Key Role of Kv1 Channels in Vasoregulation
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Tim T. Chen, Kevin D. Luykenaar, Emma J.
参见附件。
The Smooth Muscle Research Group, Faculty of Medicine, University of Calgary, Canada.
Abstract
Small arteries play an essential role in the regulation of blood pressure and organ-specific blood flow by contracting in response to increased intraluminal pressure, ie, the myogenic response. The molecular basis of the myogenic response remains to be defined. To achieve incremental changes in arterial diameter, as well as blood pressure or organ-specific blood flow, the depolarizing influence of intravascular pressure on vascular smooth muscle membrane potential that elicits myogenic contraction must be precisely controlled by an opposing hyperpolarizing influence. Here we use a dominant-negative molecular strategy and pressure myography to determine the role of voltage-dependent Kv1 potassium channels in vasoregulation, specifically, whether they act as a negative-feedback control mechanism of the myogenic response. Functional Kv1 channel expression was altered by transfection of endothelium-denuded rat middle cerebral arteries with cDNAs encoding c-myc epitope-tagged, dominant-negative mutant or wild-type rabbit Kv1.5 subunits. Expression of mutant Kv1.5 dramatically enhanced, whereas wild-type subunit expression markedly suppressed, the myogenic response over a wide range of intraluminal pressures. These effects on arterial diameter were associated with enhanced and reduced myogenic depolarization by mutant and wild-type Kv1.5 subunit expression, respectively. Expression of myc-tagged mutant and wild-type Kv1.5 subunit message and protein in transfected but not control arteries was confirmed, and isolated myocytes of transfected but not control arteries exhibited anti-c-myc immunofluorescence. No changes in message encoding other known, non-Kv1 elements of the myogenic response were apparent. These findings provide the first molecular evidence that Kv1-containing delayed rectifier K+ (KDR) channels are of fundamental importance for control of arterial diameter and, thereby, peripheral vascular resistance, blood pressure, and organ-specific blood flow.
Key Words: myogenic response delayed rectifier potassium channels vascular smooth muscle Kv1 channels
Introduction
The intrinsic ability of resistance arteries to contract in response to elevations in intraluminal (or transmural) pressure, the myogenic response, was first described over 100 years ago by Bayliss.1–4 This phenomenon is now well recognized to be an essential autoregulatory mechanism.2–4 Myogenic tone development depends on L-type Ca2+ channel (Cav1.2) activity within vascular myocytes.5 The resulting rise in intracellular free Ca2+ concentration via these Ca2+ channels6 activates cross-bridge cycling and contractile force development that may be enhanced and/or maintained by a Ca2+ sensitization of the contractile machinery.3,4
A current working hypothesis suggests that the activation of L-type Ca2+ channels during the myogenic response is the result of low amplitude, steady-state depolarization of the vascular smooth muscle (VSM) cells attributable to increased intraluminal pressure.2,3,6 However, very precise control of the extent of myogenic depolarization by an opposing hyperpolarizing influence is required to prevent regenerative Ca2+ influx and action potential initiation and to permit incremental, steady-state changes in arterial diameter that are essential for appropriate regulation of blood pressure and organ-specific blood flow.2,3,6 A negative-feedback mechanism involving the activation of smooth muscle K+ channels is thought to be required for control of myogenic depolarization.2,3,7,8 However, present understanding of the molecular basis of this fundamental example of physiological regulation in the field of vascular biology is limited. We have addressed this issue in the present study because an abnormal myogenic response contributes to several clinically relevant conditions, such as hypertension, diabetes, end-stage renal failure, coronary vasospasm, and delayed cerebral vasospasm following hemorrhagic stroke. Significant new insight into the molecular basis of the myogenic response will not only define the processes that contribute to abnormal control of arterial diameter in disease but also provide a rational basis for novel therapies aimed at restoration of normal blood pressure and organ-specific blood flow.
Previous studies provide evidence for an important role of large conductance, Ca2+-activated K+ channels (BKCa) in the negative-feedback regulation of myogenic depolarization.7,8 However, smooth muscle cells also express other types of K+ channels, for example, voltage-dependent, delayed rectifier potassium (KDR) channels, that exhibit steady-state activation within the range of membrane potential (Em) of the myogenic response (ie, approximately –55 to –30 mV).2 Specifically, expression of Kv1 and Kv2 pore-forming and modulatory Kv subunits that form KDR channels has been demonstrated for VSM cells of several vessels.9–13 Pharmacological evidence that Kv1- and Kv2-based KDR can contribute to control of Em, resting tone, and/or myogenic response of resistance arteries was previously obtained in experiments using 4-aminopyridine (4-AP)10–14 and correolide for Kv1 inhibition10–12 and stromatoxin for Kv2 inhibition.15 However, 4-AP at >1 mmol/L affects Kv2, ATP-sensitive K+ channels, and BKCa,2 as well as intracellular pH and Ca2+ release from internal stores16,17; correolide binds to Kv2.1 channels, albeit with 20-fold lower affinity compared with Kv1 subunits,18 and stromatoxin also suppresses Kv4 channel gating.19 Accordingly, direct molecular evidence is required to permit definitive conclusions regarding the role that VSM KDR channels play in the control of the myogenic response.
Here we used an in vitro strategy involving expression of cDNAs encoding dominant-negative mutant or wild-type rabbit Kv1.5 in rat middle cerebral arteries. This approach uniquely provides for a direct and specific determination of the role of Kv1-containing KDR in the myogenic response independent of channel subunit composition or stoichiometry. The findings of this study provide the first, direct molecular evidence that voltage-dependent Kv1-containing KDR channels are critical for normal vasoregulation.
Materials and Methods
Male Sprague–Dawley rats (250 to 350 g; Charles River, Montréal, Québec, Canada) were maintained and killed by halothane inhalation and exsanguination according to the standards of the Canadian Council on Animal Care and a protocol reviewed by the Animal Care Committee of the Faculty of Medicine, University of Calgary. The whole brain was carefully removed and placed in cold (4°C) Krebs buffer containing (in mmol/L): NaCl 120, NaHCO3 25, KCl 4.8, NaH2PO4 1.2, MgSO4 1.2, glucose 11, CaCl2 1.8. Left and right middle cerebral arteries were removed and cleaned of adherent fat, and the endothelium was removed (denuded) by passing a fine hair through the vessel lumen. Vessels were cannulated with fine glass pipettes mounted in a customized arteriograph chamber attached to a pressure myograph (Living Systems, Burlington, Vt) and superfused with warm (37°C) Krebs bath solution for measurement of arterial diameter with an automated edge detection system (IonOptix, Milton, Mass) (for additional details, see the online data supplement, available at http://circres.ahajournals.org). Reverse permeabilization and vessel culture were performed as in previous studies20,21 and are described in detail in the online data supplement. RT-PCR, real-time PCR, immunoblotting, immunocytochemistry, myocyte isolation, and microelectrode recordings were as previously described9,11,14 (see the online data supplement for additional details). Site-directed mutagenesis was performed using wild-type rabbit portal vein Kv1.5 (Kv1.5wt) subcloned into pcDNA3.122 to generate the dominant-negative Kv1.5W457F (Kv1.5DN) by replacing the native tryptophan at position 457 by phenylalanine. Ten residue c-myc or hemagluttinin (HA) epitope tags were added to the C terminus of Kv1.5wt and Kv1.5DN to facilitate immunoreactive detection of expressed protein (see the online data supplement for additional details). All data are shown as mean±SEM and were compared by paired Student’s t test or repeated-measures ANOVA, followed by Bonferroni post hoc test. A level of P<0.05 was considered to be statistically significant.
