E), anti-ß1.3 (F), and anti-ß1.3 plus antigenic peptide (G). Fluorescence micrograph in panel C was obtained using an identical exposure time as for panels A and B. Similar exposures were used for panels D and F as in panels E and G, respectively.

The function of our Kvß antibodies for immunocytochemical detection of subunit expression was assessed. The Kvß1.2, ß1.3, and ß2 antibodies recognized Kvß1.2, ß1.3, and ß2.1/ß2.2 expression, respectively, in HEK cells; GFP fluorescence and immunofluorescence were correlated and the immunolabeling was eliminated by preabsorption with antigenic peptide (Figure 6). The Kvß1.1 and ß2.1 antibodies were unable to identify ß1.1 or ß2.1 expression, respectively, and were not used in subsequent experiments on RPV myocytes. Exposure of RPV myocytes to Kvß1.2 and ß1.3 antibodies resulted in intense immunofluorescence that was blocked by peptide preabsorption (Figure 5). However, the anti-Kvß2 immunostaining was not blocked by peptide preabsorption. This may be due to low antibody titer for identification of the subunit in HEK cells coupled with high background fluorescence and/or a low level of Kvß2 protein expression in the myocytes compared with the overexpression in HEK cells.

View larger version (80K): [in this window] [in a new window]   Figure 6. Immunocytochemistry of Kvß of transfected HEK cells. Corresponding GFP fluorescence and immunofluorescence of HEK cells were transfected and labeled with Kvß1.2 and anti-Kvß1.2 (A and B), Kvß1.2 and anti-Kvß1.2+antigenic peptide (C and D), Kvß1.3 and anti-Kvß1.3 (E and F), Kvß1.3 and anti-KvB1.3+peptide (G and H), Kvß2.1 and anti-Kvß2 (I and J), Kvß2.2 and anti-Kvß2 (K and L), and Kvß2.1 and anti-Kvß2+peptide (M and N), respectively.

Coimmunoprecipitation of Kv Subunits From RPV
The results of the preceding experiments indicated the expression of Kv1.2, Kv1.4, Kv1.5, Kvß1.2, ß1.3, and ß2.1 transcripts and proteins in RPV. These subunits were reported to coassemble in other cell types,^17,20,21 but whether similar heteromultimeric channels are present in smooth muscle has not been determined, although data consistent with this possibility exist.³⁶ The ability of the Kv1.2, Kv1.5, and Kvß1.2 antibodies to immunoprecipitate protein from lysates of HEK cells was confirmed by Western blotting with these antibodies (Figure 7). Specific subunit bands were not apparent in untransfected HEK cell lysates immunoprecipitated with the antibodies. Additionally, no cross-reactivity of the Kv1.2 and Kv1.5 antibodies was apparent; anti-Kv1.5 did not immunoprecipitate Kv1.2 subunits from HEK cells expressing Kv1.2, and anti-Kv1.2 did not immunoprecipitate Kv1.5 subunits from HEK cells expressing Kv1.5 (Figures 7B and 7D). Anti-Kvß1.1, -ß1.3, -ß2, and -ß2.1 failed to immunoprecipitate subunit protein from transfected HEK cells and were not used for analysis of RPV protein. However, bands of appropriate molecular weight for Kv1.2, Kv1.5, and Kvß1.2 were identified in Western blots of immunoprecipitates of RPV protein obtained using Kv1.2, Kv1.5, and Kvß1.2 antibodies, respectively (Figure 8). The bands for Kv1.2 and Kv1.5 were not observed when RPV protein was immunoprecipitated with anti-Kv1.6 (Figure 8), which is not expressed in RPV (Figure 1). Significantly, immunoprecipitation of RPV protein with anti-Kv1.2 or anti-Kvß1.2, followed by blotting with anti-Kv1.5, detected the presence of Kv1.5 in both immunoprecipitates, and blotting with anti-Kvß1.2 revealed the presence of this subunit in the immunoprecipitate obtained using anti-Kv1.5 (Figure 8).

