Remodeling of Arterial Tone Regulation in Postnatal Development: Focus on Smooth Muscle Cell Potassium Channels

Maturation of the cardiovascular system is associated with crucial structural and functional remodeling. Thickening of the arterial wall, maturation of the sympathetic innervation, and switching of the mechanisms of arterial contraction from calcium-independent to calcium-dependent occur during postnatal development. All these processes promote an almost doubling of blood pressure from the moment of birth to reaching adulthood. This review focuses on the developmental alterations of potassium channels functioning as key smooth muscle membrane potential determinants and, consequently, vascular tone regulators. We present evidence that the pattern of potassium channel contribution to vascular control changes from Kir2, Kv1, Kv7 and TASK-1 channels to BKCa channels with maturation. The differences in the contribution of potassium channels to vasomotor tone at different stages of postnatal life should be considered in treatment strategies of cardiovascular diseases associated with potassium channel malfunction.

Potassium channels are transmembrane proteins which form potassium-selective pores. They are widely distributed among living organisms and are found in many cell types, including cells of the cardiovascular system. In cardiac and some vascular (mainly venous) myocytes, potassium channels mediate the repolarization phase of the action potential. In arterial smooth muscle cells, which commonly do not generate action potentials under physiological conditions, a change in potassium channel activity provides gradual shifts of the membrane potential [1,2]. This leads to alterations in the activity of voltage-gated calcium channels accompanied by changes in the calcium influx, as well as the intracellular calcium concentration and corresponding modifications of vascular tone. Of note, malfunctions of several potassium channel types are associated with arterial hypertension, diabetes mellitus and other metabolic disorders [3][4][5][6].

Potassium Channels Are Key Regulators of Arterial Tone
The membrane potential of arterial smooth muscle cells is a key determinant of vascular tone [7,8]. Indeed, a membrane potential shift by only a few millivolts towards hyperpolarization or depolarization leads to a considerable increase and decrease in arterial lumen, respectively [9]. Potassium channels of the cell membrane play a key role in the establishment of the resting membrane potential, as well as in the regulation of the membrane potential under the action of various vasoconstrictors and vasodilators [6,10].
Several functional types of potassium channels are described in arterial smooth muscle voltage-gated potassium channels (K v ), inward-rectifier potassium channels (K ir ), adenosine triphosphate-sensitive potassium channels (K ATP ), two-pore potassium channels (K 2P ), as well as calcium-activated potassium channels of large conductance (BK Ca ) [8,11].
It has been shown that potassium channel blockade leads to the depolarization of smooth muscle cells and an increase in contractile responses of the vascular wall to stimuli of different nature. Activation of potassium channels, on the contrary, counteracts the development of contraction or causes the dilation of arterial smooth muscle [8,12]. Therefore, potassium channels reduce the depolarization of arterial smooth muscle and play anticontractile or vasorelaxation roles. Importantly, potassium channel activity can limit arterial responses to the contractile influence of sympathetic nerves [13,14], a principal determinant of systemic blood pressure.

Postnatal Development Is Associated with Structural and Functional Remodeling of the Systemic Circulation
The mammalian cardiovascular system undergoes crucial structural and functional changes after birth. The arterial smooth muscle layer thickens and the amount of contractile proteins rises [15][16][17]. This increases the ability of arteries to develop a contractile response. In addition, the density of sympathetic innervation of the arteries increases gradually with age [15,18,19]. Therefore, arteries become more susceptible to procontractile influences from the sympathetic nervous system at adult age. Moreover, considerable alterations in the mechanisms governing vascular smooth muscle contraction occur with developmentthe strong contribution of mechanisms that are weakly dependent on changes in the intracellular calcium concentration (calcium sensitization) in the newborn switches to more calcium-dependent mechanisms as the organism grows [15,18,20,21]. Finally, the anticontractile influence of the vascular endothelium decreases with age [22,23]. This structural and functional remodeling leads to a dramatic rise of systemic blood pressure, which almost doubles from the time of birth [18,[23][24][25].
The impact of such important regulators of vascular tone as potassium channels also changes during postnatal life. The present review focuses on the developmental alterations of the vasomotor role of different potassium channel families in arteries of the peripheral circulation, which play a key role in the regulation of systemic blood pressure.