Results
Figure 1 illustrates the myogenic response of freshly isolated arterial segments to increased intraluminal pressure and also shows the extent of passive dilation that occurs in the absence of external Ca2+ ([Ca2+]o; ie, no added Ca2+ and EGTA at 0.2 mmol/L in Krebs saline solution). The difference in diameter in the presence and absence of [Ca2+]o represents the active, myogenic contractile response of the arteries to increased pressure. The potential involvement of Kv1-containing KDR channels in the control of the cerebral arterial myogenic response is suggested by the significantly enhanced vasoconstriction to pressure following correolide17 treatment (1 μmol/L; Figure 1).
Manipulation of functional Kv1-based KDR expression level was accomplished using c-myc–tagged wild-type rabbit Kv1.522 (Kv1.5wt) and c-myc–tagged mutant Kv1.5W457F (Kv1.5DN) constructs. Mutation of a single tryptophan (W434) to a phenylalanine residue in the pore-forming loop of the Shaker K+ channel sequence was previously shown to prevent K+ permeation without affecting expression.23 Mutation of this residue in rat Kv1.5 caused a specific dominant-negative inhibition of Kv1 but not Kv4 current in a heterologous expression system.24 Control experiments were therefore performed in this study using human embryonic kidney 293 (HEK293) cells. These results confirmed that our constructs expressed normally, that Kv1.5DN coassembled with wild-type Kv1 subunits, and that Kv1.5DN expression specifically suppressed Kv1 but not Kv2 currents (see supplemental Figures I through III).
A reverse permeabilization protocol was used to permit transient transfection of short-term cultured segments of cerebral arteries.20,21 Specifically, endothelium-denuded middle cerebral arteries were transfected with cDNAs encoding Kv1.5DN, Kv1.5wt, or empty plasmid (pcDNA3; ie, mock transfection) followed by 48 hours of culture in serum-free culture media to permit subunit protein expression.20,21 Figure 2A and 2B shows that mock-transfected, cultured arteries were myogenic and demonstrated active constrictions in response to increased intraluminal pressure. Figure 2C shows a comparison of the active constriction of mock-transfected, cultured, and freshly isolated vessels. No difference between the active response to pressure was apparent, but correolide treatment caused a significant enhancement of the active constriction. Thus, the culture and transfection procedure did not alter arterial diameter or the extent of myogenic constriction to pressures between 0 and 120 mm Hg.
Representative effects of expression of Kv1.5wt, Kv1.5DN, or plasmid alone on the myogenic response of cerebral arteries are shown in Figure 3A. All vessels in each experimental group displayed a myogenic response to increased pressure, but differences in the magnitude of the response between the groups were readily apparent. Figure 3B shows mean diameter versus pressure curves for the 3 vessel groups. Vessel diameters were similar at pressures <40 mm Hg; however, arteries transfected with Kv1.5DN showed a significantly greater decrease in diameter, and the myogenic response of arteries transfected with Kv1.5wt was markedly suppressed, compared with mock-transfected vessels at pressures 40 mm Hg (Figure 3B). In contrast, the passive responses of the vessels in the absence of Ca2+ were not different over the entire range of intraluminal pressures tested (Figure 3B). Also, exposure to 60 mmol/L KCl-containing bath solution was found to evoke vasoconstrictions to similar minimal diameters in mock-transfected (162±4 μm), Kv1.5DN-transfected (159±2 μm), and Kv1.5wt-transfected (169±3) arteries (n=3 vessels in each group).
Comparison of vessel diameters in the presence and absence of [Ca2+]o demonstrated the extent of active constriction as a function of intraluminal pressure for the 3 vessel groups, as shown in Figure 4A. The magnitude of active constriction increased proportionally with pressure in all vessels, but Kv1.5DN expression enhanced the active response compared with mock-transfected vessels, and wild-type Kv1.5 had the opposite effect (Figure 4A). Specifically, Kv1.5DN expression significantly enhanced active tone development at pressures 30 mm Hg, whereas Kv1.5wt suppressed the myogenic response at pressures 40 mm Hg compared with mock-transfected vessels. Figure 4B also shows that the levels of active constriction at 80 mm Hg were similar for freshly isolated and mock-transfected vessels but significantly greater for correolide-treated (1 μmol/L) and Kv1.5DN-transfected vessels and significantly less for Kv1.5wt-transfected arteries.
If the differences in active myogenic constriction shown in Figure 4 were owing to varied levels of Kv1-containing KDR current activation, expression of mutant and wild-type Kv1.5 subunits would be expected to alter the extent of myogenic depolarization, as well as the effect of correolide treatment on arterial diameter. Figure 5A and 5B shows representative sharp microelectrode recordings and average values of Em for mock-, Kv1.5DN-, and Kv1.5wt-transfected arteries pressurized to 80 mm Hg. We found that Em of VSM cells in vessels expressing Kv1.5DN was significantly more depolarized compared with mock-transfected arteries, whereas vessels transfected with Kv1.5wt were more hyperpolarized (Figure 5B). These data support the conclusion that manipulation of functional Kv1-containing KDR channel expression affected vasoregulation by changing the extent of myogenic depolarization evoked by increased intraluminal pressure.
Figure 6 shows the divergent effect of correolide treatment on active constriction of mock-, Kv1.5DN-, and Kv1.5wt-transfected vessels at 80 mm Hg. Correolide (1 μmol/L) caused a significant increase in active constriction of mock- and Kv1.5wt-transfected arteries, but it did not affect vessels expressing Kv1.5DN (Figure 6A). Notably, the change in active constriction induced by correolide was significantly less in vessels expressing Kv1.5DN and significantly greater for Kv1.5wt compared with mock-transfected arteries (Figure 6B). These findings are consistent with the conclusion that the contribution of correolide-sensitive Kv1-containing KDR channels to the control of the myogenic response was reduced and enhanced in Kv1.5DN and Kv1.5wt arteries, respectively, compared with mock-transfected vessels.