View larger version (31K): [in this window] [in a new window]   Figure 7. Immunoprecipitation of Kv and Kvß from HEK cell lysate. A, Kv1.2 (arrow) in an anti-Kv1.2 blot of anti-Kv1.2 immunoprecipitate of Kv1.2-Kv1.5-cotransfected, but not untransfected, HEK cell lysate. B, Kv1.2 (arrow) in anti-Kv1.2 blot of anti-Kv1.2, but not anti-Kv1.5, immunoprecipitate of Kv1.2-transfected HEK cell lysate. C, Kv1.5 (arrow) in anti-Kv1.5 blot of anti-Kv1.5 and anti-Kv1.2 immunoprecipitates of Kv1.2 and Kv1.5 cotransfected, but not untransfected (immunoprecipitation with anti-Kv1.5), HEK cell lysate. D, Kv1.5 (arrow) in anti-Kv1.5 blot of anti-Kv1.5, but not anti-Kv1.2, immunoprecipitate of Kv1.5-transfected HEK cell lysate. E, Kvß1.2 (arrow) in anti-Kvß1.2 blot of anti-Kvß1.2 immunoprecipitate of Kv1.2 and Kvß1.2-cotransfected, but not untransfected, HEK cell lysate.

View larger version (41K): [in this window] [in a new window]   Figure 8. Association of Kv1 and Kvß of RPV. A, Kv1.2 (arrow) in anti-Kv1.2 blot of anti-Kv1.2, but not anti-Kv1.6, immunoprecipitates of RPV protein. B, Kv1.5 (arrows) in anti-Kv1.5 blots of anti-Kv1.5, anti-Kvß1.2, and anti-Kv1.2, but not anti-Kv1.6, immunoprecipitates of RPV protein. C, Kvß1.2 (arrow) in anti-Kvß1.2 and anti-Kv1.5 immunoprecipitates of RPV protein.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study provides the first comprehensive examination of the expression of Kv1- and Kvß-subunit mRNAs and proteins, the first evidence for the expression of Kvß-subunit proteins, and the novel finding of the heteromultimeric association of Kv1 and Kvß proteins in vascular smooth muscle cells. This subunit association may be predicted to produce channels that possess functional properties that are distinct from homomultimeric channels, an issue that is addressed in the study by Kerr et al.²⁴ The molecular biological, biochemical, and immunocytochemical data presented in this study indicate that Kv1.2 and Kv1.5 are the only delayed rectifier-type Kv1 pore-forming subunits expressed by RPV myocytes and that these subunits associate to form heteromultimeric Kv channels. In the study by Kerr et al,²⁴ we present complementary evidence that whole-cell currents due to heteromultimeric, but not homomultimeric, channels composed of Kv1.2 and Kv1.5 possess functional identity with native 4-AP-sensitive K_DR channels of RPV.²⁴ The combination of these observations provides compelling evidence that the dominant 4-AP-sensitive K_DR channel complex of RPV vascular myocytes is a heteromultimer consisting of Kv1.2 and Kv1.5 subunits.

This study provides novel insights into vascular Kv channel subunit composition. Kv gene expression has been studied previously using vascular and nonvascular smooth muscle tissues, and distinct patterns of expression of Kv subunit transcripts were reported for vessels of different vascular beds and for different regions within the gastrointestinal tract.^{10–16,36–39} We have shown that vascular myocytes of RPV express mRNAs encoding Kv1.2, Kv1.4, and Kv1.5 pore-forming subunits, as well as the modulatory Kvß subunits ß1.1, ß1.2, ß1.3, ß2.1, and ß2.2. The expression of Kv1.2, Kv1.4, and Kv1.5 in RPV is consistent with previous reports indicating the presence of transcripts encoding these subunits in several smooth muscle tissues.^{10–16,36–39} Moreover, transcripts encoding Kvß subunits were also shown to be expressed in smooth muscle of pulmonary artery, mesenteric artery, and gastrointestinal tract.^10,12,13,38 However, Kv1.1, Kv1.3, and Kv1.6 were reported to be expressed in pulmonary¹⁰ and/or mesenteric arteries,^12,13 but were not detected in RPV. Additionally, splice variants of Kvß1 (ie, ß1.1 and ß1.2) were found in smooth muscle cells of the gastrointestinal tract, but ß2.1 expression was not detected.³⁸ It seems likely that these differences reflect the presence of vessel- or tissue-specific patterns of Kv channel subunit expression. Differences in the voltage dependence of activation and inactivation, in the rates of inactivation, and in the pharmacology (eg, IC₅₀ of 4-AP inhibition) of K_DR current of myocytes derived from different smooth muscle tissues have been reported.^1,2,3–5,12 These differences may reflect vessel-specific expression of Kv1 and Kvß subunits, the differential assembly of Kv1 family and Kvß subunits into distinct populations of heteromultimeric channels containing varied mixtures of subunits, and/or the additional expression of Kv subunits of the Kv2 to 9 families.