Developmental Alterations of Potassium Channel Functioning in Arteries of the Systemic Circulation
3.1. Voltage-Gated Potassium Channels (K v Channels) 3.1.1. K v Channels: Properties and Functions in Arteries Voltage-gated potassium channels (K v channels) are a large family of potassium channels which activate in response to membrane depolarization. A total of 12 subfamilies of K v channels (K v 1-K v 12) exist, and the functionally most important subfamilies in arterial smooth muscle are K v 1, K v 2 and K v 7 [26,27]. In arterial smooth muscle, the main isoforms of K v 1 channels are K v 1.2, K v 1.5; of K v 2 channels is K v 2.1; and of K v 7 channels are K v 7.4 and K v 7.5 [8,28]. A unique feature of K v 7 channels is their relatively low activation threshold. The V 0 . 5 value (voltage that produces 50% activation) is +5 mV for K v 1 and K v 2, and only −34 mV for K v 7, as was shown in the rat mesenteric artery smooth muscle cells [29]. In other words, the activation threshold of K v 7 channels is close to the level of the resting membrane potential of arterial smooth muscle cells, which varies in the range from -40 to −65 mV [6,8,9]. A number of studies have demonstrated the important role of K v 1, K v 2 and, especially, K v 7 channels in maintaining basal tone and regulating contractile responses of arteries, since the blockade of these channels caused depolarization of arterial smooth muscle cells and the development of strong basal tone and enhanced vasoconstrictor responses [26,[30][31][32][33][34][35][36].

The Vasomotor Role of K v Channels Decreases with Maturation
In earlier studies, 4-aminopyridine (4-AP) was used as a blocker of K v channels [37]. It was demonstrated that the cumulative addition of 4-AP caused contraction of isolated aorta segments from newborn rats, while its effects on adult aorta were considerably less pronounced [38]. In accordance with this, 4-AP inhibited [38] or completely blocked [39] the outward potassium current in isolated smooth muscle cells from newborn, but not adult, aorta. In contrast, the effects of 4-AP on resting tone and 5-HT (5-hydroxytryptamine or serotonin)-induced contractions were more pronounced in cerebral arteries of adult sheep than in fetuses [40], suggesting an increase in K v -functional contribution with maturation. However, in recent studies, 4-AP was shown to activate K v 7.4 channels [41] and increase the intracellular pH affecting BK Ca currents [42], which complicates the interpretation of the results obtained using this inhibitor.
Recently, selective blockers for K v 1, K v 2 and K v 7 channels have become available: DPO-1, stromatoxin and XE991, respectively [33,43,44], which made it possible to evaluate the contribution of these K v channel subfamilies to the regulation of arterial tone separately.
With the use of DPO-1 (1 µM), it was shown that the contribution of the K v 1 subfamily decreases with maturation [45]. DPO-1 caused the development of basal tone, increased contractile responses and sensitivity to the α 1 -adrenoceptor agonist methoxamine in saphenous arteries of young rats (10 to 15 days old), while no effect was observed in arteries of adult animals (2 to 3 months old) [45]. The decrease in the functional impact of K v 1 channels during development may be associated with a decrease in their expression. This assumption is supported by data showing a decrease in K v 1.2 channel protein expression in rat aorta in the period between birth and adulthood [39].
Stromatoxin, a blocker of the K v 2 subfamily, did not affect the contractile responses of saphenous arteries, either in young or in adult rats [45], although an increase in K v 2.1 channel protein expression with maturation was previously demonstrated in rat aorta [39]. Perhaps the levels of expression and, consequently, functional contribution of K v 2 channels may differ between the aorta and saphenous arteries.
The functional role of K v 7 channels strongly declines with maturation. Blockade of these channels with XE991 and linopirdine led to the development of basal tone, increased contractile responses and sensitivity to the α 1 -adrenoreceptor agonist methoxamine and the thromboxane A2 receptor agonist U46619 in saphenous arteries of young rats [45,46]. These effects were considerably less pronounced in arteries of adult rats. The differences found were associated with a smaller effect of K v 7 channel blockade on the membrane potential of smooth muscle in adult rats [45]. These effects are probably associated with a decrease in channel expression in arterial smooth muscle with development. Indeed, a decrease in K v 7.4 channel protein expression was demonstrated with maturation. Importantly, the protein expression of the accessory KCNE4 subunit, which was shown to strongly augment K v 7.4 channel protein expression and K v 7.4 channel activity [47], also declined with age [45]. In addition to different expression levels, the functional impact of K v 7 channels depends on the activity of other potassium channels. A recent study demonstrated that BK Ca channel activity (which increases with age, see further in detail) suppresses K v 7 channel functional availability in saphenous arteries of adult, but not young, rats [46].
Thus, the results with the use of selective K v channel subfamily blockers allow the suggestion that the vasomotor role of K v 1 and, especially, K v 7 channels in systemic arteries decreases from the early postnatal period to adulthood. Moreover, their functional availability can be suppressed by the increasing contribution of other potassium channels, for example BK Ca channels, with age.