Confirmation of expression of c-myc–tagged mutant and wild-type Kv1.5 message and protein by arteries after 48 hours of in vitro culture was obtained by RT-PCR, immunoblotting, and immunocytochemistry. The primers used were specifically designed to amplify c-myc–tagged rabbit Kv1.5 but not endogenous rat Kv1.5. PCR reactions were also conducted before the RT reaction in 3 sets of experiments to control for contamination of DNase-treated RNA samples by the cDNAs used in the transfection procedure. No evidence of contamination was detected (Figure 7A, top). However, amplicons of appropriate size (224 bp) were detected after RT-PCR in RNA samples from Kv1.5wt- and Kv1.5DN-transfected but not freshly isolated or mock-transfected arteries in 3 experiments (Figure 7A, bottom). The identity of the amplicons was confirmed by sequencing. We also detected anti–c-myc immunoreactive protein with an apparent molecular mass of &77 kDa in immunoblots of Kv1.5wt- and Kv1.5DN-transfected, but not freshly isolated or mock-transfected, vessels in 3 separate experiments (Figure 7B, left). Confirmation of these results was provided by 3 additional experiments in which the c-myc tag on both constructs was replaced with a hemagglutinin (HA) epitope and immunoblotting performed with an anti-HA (Figure 7B, right). Finally, 30% to 40% of single smooth muscle cells isolated from Kv1.5DN- and Kv1.5wt-transfected, but not mock-transfected, arteries (3 vessels per group) demonstrated immunofluorescence when exposed to anti–c-myc followed by cy3-tagged secondary antibody (Figure 7C). Taken together, these data confirm the expression of exogenous rabbit Kv1.5 message and protein by VSM cells of Kv1.5DN- and Kv1.5wt-transfected, but not control, arteries.
A final set of control experiments using quantitative PCR analysis was conducted to confirm that expression of Kv1.5DN, Kv1.5wt, and empty plasmid did not affect the levels of mRNAs encoding proteins previously implicated to be components of the myogenic mechanism of arterial smooth muscle. Figure 8 shows that the levels of transcripts encoding the transient receptor potential nonselective cation channel subunit TRPC6,21 the voltage-dependent Ca2+ channel subunit, Cav1.2,5 the pore-forming, Slo1 subunit of BKCa,7,8 and an element of the Ca2+ sensitization pathway, RhoA,3,4 were not statistically different in mock-, Kv1.5DN-, and Kv1.5wt-transfected compared with freshly isolated middle cerebral arteries. These data are consistent with the view that the molecular strategy used specifically affected the Kv1-containing KDR channel component of the myogenic mechanism.
Discussion
Our findings provide the first direct molecular evidence of a key role for voltage-dependent channel pore-forming Kv1 subunits in control of the arterial myogenic response. Thus, VSM KDR channels containing Kv1 subunits are an important determinant of arterial diameter and, thereby, blood pressure regulation and organ-specific blood flow. Expression of Kv1.5DN-myc enhanced, whereas overexpression of wild-type Kv1.5 suppressed, myogenic contraction without affecting passive dilation to increased pressure. These findings provide compelling evidence that manipulating functional Kv1 channel expression by VSM cells alters vasoregulation. The validation of a simple molecular approach for specific determination of the contribution of heteromultimeric Kv1-containing KDR in this study will facilitate direct analysis of their additional roles (eg, in the actions of vasoactive agonists) and via a similar approach, the roles of other K+ channels (eg, Kv2 and inward rectifier K+ channels), in control of arterial diameter in health and various models of vascular disease.
We used a dominant-negative strategy to evaluate the role of Kv1-containing KDR channels in vasoregulation because they are thought to be heteromultimeric channels composed of multiple types of pore-forming Kv1 subunits. For example, rat mesenteric arterial KDR were shown to contain 3 different Kv1 subunits, ie, Kv1.2, Kv1.5, and Kv1.6, as well as modulatory Kv subunits,11 and coassembly of at least Kv1.2 and Kv1.5 has been demonstrated for rat cerebral arteries.10 Application of alternative genetic/molecular approaches, such as knock-out, antisense, or siRNA strategies, was viewed to be limited by the potential need for independent suppression of each of the Kv1 subunits to manipulate the Kv1-based KDR current. In contrast, the strategy of using Kv1.5DN is based on the assumption that the mutant subunits coassemble with endogenous, wild-type subunits and suppress K+ permeation through the pore of the resultant heterotetrameric channels irrespective of Kv1 subunit composition or stoichiometry. Mutation of the tryptophan residue at position 434 to phenylalanine within the pore region of the Shaker K+ channel was previously shown to prevent K+ permeation (hence, ionic current flow through the channel pore) without affecting expression or gating currents.23 Additionally, expression of mammalian Kv1.5 subunits with a similar mutation was shown to cause a dominant-negative suppression of currents attributable to heterologous expression of wild-type Kv1.5 but not Kv4 subunits in mammalian cells, as well as native K+ current of rat cardiac myocytes.24 We opted to use mutant and wild-type Kv1.5, rather than Kv1.2, based on previous observations indicating the presence of a C-terminal motif in Kv1.5 that promotes cell surface expression and a suboptimal motif in Kv1.2.25 We confirmed that the Kv1.5DN used in this study coassembled with wild-type Kv1.2 and Kv1.5 subunits via coimmunoprecipitation experiments and that it suppressed ionic current attributable to Kv1.2 and Kv1.5, but not Kv2.1 channels expressed in human embryonic kidney cells by whole-cell voltage-clamp technique (see the online data supplement for these additional data). Moreover, expression of c-myc–tagged mutant and wild-type Kv1.5 subunit message and protein following transient transfection was confirmed by RT-PCR and immunoblotting using intact arteries and by immunocytochemistry using myocytes isolated from transfected arteries. Taken together, the data from these control experiments indicate the presence of exogenous subunit expression by smooth muscle cells of the transfected, cultured arteries and the ability of the mutant Kv1.5 subunit to coassemble with and specifically suppress K+ current mediated by wild-type Kv1, but not Kv2 subunits.