However, substantive conclusions concerning Kv subunit expression in cells can only be achieved through the use of complementary techniques to identify the expression of mRNAs encoding the subunits and the subunit proteins themselves. In this study, RT-PCR was used to identify the expression in RPV of Kv1 and Kvß subunits at the mRNA level. This identification was followed by Western blotting and/or immunoprecipitation experiments to identify the expression of subunit proteins and to demonstrate the ability of the antibodies to identify proteins of appropriate molecular weight. Finally, immunocytochemistry was used to identify the specific expression of Kv subunit proteins by freshly isolated myocytes. Parallel control experiments were also conducted using a heterologous cell type expressing the subunits to confirm the specificity of the antibodies used. This complementary approach minimizes the possibility of artifactual identification of subunit expression due to (1) contaminating mRNA derived from cell types, such as neurons (eg, Kv1.1) and lymphocytes derived from the blood (eg, Kv1.3); (2) nonspecific immunolabeling by Kv subunit antibodies; and (3) the use of phenotypically distinct, cell-cultured myocytes. In addition, in the Kerr et al²⁴ study, we exploited a feature of 4-AP inhibition and lack of charybdotoxin sensitivity of native K_DR current to provide the first functional evidence for the presence of heteromultimeric channels composed of Kv1.2 and Kv1.5 subunits in vascular myocytes.

Vascular myocytes exhibit the following two components of Kv current that are suppressed by submillimolar 4-AP: rapidly inactivating K_TO and slowly inactivating K_DR currents.^1–6,11,24 We attribute the K_TO component to the expression of Kv1.4, but it is unlikely that this subunit contributes substantially to the K_DR current. Whole-cell currents, as a result of the expression of Kv1.4 in heterologous cell types,^25,40 exhibit several characteristics consistent with those of vascular K_TO currents.^1,6 For example, the inactivation kinetics are identical and the activation threshold of the K_TO current at -65 mV⁶ is consistent with that reported for Kv1.4 current,^25,40 but inconsistent with the properties of fast-inactivating Kv3 and Kv4 channels that activate at more positive voltages.^27–30 The view that Kv1.4 subunits have a minimal contribution to K_DR channel complex(es) of RPV myocytes is suggested by the ability of even a single Kv1.4 subunit to induce rapid inactivation of channels involving the association of Kv1.4 with Kv1.2 or Kv1.5 subunits.²⁵