K 2P Channels: Properties and Functions in Arteries
These channels were discovered in the vasculature relatively recently [48,49]. The channel is formed by two subunits, each has two pore-forming loops (P-loop, hence the name of the channel, K 2P ). Originally, K 2P channels were described as leakage channels, since at resting conditions they carry an outward potassium current, and the probability of channel opening does not depend on the membrane potential [11,50,51]. Research over the last few years has changed the image of these channels from being simply leak channels to regulatory channels having important roles in the control of cell excitability [52]. These facts indicate the involvement of K 2P channels in the maintenance of the resting potential, denoting their importance for the regulation of arterial tone.
There are six functional classes of K 2P channels. One of them, the acid-sensitive TASK-1 channel, is of particular interest. Recently, it was shown that a mutation in the KCNK3 gene encoding the TASK-1 channel is associated with familial and idiopathic forms of pulmonary hypertension [53].

The Impact of TASK-1 Channels on the Regulation of Vascular Tone and Blood Pressure Dramatically Decreases with Maturation
The functional role of these channels is important in pulmonary circulation [53][54][55], but very little is known about their function in systemic arteries. The ontogenetic aspect of TASK-1 channel function in systemic arteries has been studied very little. The only work devoted to this issue indicated a decrease in their vasomotor role in early postnatal ontogenesis [24].
Blockade of TASK-1 channels was shown to increase basal tone and contractile responses to methoxamine in the saphenous artery of young, but not adult, rats [24]. In arteries of adult rats, TASK-1 channel blockade did not lead to such a pronounced depolarization of the smooth muscle cells as was observed in young animals. Intravenous administration of AVE1231, a TASK-1 channel blocker [56], led to a considerable increase in mean blood pressure at young age, but not in adulthood, which indicates a decrease in the role of TASK-1 channels in blood pressure control with maturation. Finally, both TASK-1 mRNA and protein content in arterial tissue decreased as the rats grew older [24].
Therefore, the anticontractile role of TASK-1 channels in the arteries of systemic circulation and their impact to the lowering of blood pressure most likely declines with maturation.

K ir Channels: Properties and Functions in Arteries
Inward-rectifier potassium channels mediate an inward potassium current at membrane potentials more negative than the equilibrium potassium potential (E M > E K ) and a less pronounced outward potassium current at membrane potentials more positive than the equilibrium potassium potential (E M < E K ) [57]. The property of inward rectification is determined by the influence of intracellular substances-magnesium ions and polyamines, which block the outward potassium current at E M < E K [58,59]. There are seven subfamilies of K ir channels, the second (K ir 2) and sixth subfamilies (K ir 6) are expressed in arterial smooth muscle [8]. The sixth subfamily of K ir channels has distinct functional and regulatory features and therefore is classified into a class of ATP-dependent potassium channels (K ATP channels) which will be discussed separately (see Section 3.4 below).
Arterial smooth muscle cells express K ir 2.1, K ir 2.2 and K ir 2.4 isoforms. Using K ir 2.1 gene knockout mice, it was demonstrated that this isoform is primarily functionally important in cerebral arteries at least in the early postnatal period, since the data were obtained on young animals [60]. When the level of the membrane potential of arterial smooth muscle is more positive than E K (at physiological resting membrane potentials), K ir channels mediate an outward hyperpolarizing current which counteracts the contraction of arteries. Indeed, a number of studies have demonstrated that K ir channels are involved in maintaining the resting potential since their blocker barium chloride caused a depolarization of arterial smooth muscle [61][62][63]. Moreover, pretreatment of the rat coronary, cerebral and tail small arteries with barium chloride inhibited KCl-induced vasodilation, indicating that K ir 2 channels mediate this reaction [61,63].