We attribute the alterations in the myogenic response accompanying the expression of mutant or wild-type Kv1.5 subunits to differences in the contribution of Kv1-containing KDR channels to control of pressure-induced myogenic depolarization and vasoconstriction. We do not believe that the alterations in the myogenic response were the result of nonspecific effects on the vessels resulting from the reverse-permeabilization, transfection, or culture procedures, or to non–KDR-related variations in expression of other components of the myogenic mechanism. This view is based on the absence of any differences in the extent of: (1) passive dilation to pressure; (2) active myogenic contraction to 60 mmol/L KCl; and (3) expression of message encoding proteins previously implicated in the myogenic response, specifically TRPC6, Cav1.2, BKCa, and RhoA,2–5,21,26 in mock-, Kv1.5DN-, and Kv1.5wt-transfected vessels compared with freshly isolated arteries.
On the other hand, we found that Kv1.5DN and Kv1.5wt expression affected the level of Em and caused pressure-dependent alterations in arterial diameter. Specifically, Em was more depolarized at approximately –29±2 mV in vessels expressing Kv1.5DN compared with mock-transfected arteries at –44±2 mV, whereas expression of Kv1.5wt had the opposite effect, and Em was less depolarized at –48±1 mV. These values are consistent with those previously reported for rat cerebral and skeletal arteries/arterioles in studies that indicate that the myogenic response is activated over a narrow range of Em between approximately –55 and –30 mV and that small changes of Em within this range are associated with substantial changes in intracellular Ca2+ concentration and arterial diameter.6,26 The myogenic response is thought to involve VSM cell depolarization attributable to nonselective cation,21,27 chloride,28,29 and/or L-type Ca2+ channel activity,30 with the amplitude of current activation and extent of depolarization increasing proportionally with increasing intraluminal pressure. The contribution of KDR channels would also be expected to increase with increasing pressure if they are involved in negative-feedback regulation of myogenic depolarization. That Kv1-containing KDR should activate more strongly with progressive depolarization to oppose myogenic changes in Em may be predicted based on their voltage-dependent activation that overlaps with the Em range of the myogenic response.2,11 Direct experimental evidence of a pressure-dependent contribution to control of diameter is evident from our finding that the diameters of Kv1.5DN-, Kv1.5wt-, and mock-transfected arteries were similar at <40 mm Hg, but, at 40 mm Hg, the vasoconstriction of Kv1.5DN-transfected vessels was greater than that of mock-transfected vessels and significantly less than the mock-transfected vessels in arteries expressing myc-tagged wild-type Kv1.5 subunits. Thus, as we found for the level of Em at 80 mm Hg, pressure-dependent alterations in control of arterial diameter were apparent regardless of whether functional Kv1 channel expression was manipulated to decrease or increase KDR activity. Taken together, these electrophysiological and pressure myography results provide a direct indication of the important role that Kv1 channels serve in providing a negative-feedback control mechanism that limits myogenic depolarization and opposes myogenic alterations in arterial diameter, vascular resistance and blood flow (see supplemental Figure IV for schematic illustration of the role of Kv1 channels). To place our observations in this physiological context, Poiseuille’s Law predicts that the decrease in diameter at 100 mm Hg from 199±2 in mock- to 162±4 μm in Kv1.5DN-transfected vessels would be expected to cause a &2.4-fold increase in resistance and &60% reduction in blood flow and the increase in diameter of Kv1.5wt vessels to 227±3 μm an almost equivalent but opposite increase in flow. Clearly, altering the contribution of VSM Kv1-containing KDR channels to the control of myogenic depolarization has the potential to exert a profound effect on blood flow in vivo.
The results of this study are consistent with the view that the level of Kv1 subunit expression and magnitude of functional KDR current within a specific vessel bed may be an important determinant of the magnitude of the myogenic response. A lower relative contribution of Kv1-containing KDR caused by reduced subunit expression or channel gating as a result of phosphorylation by protein kinase C (PKC) or Rho-associated kinase (see Cole et al13) would permit greater myogenic depolarization and tone development, whereas increased expression or gating (attributable to PKA13) would limit depolarization and myogenic constriction. This view is consistent with our previous findings that the relative abundance of Kv1 message detected by real-time PCR was greater in first-order conduit versus fourth-order resistance mesenteric arteries and that 4-AP or correolide converted apparently nonmyogenic first-order arteries into vessels that exhibited active tone development in response to increases in intraluminal pressure.11 However, these data do not rule out the possibility that differences in functional contribution of other K+ channels (eg, Kv215 and BKCa26) and/or inward currents act in concert with Kv1-containing KDR in determining the pressure-dependence of VSM Em. Defining the relative contributions of differences in ion channel expression and function, as well as varied levels of Ca2+ sensitization to vessel- and vascular bed-specific differences in the magnitude and pressure dependence of the myogenic response, as are apparent between cerebral and skeletal muscle arteries/arterioles,6,26 will require considerable further study.
Previous findings provide compelling pharmacological and molecular evidence of a mechanism for control of arterial diameter involving BKCa of vascular myocytes.7,8 For example, enhanced cerebral arterial myogenic depolarization and contraction were observed in the presence of the specific blocker of BKCa channels, iberiotoxin.7 This BKCa-dependent mechanism is initiated by release of Ca2+ via ryanodine receptors in the sarcoplasmic reticulum (ie, "sparks")8 as well as Ca2+ influx through L-type Ca2+ channels in close proximity to the BKCa.31 Targeted deletion of the 1 subunit to reduce the Ca2+ sensitivity of BKCa in mice increased peripheral blood pressure by 15 to 20 mm Hg,32 and reduced 1 expression is associated with the development of genetic hypertension in spontaneously hypertensive rats.33 Reduced Kv1 and/or Kv2 subunit expression and/or KDR current density were previously shown to be associated with pulmonary, cerebral, and/or peripheral hypertension,15,34,35 and currently available pharmacological evidence is consistent with the view that suppression of Kv1- and/or Kv2-containing KDR channels leads to vasoconstriction and an enhanced myogenic response.10–15 However, until now, definitive molecular evidence that vascular KDR contribute to the myogenic response has been lacking. Our results demonstrate that Kv1-based KDR channels provide a key voltage-activated mechanism for control of myogenic constriction in cerebral arteries. The data do not exclude a parallel role for Kv2-containing channels in vasoregulation, as was recently suggested by Amberg and Santana15 based on experiments using the tarantula venom component stromatoxin. According to our data and the findings of others, appropriate regulation of arterial diameter through the myogenic response requires the involvement of KDR in addition to BKCa channels, and alterations in the behavior of negative-feedback mechanisms involving either channel type may be expected to contribute to dysfunctional control of arterial diameter, blood pressure, and organ-specific blood flow in disease.
Acknowledgments
Sources of Funding
This study was supported by an operating grant from the Canadian Institutes of Health Research (MT-13505) and salary awards from the Alberta Heritage Foundation for Medical Research (to T.T.C. and M.P.W.).
Disclosures
None.