This study provides evidence that Kv1.2 and Kv1.5 are the primary Kv1 subunits contributing to 4-AP-sensitive K_DR channels of RPV myocytes. Kv1.2 and Kv1.5 are the only Kv1 subunits expressed in RPV that display delayed activation and slow inactivation.^{11,17,22–25,36} The dominant component of Kv current in RPV myocytes exhibits half-maximal activation at -15 mV and inactivates over a slow time course that occurs with time constants of 0.5 and 3 seconds.^2,4,5,8,9,11 These properties are similar to those of homo- and heterotetrameric Kv1.2 and Kv1.5 channels.^{11,17,22–26,36} The coimmunoprecipitation of Kv1.2 and Kv1.5 from RPV provides the first evidence for the association of these subunits in vascular myocytes. The data presented in this study indicate, therefore, the potential contribution of homomultimers of Kv1.2 and Kv1.5, as well as Kv1.2 and Kv1.5 heteromultimeric channels. However, the data cannot determine whether both homo- and heteromultimeric channels are present in the myocytes. In the Kerr et al²⁴ study, we use 4-AP and charybdotoxin to demonstrate that the dominant K_DR channel complex of RPV myocytes is due to the coassembly of Kv1.2 and Kv1.5. The electrophysiological data indicate the functional identity of vascular K_DR channels and heteromultimeric Kv1.2-Kv1.5, but not homomultimeric Kv1.2 or Kv1.5 channels. The present study provides direct molecular evidence of the presence of this coassembly of Kv1.2 and Kv1.5 in native K_DR channels of vascular myocytes.

The expression of modulatory Kvß1.2, ß1.3, and ß2.1 proteins in RPV was detected by Western blotting and/or immunocytochemistry, and ß1.2 was demonstrated to associate with Kv1 subunits by coimmunoprecipitation. A similar association was previously reported to occur in neurons,^20,21 but our finding of Kvß subunit protein expression and coassembly with Kv1 subunits is novel for vascular smooth muscle. We found that the presence of Kvß subunit was necessary to shift the voltage dependence of activation of RPV Kv1.5 channels to potentials consistent with those exhibited by native RPV K_DR channels.¹¹ Previous studies show that both Kvß1 and ß2 subunits are capable of conferring this shift in voltage dependence to Kv1 subunit-containing channels.^22,23,35 However, the Kvß1 subunits also possess an N-terminal ball motif that interacts with residues at the intracellular surface of the pore of Kv1 channels and induces rapid inactivation at voltages positive to 0 mV.^20,22,23 The inactivation rate of native whole-cell K_DR currents of smooth muscle cells is inconsistent with the kinetics of Kvß1-containing Kv channels. It is possible that Kvß1 subunits are not uniformly present in all vascular K_DR channels or that the inactivation ball is immobilized. For example, Kv1.6 subunits possess a unique motif that immobilizes the Kvß1 ball motif,⁴¹ and Epperson et al³⁸ proposed that this interaction may prevent fast inactivation of K_DR channels of gastrointestinal myocytes that express Kv1.6 and Kvß1 subunits. Although Kv1.6 is not expressed in RPV, it is possible that the ball motif of Kvß1 subunits of RPV myocytes is immobilized by some other-as-yet unidentified protein(s).

In conclusion, we have identified the expression of Kv1.2, Kv1.4, Kv1.5, ß1.2, ß1.3, and ß2.1 mRNA and protein in RPV smooth muscle cells. We also provide the first evidence for the formation of heteromultimeric Kv channels in vascular myocytes. The data presented here combined with the pharmacological evidence in the Kerr et al²⁴ study, indicate that the 4-AP-sensitive K_DR current of RPV is predominantly due to heteromultimeric channels composed of Kv1.2 and Kv1.5. In light of the uniform expression of these subunits by myocytes of several vascular and nonvascular smooth muscle tissues, as well as cardiac muscle,^{10–14,16,17,37–39} it seems likely that there is a widespread distribution of K_DR channels with identical Kv1.2 and Kv1.5 subunit composition and functional properties in these cell types.

	Acknowledgments

 
This study was supported by a grant from the Canadian Institutes of Health Research Group (CIHR, Grant MT-10569). W.C.C. is a Senior Scholar and M.P.W. is a Senior Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR). M.P.W. holds a Canada Research Chair (Tier 1) in Biochemistry. K.S.T. and T.T.C. were recipients of Graduate Studentships from the AHFMR; K.S.T. held a CIHR Doctoral Research Award. We thank Dr Kuniaki Ishii for his generous gift of the Kv1.4 cDNA used in this study and C. Sutherland and C. Veillette for their expert assistance.

Received July 6, 2001; revision received October 8, 2001; accepted October 16, 2001.

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