Cycling matters: Sex hormone regulation of vascular potassium channels

ABSTRACT Sex hormones and the reproductive cycle (estrus in rodents and menstrual in humans) have a known impact on arterial function. In spite of this, sex hormones and the estrus/menstrual cycle are often neglected experimental factors in vascular basic preclinical scientific research. Recent research by our own laboratory indicates that cyclical changes in serum concentrations of sex -hormones across the rat estrus cycle, primarily estradiol, have significant consequences for the subcellular trafficking and function of KV7. Vascular potassium channels, including KV7, are essential components of vascular reactivity. Our study represents a small part of a growing field of literature aimed at determining the role of sex hormones in regulating arterial ion channel function. This review covers key findings describing the current understanding of sex hormone regulation of vascular potassium channels, with a focus on KV7 channels. Further, we highlight areas of research where the estrus cycle should be considered in future studies to determine the consequences of physiological oscillations in concentrations of sex hormones on vascular potassium channel function.


Introduction
Cyclical changes in sex hormones driven by the human or rodent reproductive cycle coincide with changes in vascular function [1].To date, research regarding the impact of sex hormones on vascular function has focused on sex-hormone-mediated changes in the production of endothelial-derived relaxing factors such as nitric oxide (NO), prostaglandin I 2 (PGI 2 ), or endothelial-derived hyperpolarization (EDH) [2][3][4][5].These pathways depend on the activation of vascular smooth muscle cell (VSMC) potassium (K + ) channels to elicit their effect.For example, large conductance calciumactivated potassium channels (BK Ca ), voltage-gated potassium channels (K V ), and inwardly rectifying potassium channels are activated in response to NO [6,7], PGI 2 [8][9][10][11][12], and EDH [13,14], respectively.Yet, comparably little is known about the influence of sex hormones on vascular smooth muscle K + channels.Recently, Baldwin et al. 2022 and2023 showed the functional impact of arterial KCNQ-encoded voltage-dependent potassium channels (termed Kv7s) changed considerably across the rat estrus cycle [12,15].These works are in the vanguard of research identifying estrus-cycle-dependent oscillations in smooth muscle functionality outside of the sex-dependent differences in arterial responsiveness conventionally reported [16].Alterations in smooth muscle ion channel activity driven by sex hormone oscillations have potential implications in disease manifestations and pharmacological treatments, highlighting why estrus "cycling" matters.This review provides an overview of this nascent and emerging field of research and draws comparisons with known work in other cell types.It focuses primarily on Kv7 channels, but also includes aspects on how sex and sex hormones regulate other K + channels.

The estrus cycle
Sex hormones, principally estrogens, progesterone, and androgens, fluctuate throughout the course of the estrus (rodent) and menstrual (human) cycles.A direct comparison between rodent and human cycles is difficult as the rodent estrus cycle last only 4-5 days and is split into four stages (proestrus, estrus, metestrus and diestrus); whereas the human cycle lasts roughly 28 days and has three phases (follicular, ovulation and luteal), followed by menses.Deducing the impact of sex hormones on vascular reactivity throughout the menstrual cycle is challenging, not least because of additional genetic and environmental factors that will also drive significant changes in human vascular reactivity.Thus, there is a paucity of human data describing arterial function changes throughout the menstrual cycle.As such, this review will focus primarily on the rodent estrus cycle, which is described comprehensively by Nilsson et al. (2015) [17].A brief overview of each hormone and their vascular receptors will be given here.

Estrogen
The bioactive form of estrogen, 17-β estradiol (E2), is synthesized in the theca and granulosa cells of the ovaries, the adrenal glands and locally within the vasculature by aromatase enzymes.E2 produces both genomic and fast acting non-genomic vascular effects through nuclear-and membrane-bound estrogen receptors (ERs).ERα/ERβ, which are canonically considered cytoplasmic nuclear receptors, dimerize to form homo/hetromers upon binding E2, which translocate to the nucleus.Active nuclear ERs bind estrogen response elements within the genome and regulate transcription [18,19].E2 also exerts rapid non-genomic effects that derive from membrane bound ERα/β [20], which evoke a myriad of intracellular signaling cascades attributed to mitogen activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK), serine and threonine kinases, phosphoinositide 3-kinase (PI3K), and cAMP [21][22][23][24].ERα/ERβ are ubiquitously expressed across the vasculature [19,25].A G protein-coupled estrogen receptor (GPER1), designated GPR30 initially, is also expressed in the membrane of the sarcoplasmic reticulum, the golgi apparatus and the plasma membrane of VSMCs and endothelial cells (ECs) [19].GPER1 couples to a complex array of signaling cascades via G αs,i,q/ll and is further complicated via receptor cross talk with the mineralocorticoid and epidermal growth factor receptors [26,27].