Footnotes
Original received January 24, 2006; resubmission received May 11, 2006; accepted May 19, 2006.
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The Smooth Muscle Research Group, Faculty of Medicine, University of Calgary, Canada.
Abstract
Small arteries play an essential role in the regulation of blood pressure and organ-specific blood flow by contracting in response to increased intraluminal pressure, ie, the myogenic response. The molecular basis of the myogenic response remains to be defined. To achieve incremental changes in arterial diameter, as well as blood pressure or organ-specific blood flow, the depolarizing influence of intravascular pressure on vascular smooth muscle membrane potential that elicits myogenic contraction must be precisely controlled by an opposing hyperpolarizing influence. Here we use a dominant-negative molecular strategy and pressure myography to determine the role of voltage-dependent Kv1 potassium channels in vasoregulation, specifically, whether they act as a negative-feedback control mechanism of the myogenic response. Functional Kv1 channel expression was altered by transfection of endothelium-denuded rat middle cerebral arteries with cDNAs encoding c-myc epitope-tagged, dominant-negative mutant or wild-type rabbit Kv1.5 subunits. Expression of mutant Kv1.5 dramatically enhanced, whereas wild-type subunit expression markedly suppressed, the myogenic response over a wide range of intraluminal pressures. These effects on arterial diameter were associated with enhanced and reduced myogenic depolarization by mutant and wild-type Kv1.5 subunit expression, respectively. Expression of myc-tagged mutant and wild-type Kv1.5 subunit message and protein in transfected but not control arteries was confirmed, and isolated myocytes of transfected but not control arteries exhibited anti-c-myc immunofluorescence. No changes in message encoding other known, non-Kv1 elements of the myogenic response were apparent. These findings provide the first molecular evidence that Kv1-containing delayed rectifier K+ (KDR) channels are of fundamental importance for control of arterial diameter and, thereby, peripheral vascular resistance, blood pressure, and organ-specific blood flow.
Key Words: myogenic response delayed rectifier potassium channels vascular smooth muscle Kv1 channels
Introduction
The intrinsic ability of resistance arteries to contract in response to elevations in intraluminal (or transmural) pressure, the myogenic response, was first described over 100 years ago by Bayliss.1–4 This phenomenon is now well recognized to be an essential autoregulatory mechanism.2–4 Myogenic tone development depends on L-type Ca2+ channel (Cav1.2) activity within vascular myocytes.5 The resulting rise in intracellular free Ca2+ concentration via these Ca2+ channels6 activates cross-bridge cycling and contractile force development that may be enhanced and/or maintained by a Ca2+ sensitization of the contractile machinery.3,4
A current working hypothesis suggests that the activation of L-type Ca2+ channels during the myogenic response is the result of low amplitude, steady-state depolarization of the vascular smooth muscle (VSM) cells attributable to increased intraluminal pressure.2,3,6 However, very precise control of the extent of myogenic depolarization by an opposing hyperpolarizing influence is required to prevent regenerative Ca2+ influx and action potential initiation and to permit incremental, steady-state changes in arterial diameter that are essential for appropriate regulation of blood pressure and organ-specific blood flow.2,3,6 A negative-feedback mechanism involving the activation of smooth muscle K+ channels is thought to be required for control of myogenic depolarization.2,3,7,8 However, present understanding of the molecular basis of this fundamental example of physiological regulation in the field of vascular biology is limited. We have addressed this issue in the present study because an abnormal myogenic response contributes to several clinically relevant conditions, such as hypertension, diabetes, end-stage renal failure, coronary vasospasm, and delayed cerebral vasospasm following hemorrhagic stroke. Significant new insight into the molecular basis of the myogenic response will not only define the processes that contribute to abnormal control of arterial diameter in disease but also provide a rational basis for novel therapies aimed at restoration of normal blood pressure and organ-specific blood flow.
Previous studies provide evidence for an important role of large conductance, Ca2+-activated K+ channels (BKCa) in the negative-feedback regulation of myogenic depolarization.7,8 However, smooth muscle cells also express other types of K+ channels, for example, voltage-dependent, delayed rectifier potassium (KDR) channels, that exhibit steady-state activation within the range of membrane potential (Em) of the myogenic response (ie, approximately –55 to –30 mV).2 Specifically, expression of Kv1 and Kv2 pore-forming and modulatory Kv subunits that form KDR channels has been demonstrated for VSM cells of several vessels.9–13 Pharmacological evidence that Kv1- and Kv2-based KDR can contribute to control of Em, resting tone, and/or myogenic response of resistance arteries was previously obtained in experiments using 4-aminopyridine (4-AP)10–14 and correolide for Kv1 inhibition10–12 and stromatoxin for Kv2 inhibition.15 However, 4-AP at >1 mmol/L affects Kv2, ATP-sensitive K+ channels, and BKCa,2 as well as intracellular pH and Ca2+ release from internal stores16,17; correolide binds to Kv2.1 channels, albeit with 20-fold lower affinity compared with Kv1 subunits,18 and stromatoxin also suppresses Kv4 channel gating.19 Accordingly, direct molecular evidence is required to permit definitive conclusions regarding the role that VSM KDR channels play in the control of the myogenic response.
Here we used an in vitro strategy involving expression of cDNAs encoding dominant-negative mutant or wild-type rabbit Kv1.5 in rat middle cerebral arteries. This approach uniquely provides for a direct and specific determination of the role of Kv1-containing KDR in the myogenic response independent of channel subunit composition or stoichiometry. The findings of this study provide the first, direct molecular evidence that voltage-dependent Kv1-containing KDR channels are critical for normal vasoregulation.
Materials and Methods
Male Sprague–Dawley rats (250 to 350 g; Charles River, Montréal, Québec, Canada) were maintained and killed by halothane inhalation and exsanguination according to the standards of the Canadian Council on Animal Care and a protocol reviewed by the Animal Care Committee of the Faculty of Medicine, University of Calgary. The whole brain was carefully removed and placed in cold (4°C) Krebs buffer containing (in mmol/L): NaCl 120, NaHCO3 25, KCl 4.8, NaH2PO4 1.2, MgSO4 1.2, glucose 11, CaCl2 1.8. Left and right middle cerebral arteries were removed and cleaned of adherent fat, and the endothelium was removed (denuded) by passing a fine hair through the vessel lumen. Vessels were cannulated with fine glass pipettes mounted in a customized arteriograph chamber attached to a pressure myograph (Living Systems, Burlington, Vt) and superfused with warm (37°C) Krebs bath solution for measurement of arterial diameter with an automated edge detection system (IonOptix, Milton, Mass) (for additional details, see the online data supplement, available at http://circres.ahajournals.org). Reverse permeabilization and vessel culture were performed as in previous studies20,21 and are described in detail in the online data supplement. RT-PCR, real-time PCR, immunoblotting, immunocytochemistry, myocyte isolation, and microelectrode recordings were as previously described9,11,14 (see the online data supplement for additional details). Site-directed mutagenesis was performed using wild-type rabbit portal vein Kv1.5 (Kv1.5wt) subcloned into pcDNA3.122 to generate the dominant-negative Kv1.5W457F (Kv1.5DN) by replacing the native tryptophan at position 457 by phenylalanine. Ten residue c-myc or hemagluttinin (HA) epitope tags were added to the C terminus of Kv1.5wt and Kv1.5DN to facilitate immunoreactive detection of expressed protein (see the online data supplement for additional details). All data are shown as mean±SEM and were compared by paired Student’s t test or repeated-measures ANOVA, followed by Bonferroni post hoc test. A level of P<0.05 was considered to be statistically significant.