The Vasomotor Role of K ir 2 Channels Decreases with Maturation
The contribution of K ir 2 channels on the regulation of vascular tone, as well as its expression pattern during ontogenesis, is not fully understood. It was shown that K ir 2.1 and K ir 2.4 mRNA expression decreased with age, while the expression of K ir 2.2 increased during postnatal maturation in smooth muscle cells of the rat saphenous artery [45]. In functional experiments on the sheep middle cerebral artery, blockade of K ir 2 channels with barium chloride did not affect basal tone and contractile responses to 5-HT in either adult sheep or sheep fetuses [40]. However, another agonist, noradrenaline, enhanced the contractile responses of middle cerebral arteries of sheep fetuses in the presence of barium chloride, which was not observed in arteries of adult animals [64]. In addition, barium chloride increased the contractile responses to the α 1 -adrenoceptor agonist methoxamine in rat saphenous arteries at different stages of development and the extent of this effect declined with age [45]. A greater contraction during the blockade of K ir channels in young rats was accompanied by a greater depolarization of the smooth muscle membrane potential compared to adults [45].
Therefore, results of a few studies suggest a decrease in the functional role of K ir 2 channels with age [45,64]. The decline of K ir 2.1 and K ir 2.4 expression may result in the decline of their vasomotor role. However, more studies on this issue should be conducted.

K ATP Channels: Properties and Functions in Arteries
K ATP channels are sensitive to the concentration of ATP in cell cytoplasm. The intracellular ATP concentration is quite high at physiological resting conditions and K ATP channels are closed. However, under conditions of limited oxygen and substrates, ADP accumulates, which activates the K ATP channel and leads to an efflux of potassium from the cell, hyperpolarizing the membrane potential. Thus, the ADP/ATP ratio is a key physiological regulator of K ATP channel activity [65,66]. Relaxation of arteries under functional and reactive hyperemia occurs largely due to the activation of K ATP channels [67,68]. It is important to note that under such pathological conditions as hypoxia, ischemia, and sepsis, the role of K ATP channels in the regulation of arterial tone is extremely large [69,70].

The Vasomotor Role of K ATP Channels under Normal Physiological Conditions Does Not Change during Postnatal Development
The functional role of K ATP channels during ontogenesis has been poorly studied. Of note, the results of these studies qualitatively depend on the method employed to assess the vasomotor role of K ATP channels-using an activator or a blocker.
In one of the early studies, lemakalim, an activator of K ATP channels, relaxed the middle cerebral artery in both adult sheep and fetuses [71]. At the same time, the sensitivity to lemakalim was considerably higher in adult sheep, which suggests an increase in the functional impact of K ATP channels in the regulation of vascular tone with age. Later on, this group demonstrated a qualitatively similar result using another K ATP channel activator-pinacidil. The incubation of segments of the middle cerebral artery with pinacidil resulted in a weakening of contractile responses to noradrenaline in adult sheep, but not in fetuses [64].
The K ATP channel blocker, glibenclamide, did not increase the basal tone and contractile responses to noradrenaline of the middle cerebral artery in either adult sheep or fetuses [64]. Similarly, no effect of glibenclamide on basal tone and contractile responses to the α 1 -adrenoceptor agonist methoxamine was found in the rat saphenous artery at different stages of postnatal development [45].
Apparently, under normal physiological conditions, K ATP channels are not active in the vasculature of either young or adult animals, and, therefore, the effect of their blockade on vascular tone cannot be observed. At the same time, results obtained with the use of activators suggest that the functional contribution of these channels grows as the organism ages. It is possible that the number of these channels increases with age, as it has been shown for other tissues, for example the rat myocardium [71].