Progesterone
Progesterone is a steroidal hormone produced within the adrenal cortex, gonads and the corpus luteum of the pregnant rat.Its receptors have been found within both ECs and VSMCs of rats, mice and humans [28].Compared to E2, the literature on progesterone within the vasculature is sparse, though it can be broken down into nuclear and non-nuclear signaling.Progesterone binds two distinct cytoplasmic nuclear receptors termed progesterone receptor (PR) A/B, which are derived from a single gene.Upon binding progesterone, the receptors dimerize, forming homo/heteromers, which translocate to the nucleus and bind progesterone response elements that subsequently regulate gene transcription [29,30].Moreover, a novel class of membranebound progesterone receptor (mPR) has been discovered and includes three subtypes, α, β and γ.The activation of mPR is associated with G αi and subsequent decrease in cAMP, as well as the activation of MAPKs, ERK1/2, and c-Jun N-terminal kinase (JNK1/2) [31][32][33].

Testosterone
Testosterone, and its more potent analogue dihydro testosterone, are produced within the gonads and adrenal glands of both sexes.Like E2 and progesterone, testosterone activates both nuclear-and membrane-bound androgen receptors (ARs).Classic ARs include two isoforms, A/ B, which are cytosolic nuclear transcription factors that dimerize and translocate to the nucleus where they bind androgen response elements, mediating a change in gene transcription [24].Recently, a novel class of testosterone-activated G proteincoupled receptor was discovered [34,35], in addition to membrane-associated nuclear ARs.Upon binding testosterone, activated membraneassociated nuclear, and membrane-bound G protein-coupled ARs undergo a conformational change in shape, and can mediated rapid nongenomic effects associated with the activation of a protein kinase C (PKC), protein kinase A (PKA), MAPK, cAMP, ERK, and protein kinase G (PKG) [24,34,35].Testosterone can also be converted to E2 locally within the vasculature by aromatase activity, which complicates delineation between vascular testosterone and E2 signaling.