Results
Figure 1 illustrates the myogenic response of freshly isolated arterial segments to increased intraluminal pressure and also shows the extent of passive dilation that occurs in the absence of external Ca2+ ([Ca2+]o; ie, no added Ca2+ and EGTA at 0.2 mmol/L in Krebs saline solution). The difference in diameter in the presence and absence of [Ca2+]o represents the active, myogenic contractile response of the arteries to increased pressure. The potential involvement of Kv1-containing KDR channels in the control of the cerebral arterial myogenic response is suggested by the significantly enhanced vasoconstriction to pressure following correolide17 treatment (1 μmol/L; Figure 1).
Manipulation of functional Kv1-based KDR expression level was accomplished using c-myc–tagged wild-type rabbit Kv1.522 (Kv1.5wt) and c-myc–tagged mutant Kv1.5W457F (Kv1.5DN) constructs. Mutation of a single tryptophan (W434) to a phenylalanine residue in the pore-forming loop of the Shaker K+ channel sequence was previously shown to prevent K+ permeation without affecting expression.23 Mutation of this residue in rat Kv1.5 caused a specific dominant-negative inhibition of Kv1 but not Kv4 current in a heterologous expression system.24 Control experiments were therefore performed in this study using human embryonic kidney 293 (HEK293) cells. These results confirmed that our constructs expressed normally, that Kv1.5DN coassembled with wild-type Kv1 subunits, and that Kv1.5DN expression specifically suppressed Kv1 but not Kv2 currents (see supplemental Figures I through III).
A reverse permeabilization protocol was used to permit transient transfection of short-term cultured segments of cerebral arteries.20,21 Specifically, endothelium-denuded middle cerebral arteries were transfected with cDNAs encoding Kv1.5DN, Kv1.5wt, or empty plasmid (pcDNA3; ie, mock transfection) followed by 48 hours of culture in serum-free culture media to permit subunit protein expression.20,21 Figure 2A and 2B shows that mock-transfected, cultured arteries were myogenic and demonstrated active constrictions in response to increased intraluminal pressure. Figure 2C shows a comparison of the active constriction of mock-transfected, cultured, and freshly isolated vessels. No difference between the active response to pressure was apparent, but correolide treatment caused a significant enhancement of the active constriction. Thus, the culture and transfection procedure did not alter arterial diameter or the extent of myogenic constriction to pressures between 0 and 120 mm Hg.
Representative effects of expression of Kv1.5wt, Kv1.5DN, or plasmid alone on the myogenic response of cerebral arteries are shown in Figure 3A. All vessels in each experimental group displayed a myogenic response to increased pressure, but differences in the magnitude of the response between the groups were readily apparent. Figure 3B shows mean diameter versus pressure curves for the 3 vessel groups. Vessel diameters were similar at pressures <40 mm Hg; however, arteries transfected with Kv1.5DN showed a significantly greater decrease in diameter, and the myogenic response of arteries transfected with Kv1.5wt was markedly suppressed, compared with mock-transfected vessels at pressures 40 mm Hg (Figure 3B). In contrast, the passive responses of the vessels in the absence of Ca2+ were not different over the entire range of intraluminal pressures tested (Figure 3B). Also, exposure to 60 mmol/L KCl-containing bath solution was found to evoke vasoconstrictions to similar minimal diameters in mock-transfected (162±4 μm), Kv1.5DN-transfected (159±2 μm), and Kv1.5wt-transfected (169±3) arteries (n=3 vessels in each group).
Comparison of vessel diameters in the presence and absence of [Ca2+]o demonstrated the extent of active constriction as a function of intraluminal pressure for the 3 vessel groups, as shown in Figure 4A. The magnitude of active constriction increased proportionally with pressure in all vessels, but Kv1.5DN expression enhanced the active response compared with mock-transfected vessels, and wild-type Kv1.5 had the opposite effect (Figure 4A). Specifically, Kv1.5DN expression significantly enhanced active tone development at pressures 30 mm Hg, whereas Kv1.5wt suppressed the myogenic response at pressures 40 mm Hg compared with mock-transfected vessels. Figure 4B also shows that the levels of active constriction at 80 mm Hg were similar for freshly isolated and mock-transfected vessels but significantly greater for correolide-treated (1 μmol/L) and Kv1.5DN-transfected vessels and significantly less for Kv1.5wt-transfected arteries.
If the differences in active myogenic constriction shown in Figure 4 were owing to varied levels of Kv1-containing KDR current activation, expression of mutant and wild-type Kv1.5 subunits would be expected to alter the extent of myogenic depolarization, as well as the effect of correolide treatment on arterial diameter. Figure 5A and 5B shows representative sharp microelectrode recordings and average values of Em for mock-, Kv1.5DN-, and Kv1.5wt-transfected arteries pressurized to 80 mm Hg. We found that Em of VSM cells in vessels expressing Kv1.5DN was significantly more depolarized compared with mock-transfected arteries, whereas vessels transfected with Kv1.5wt were more hyperpolarized (Figure 5B). These data support the conclusion that manipulation of functional Kv1-containing KDR channel expression affected vasoregulation by changing the extent of myogenic depolarization evoked by increased intraluminal pressure.
Figure 6 shows the divergent effect of correolide treatment on active constriction of mock-, Kv1.5DN-, and Kv1.5wt-transfected vessels at 80 mm Hg. Correolide (1 μmol/L) caused a significant increase in active constriction of mock- and Kv1.5wt-transfected arteries, but it did not affect vessels expressing Kv1.5DN (Figure 6A). Notably, the change in active constriction induced by correolide was significantly less in vessels expressing Kv1.5DN and significantly greater for Kv1.5wt compared with mock-transfected arteries (Figure 6B). These findings are consistent with the conclusion that the contribution of correolide-sensitive Kv1-containing KDR channels to the control of the myogenic response was reduced and enhanced in Kv1.5DN and Kv1.5wt arteries, respectively, compared with mock-transfected vessels.