BK Ca Channels: Properties and Functions in Arteries
Calcium-activated potassium channels of large conductance (BK Ca channels) are regulated by both the membrane potential and the intracellular calcium concentration. The activity of BK Ca channels increases with membrane depolarization with an increase in the intracellular calcium concentration [72].
An important physiological role of BK Ca channels is that they provide a negative feedback regulation of arterial myogenic tone. It is known that ryanodine receptors (RyRs) of the sarcoplasmic reticulum adjoin to BK Ca channels in the smooth muscle cell outer membrane [73,74]. Moreover, BK Ca channels are co-localized with voltage-dependent calcium channels in the caveolae of arterial smooth muscle cells [75,76]. Thus, a local increase in the calcium concentration as a result of calcium sparks or activation of voltagedependent calcium channels causes a short-term increase in the activity of BK Ca channels, the efflux of potassium ions from the cell, hyperpolarization and, as a result, relaxation.

The Vasomotor Role of BK Ca Channels Increases during Postnatal Development
Most studies have demonstrated that the contribution of BK Ca channels to the regulation of vascular tone in systemic circulation increases with age.
In rat cerebral arteries, the effects of BK Ca channel blockade became more pronounced with age-the BK Ca channel blocker, iberiotoxin, caused a contraction and an increase in the intracellular calcium concentration in arteries of adult (12-14 weeks old), but not newborn, rats (1-2 days old) [77]. Similarly, iberiotoxin led to an increase in basal tone and sensitivity to the α 1 -adrenoceptor agonist methoxamine in saphenous arteries of adult (2-3 months old), but not of young (10-15 day old), rats [45,46]. The effects of iberiotoxin on the contraction of sheep middle cerebral arteries also increased with age [40]. Later, this group also demonstrated an increase in the contractile responses of the carotid artery of adult sheep, but not fetuses, to 5-HT in the presence of iberiotoxin [78]. The effects of tetraethylammonium (TEA), another-albeit less selective-blocker of BK Ca channels, also intensified with age: TEA caused more pronounced contractions of the aortic segments of 8-and 12-week-old rats compared to newborns and 4-week-old animals [38]. In addition, TEA enhanced contractile responses to the noradrenaline of the aorta of 8-12 week old, but not neonatal, rats [38]. These authors also demonstrated that the average density of the outward potassium current in isolated smooth muscle cells from the rat aorta increased with age. This was achieved by a considerable increase in the contribution of the calciumdependent potassium current to the total outward potassium current between the age of 4 and 8 weeks [38]. In accordance with this, various blockers of BK Ca channels (TEA, charybdotoxin and paxillin) had a suppressive effect on the outward potassium current in isolated smooth muscle cells from adult, but not newborn, aorta [38,39].
The more pronounced contribution of BK Ca channels to the regulation of arterial tone in the adult organism may be due to several reasons. First, negative feedback regulation of vascular tone, as described above, requires co-localization of sarcoplasmic reticulum ryanodine receptors, voltage-gated calcium channels and BK Ca channels in the plasma membrane. Although ryanodine receptors and BK Ca channels are expressed in smooth muscle cells at newborn age, they are not yet organized into clusters; therefore, BK Ca channels cannot function properly [77]. Indeed, a considerable part of calcium sparks does not activate BK Ca channels in smooth muscle cells of newborn animals [79].
Second, the frequency of calcium sparks in the smooth muscle cells of newborn animals is considerably lower than in adults [77] and the amount of ryanodine receptors increases with age in, at least, the rat saphenous artery [80]. Probably, as a compensation for limited calcium availability, BK Ca channels of immature arteries have a lower set point for calcium (the calcium concentration required for half-maximum activation at a membrane potential of 0 mV) as it was demonstrated in isolated smooth muscle cells of sheep cerebral arteries [81]. It was suggested that BK Ca channels of fetal sheep arteries have a greater sensitivity to calcium because they are more phosphorylated by the protein kinase G [82,83].
Third, the expression of BK Ca channels in smooth muscle cells can change as the organism matures. It was demonstrated that the mRNA expression of both the poreforming α1and the regulatory β1-subunits, where the latter has a positive effect on channel activity [84,85], increases with age [45,46]. In addition, the level of labeled charybdotoxin (a ligand of BK Ca channels) binding was considerably lower in the aorta of 4-week-old compared to 12-week-old rats [38].
Finally, the biophysical properties of BK Ca channels may vary at different developmental stages. BK Ca channels of adult aortic smooth muscle cells remain open almost 10 times longer than the channels of fetal cells [86]. In addition, currents mediated by BK Ca channels in isolated smooth muscle cells of the middle cerebral artery of adult female baboons activated faster at lower membrane potential values than in fetal cells [87]. Probably the differences in the biophysical properties of the BK Ca channel between an adult and an immature organism correlate with the expression of the regulatory β1-subunit, which has a positive regulatory effect on BK Ca channel function [84,85].