Confirmation of expression of c-myc–tagged mutant and wild-type Kv1.5 message and protein by arteries after 48 hours of in vitro culture was obtained by RT-PCR, immunoblotting, and immunocytochemistry. The primers used were specifically designed to amplify c-myc–tagged rabbit Kv1.5 but not endogenous rat Kv1.5. PCR reactions were also conducted before the RT reaction in 3 sets of experiments to control for contamination of DNase-treated RNA samples by the cDNAs used in the transfection procedure. No evidence of contamination was detected (Figure 7A, top). However, amplicons of appropriate size (224 bp) were detected after RT-PCR in RNA samples from Kv1.5wt- and Kv1.5DN-transfected but not freshly isolated or mock-transfected arteries in 3 experiments (Figure 7A, bottom). The identity of the amplicons was confirmed by sequencing. We also detected anti–c-myc immunoreactive protein with an apparent molecular mass of &77 kDa in immunoblots of Kv1.5wt- and Kv1.5DN-transfected, but not freshly isolated or mock-transfected, vessels in 3 separate experiments (Figure 7B, left). Confirmation of these results was provided by 3 additional experiments in which the c-myc tag on both constructs was replaced with a hemagglutinin (HA) epitope and immunoblotting performed with an anti-HA (Figure 7B, right). Finally, 30% to 40% of single smooth muscle cells isolated from Kv1.5DN- and Kv1.5wt-transfected, but not mock-transfected, arteries (3 vessels per group) demonstrated immunofluorescence when exposed to anti–c-myc followed by cy3-tagged secondary antibody (Figure 7C). Taken together, these data confirm the expression of exogenous rabbit Kv1.5 message and protein by VSM cells of Kv1.5DN- and Kv1.5wt-transfected, but not control, arteries.
A final set of control experiments using quantitative PCR analysis was conducted to confirm that expression of Kv1.5DN, Kv1.5wt, and empty plasmid did not affect the levels of mRNAs encoding proteins previously implicated to be components of the myogenic mechanism of arterial smooth muscle. Figure 8 shows that the levels of transcripts encoding the transient receptor potential nonselective cation channel subunit TRPC6,21 the voltage-dependent Ca2+ channel subunit, Cav1.2,5 the pore-forming, Slo1 subunit of BKCa,7,8 and an element of the Ca2+ sensitization pathway, RhoA,3,4 were not statistically different in mock-, Kv1.5DN-, and Kv1.5wt-transfected compared with freshly isolated middle cerebral arteries. These data are consistent with the view that the molecular strategy used specifically affected the Kv1-containing KDR channel component of the myogenic mechanism.
Discussion
Our findings provide the first direct molecular evidence of a key role for voltage-dependent channel pore-forming Kv1 subunits in control of the arterial myogenic response. Thus, VSM KDR channels containing Kv1 subunits are an important determinant of arterial diameter and, thereby, blood pressure regulation and organ-specific blood flow. Expression of Kv1.5DN-myc enhanced, whereas overexpression of wild-type Kv1.5 suppressed, myogenic contraction without affecting passive dilation to increased pressure. These findings provide compelling evidence that manipulating functional Kv1 channel expression by VSM cells alters vasoregulation. The validation of a simple molecular approach for specific determination of the contribution of heteromultimeric Kv1-containing KDR in this study will facilitate direct analysis of their additional roles (eg, in the actions of vasoactive agonists) and via a similar approach, the roles of other K+ channels (eg, Kv2 and inward rectifier K+ channels), in control of arterial diameter in health and various models of vascular disease.
We used a dominant-negative strategy to evaluate the role of Kv1-containing KDR channels in vasoregulation because they are thought to be heteromultimeric channels composed of multiple types of pore-forming Kv1 subunits. For example, rat mesenteric arterial KDR were shown to contain 3 different Kv1 subunits, ie, Kv1.2, Kv1.5, and Kv1.6, as well as modulatory Kv subunits,11 and coassembly of at least Kv1.2 and Kv1.5 has been demonstrated for rat cerebral arteries.10 Application of alternative genetic/molecular approaches, such as knock-out, antisense, or siRNA strategies, was viewed to be limited by the potential need for independent suppression of each of the Kv1 subunits to manipulate the Kv1-based KDR current. In contrast, the strategy of using Kv1.5DN is based on the assumption that the mutant subunits coassemble with endogenous, wild-type subunits and suppress K+ permeation through the pore of the resultant heterotetrameric channels irrespective of Kv1 subunit composition or stoichiometry. Mutation of the tryptophan residue at position 434 to phenylalanine within the pore region of the Shaker K+ channel was previously shown to prevent K+ permeation (hence, ionic current flow through the channel pore) without affecting expression or gating currents.23 Additionally, expression of mammalian Kv1.5 subunits with a similar mutation was shown to cause a dominant-negative suppression of currents attributable to heterologous expression of wild-type Kv1.5 but not Kv4 subunits in mammalian cells, as well as native K+ current of rat cardiac myocytes.24 We opted to use mutant and wild-type Kv1.5, rather than Kv1.2, based on previous observations indicating the presence of a C-terminal motif in Kv1.5 that promotes cell surface expression and a suboptimal motif in Kv1.2.25 We confirmed that the Kv1.5DN used in this study coassembled with wild-type Kv1.2 and Kv1.5 subunits via coimmunoprecipitation experiments and that it suppressed ionic current attributable to Kv1.2 and Kv1.5, but not Kv2.1 channels expressed in human embryonic kidney cells by whole-cell voltage-clamp technique (see the online data supplement for these additional data). Moreover, expression of c-myc–tagged mutant and wild-type Kv1.5 subunit message and protein following transient transfection was confirmed by RT-PCR and immunoblotting using intact arteries and by immunocytochemistry using myocytes isolated from transfected arteries. Taken together, the data from these control experiments indicate the presence of exogenous subunit expression by smooth muscle cells of the transfected, cultured arteries and the ability of the mutant Kv1.5 subunit to coassemble with and specifically suppress K+ current mediated by wild-type Kv1, but not Kv2 subunits.
We attribute the alterations in the myogenic response accompanying the expression of mutant or wild-type Kv1.5 subunits to differences in the contribution of Kv1-containing KDR channels to control of pressure-induced myogenic depolarization and vasoconstriction. We do not believe that the alterations in the myogenic response were the result of nonspecific effects on the vessels resulting from the reverse-permeabilization, transfection, or culture procedures, or to non–KDR-related variations in expression of other components of the myogenic mechanism. This view is based on the absence of any differences in the extent of: (1) passive dilation to pressure; (2) active myogenic contraction to 60 mmol/L KCl; and (3) expression of message encoding proteins previously implicated in the myogenic response, specifically TRPC6, Cav1.2, BKCa, and RhoA,2–5,21,26 in mock-, Kv1.5DN-, and Kv1.5wt-transfected vessels compared with freshly isolated arteries.
On the other hand, we found that Kv1.5DN and Kv1.5wt expression affected the level of Em and caused pressure-dependent alterations in arterial diameter. Specifically, Em was more depolarized at approximately –29±2 mV in vessels expressing Kv1.5DN compared with mock-transfected arteries at –44±2 mV, whereas expression of Kv1.5wt had the opposite effect, and Em was less depolarized at –48±1 mV. These values are consistent with those previously reported for rat cerebral and skeletal arteries/arterioles in studies that indicate that the myogenic response is activated over a narrow range of Em between approximately –55 and –30 mV and that small changes of Em within this range are associated with substantial changes in intracellular Ca2+ concentration and arterial diameter.6,26 The myogenic response is thought to involve VSM cell depolarization attributable to nonselective cation,21,27 chloride,28,29 and/or L-type Ca2+ channel activity,30 with the amplitude of current activation and extent of depolarization increasing proportionally with increasing intraluminal pressure. The contribution of KDR channels would also be expected to increase with increasing pressure if they are involved in negative-feedback regulation of myogenic depolarization. That Kv1-containing KDR should activate more strongly with progressive depolarization to oppose myogenic changes in Em may be predicted based on their voltage-dependent activation that overlaps with the Em range of the myogenic response.2,11 Direct experimental evidence of a pressure-dependent contribution to control of diameter is evident from our finding that the diameters of Kv1.5DN-, Kv1.5wt-, and mock-transfected arteries were similar at <40 mm Hg, but, at 40 mm Hg, the vasoconstriction of Kv1.5DN-transfected vessels was greater than that of mock-transfected vessels and significantly less than the mock-transfected vessels in arteries expressing myc-tagged wild-type Kv1.5 subunits. Thus, as we found for the level of Em at 80 mm Hg, pressure-dependent alterations in control of arterial diameter were apparent regardless of whether functional Kv1 channel expression was manipulated to decrease or increase KDR activity. Taken together, these electrophysiological and pressure myography results provide a direct indication of the important role that Kv1 channels serve in providing a negative-feedback control mechanism that limits myogenic depolarization and opposes myogenic alterations in arterial diameter, vascular resistance and blood flow (see supplemental Figure IV for schematic illustration of the role of Kv1 channels). To place our observations in this physiological context, Poiseuille’s Law predicts that the decrease in diameter at 100 mm Hg from 199±2 in mock- to 162±4 μm in Kv1.5DN-transfected vessels would be expected to cause a &2.4-fold increase in resistance and &60% reduction in blood flow and the increase in diameter of Kv1.5wt vessels to 227±3 μm an almost equivalent but opposite increase in flow. Clearly, altering the contribution of VSM Kv1-containing KDR channels to the control of myogenic depolarization has the potential to exert a profound effect on blood flow in vivo.
The results of this study are consistent with the view that the level of Kv1 subunit expression and magnitude of functional KDR current within a specific vessel bed may be an important determinant of the magnitude of the myogenic response. A lower relative contribution of Kv1-containing KDR caused by reduced subunit expression or channel gating as a result of phosphorylation by protein kinase C (PKC) or Rho-associated kinase (see Cole et al13) would permit greater myogenic depolarization and tone development, whereas increased expression or gating (attributable to PKA13) would limit depolarization and myogenic constriction. This view is consistent with our previous findings that the relative abundance of Kv1 message detected by real-time PCR was greater in first-order conduit versus fourth-order resistance mesenteric arteries and that 4-AP or correolide converted apparently nonmyogenic first-order arteries into vessels that exhibited active tone development in response to increases in intraluminal pressure.11 However, these data do not rule out the possibility that differences in functional contribution of other K+ channels (eg, Kv215 and BKCa26) and/or inward currents act in concert with Kv1-containing KDR in determining the pressure-dependence of VSM Em. Defining the relative contributions of differences in ion channel expression and function, as well as varied levels of Ca2+ sensitization to vessel- and vascular bed-specific differences in the magnitude and pressure dependence of the myogenic response, as are apparent between cerebral and skeletal muscle arteries/arterioles,6,26 will require considerable further study.
Previous findings provide compelling pharmacological and molecular evidence of a mechanism for control of arterial diameter involving BKCa of vascular myocytes.7,8 For example, enhanced cerebral arterial myogenic depolarization and contraction were observed in the presence of the specific blocker of BKCa channels, iberiotoxin.7 This BKCa-dependent mechanism is initiated by release of Ca2+ via ryanodine receptors in the sarcoplasmic reticulum (ie, "sparks")8 as well as Ca2+ influx through L-type Ca2+ channels in close proximity to the BKCa.31 Targeted deletion of the 1 subunit to reduce the Ca2+ sensitivity of BKCa in mice increased peripheral blood pressure by 15 to 20 mm Hg,32 and reduced 1 expression is associated with the development of genetic hypertension in spontaneously hypertensive rats.33 Reduced Kv1 and/or Kv2 subunit expression and/or KDR current density were previously shown to be associated with pulmonary, cerebral, and/or peripheral hypertension,15,34,35 and currently available pharmacological evidence is consistent with the view that suppression of Kv1- and/or Kv2-containing KDR channels leads to vasoconstriction and an enhanced myogenic response.10–15 However, until now, definitive molecular evidence that vascular KDR contribute to the myogenic response has been lacking. Our results demonstrate that Kv1-based KDR channels provide a key voltage-activated mechanism for control of myogenic constriction in cerebral arteries. The data do not exclude a parallel role for Kv2-containing channels in vasoregulation, as was recently suggested by Amberg and Santana15 based on experiments using the tarantula venom component stromatoxin. According to our data and the findings of others, appropriate regulation of arterial diameter through the myogenic response requires the involvement of KDR in addition to BKCa channels, and alterations in the behavior of negative-feedback mechanisms involving either channel type may be expected to contribute to dysfunctional control of arterial diameter, blood pressure, and organ-specific blood flow in disease.
Acknowledgments
Sources of Funding
This study was supported by an operating grant from the Canadian Institutes of Health Research (MT-13505) and salary awards from the Alberta Heritage Foundation for Medical Research (to T.T.C. and M.P.W.).
Disclosures
None.
Footnotes
Original received January 24, 2006; resubmission received May 11, 2006; accepted May 19, 2006.
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