Conclusions
Taken together, the data described above demonstrate that the pattern of potassium channel expression and function in arterial smooth muscle changes dramatically while the organism matures ( Figure 1, Table 1). Importantly, such alterations do not seem to be dependent on the embryological origin of the particular vascular bed, since similar patterns of changes were observed in the proximal part of the aorta and smaller arteries [88]. BK Ca channels acquire an important role in the regulation of vascular tone with maturation, but their role in developing arteries is limited. BK Ca channel activation by calcium sparks is weak in the newborn organism due to an unfinished co-localization process with RyRs of the sarcoplasmic reticulum and a low calcium spark frequency. Less calcium-dependent potassium channel types (K v 1, K v 7, TASK-1 and K ir 2) play the main role in counteracting vasocontraction at early postnatal life. Indeed, contraction of the arterial smooth muscle depends much more on calcium in the adult age than in the early postnatal period. Therefore, while the vascular remodeling in postnatal development proceeds (including the formation of BK Ca /RyRs clusters), a switch of the potassium channels from less to more calcium-dependent ones occurs. Conditions affecting arterial smooth muscle potassium channels expressed at high levels during the early postnatal period, especially Kv7 and TASK-1 channels, may disturb the developmental switch of potassium channel contribution to arterial tone regulation. This may possibly lead to disturbances in potassium channel function in childhood. The latter in its turn may cause the development of cardiovascular disorders, including hypertension [3,[89][90][91][92][93][94], the occurrence of which is steadily increasing in childhood [95]. Decrease SMCs from sheep cerebral arteries IbTx (100 nM) [81] No change Sheep middle cerebral artery IbTx (100 nM) [64] * An increase in K ATP functional contribution was shown with the use of the activators lemakalim and pinacidil. All other substances listed above are blockers of potassium channels: 4-AP-4-aminopyridine; DPO-1-diphenyl phosphine oxide-1; Glib-glibenclamide; IbTx-iberiotoxin; ScTx-stromatoxin; TEA-tetraethylammonium.  [3,[89][90][91][92][93][94], the occurrence of which is steadily increasing in childhood [95]. Figure 1. The switch of the leading role in the regulation of vascular tone from Kir2, Kv1, Kv7 and TASK-1 channels to BKCa channels with maturation. The activation of potassium channels leads to K + efflux and hyperpolarization of the membrane, which in turn suppresses the opening of voltage-dependent calcium channels (VDCC) and counteracts vasocontraction. The arrow thickness represents the functional impact of the channel in this process. Figure 1. The switch of the leading role in the regulation of vascular tone from K ir 2, K v 1, K v 7 and TASK-1 channels to BK Ca channels with maturation. The activation of potassium channels leads to K + efflux and hyperpolarization of the membrane, which in turn suppresses the opening of voltage-dependent calcium channels (VDCC) and counteracts vasocontraction. The arrow thickness represents the functional impact of the channel in this process.

Conflicts of Interest:
The authors declare no conflict of interests.