K + channels
K + channels are a fundamental factor in the generation of VSMC resting membrane potential, which is roughly −70 to −55 mV [36].As K + channels open, the cell becomes permeable to K + ions, which move down their electrochemical gradient, exiting the cell, thus moving VSMC membrane potential (V m ) toward the equilibrium potential for K + (E k ).As E k within VSMC is roughly −90 mV, the cell membrane becomes hyperpolarized.Despite the known role for K + channels in VSMC reactivity within the male, their role within the female vasculature and how their activity is affected by hormonal fluctuations is poorly understood.K + channels are principally comprised of homo or heterotetrameric configurations of αsubunits that exist within the membrane in associated with β-auxiliary subunits.β-auxiliary subunits regulate the biophysical, physiological and pharmacological properties of the channel.Both the α-and β-subunits are candidate targets for sex hormone regulation of ion channel function, and will be discussed below.See Table 1 for a summary of the effect of sex hormones on vascular and non-vascular K + channels.K V 7 channels K V 7 channels are a K V channel subfamily encoded for by the genes KCNQ1-5, which give rise to distinct α-subunit proteins, termed K V 7.1-7.5,respectively.K V 7 channels activate slowly upon depolarization with a relatively negative threshold compared to other K V channels (~-40 mV) and exhibit little inactivation.Every human or rodent vascular bed studied expresses K V 7 channel transcripts, with Kcnq1, Kcnq4, and Kcnq5 being the principally expressed genes [92,93].There is comparably little contribution from Kcnq3 and rarely expression of Kcnq2.K V 7 channel stoichiometry varies between systems.Vascular K V 7.1 is commonly thought of as exclusively homotetrameric [94], although Oliveras et al. (2014) demonstrated a K V 7.1/7.5 heteromer within coronary artery VSMCs from rats [95].Vascular K V 7.4 readily forms heterotetrameric channels with Kv7.5, but are also found as homotetrameric channels, whereas K V 7.5 exists predominantly with K V 7.4 in native VSMCs [96,97].Within rat [98] and murine [99] mesenteric VSMCs, K V 7.4 and Kv7.4/7.5 channels exist in close proximity with the KCNE4 β-auxiliary subunit protein, which positively regulates channel trafficking and biophysical/pharmacological properties [99].K V 7 channels regulate arterial smooth muscle contractility and are a key component of receptormediated vasodilations in arteries from male animals.Application of the pan-K V 7 channel blockers, linopirdine or XE991, depolarizes rat V m [100,101] and mediates a rise in baseline tension in both human and rodent arteries [92,[102][103][104][105].In contrast, K V 7.1-specific blockers like HMR-1556 or chromanol 293B do not effect baseline tension in rat aorta, mesenteric nor intrapulmonary arteries [106].Functional vascular K V 7 channel activity is further evidenced by relaxation of precontracted arterial tone in response to several K V 7.2-7.5 channel activators (e.g.retigabine, acrylamide S-1, ML213) across a number of rodent arteries [93,105,107,108].Similarly, activators of K V 7.1, including (R)-L3, Mefenamic acid and ML277 [106,108], are effective and reversible relaxants of contracted arteries.These data suggest that K V 7.4/K V 7.5 are regulators of resting arterial smooth muscle V m due to their low activation threshold and that their activity hyperpolarizes the membrane potential reducing the open probability of voltage-gated calcium channels, thereby negating Ca 2+ influx.Whereas K V 7.1 channels do not contribute to resting V m , but are functionally expressed.
Pan-K V 7 blockers and molecular knockdown of K V 7.4/K V 7.5 channels also impair receptormediated vasorelaxation generated by β-adrenoreceptor, adenosine, calcitonin-gene related peptide, and prostacyclin (IP) receptor agonists (cAMPlinked [12,96,104,109,110]), as well as atrial natriuretic peptide and nitric oxide-mediated responses (cGMP-linked [111][112][113]) in many arteries from male rats.Interestingly, βadrenoreceptor mediated relaxation differentially Testosterone K Cultured rat hippocampal neurons [51] Guinea pig heat (Continued ) couples to K V 7 in a vascular bed specific manner, via PKA in renal arteries, and a novel alternate signaling cascade mediated by exchange protein activated by cAMP (EPAC) in mesenteric arteries [109].K V 7.1-specific blockers do not affect an array of receptor-mediated vasorelaxations, although Baldwin et al. (2022) revealed that relaxations of rat mesenteric artery induced by activation of IP receptors, either with iloprost or MRE-269, were attenuated by HMR1556 to the same degree as the pan-Kv7 blocker linopirdine.
A growing body of literature indicates sexual dimorphisms in vascular K V 7 physiology.Abbott and Jepps (2015) observed that mesenteric arteries from male Kcne4-/-knockout mice showed an increased sensitivity to α-1 adrenoreceptor agonist methoxamine and decreased sensitivity to K V 7.2-7.5 activator ML213, which was not observed in females [98].Further, Berg (2018) observed that vascular K V 7 channel function was preserved in female hypertensive animals when compared to males [114].As above, Baldwin et al. (2022) demonstrated a novel role for K V 7.1 as the downstream target of IP receptor evoked relaxation in mesenteric arteries from both male and female rats [12].However, the sensitivity of IP receptormediated relaxation to inhibition of K V 7.1 by preincubation of HMR-1556 (10 µmol-L −1 ) was absent in arteries from animals harvested during proestrus or estrus stages of the estrus cycle [12], stages associated with an initial spike in serum concentration of E2 [17].This correlated with a significant decrease in sensitivity to IP receptormediated relaxation when compared to mesenteric arteries from female animals harvested from diestrus/met-estrus, where HMR-1556 still had an effect.These findings were the first demonstration of an estrus cycle-sensitive regulation of arterial K V 7 function.
Subsequently, Baldwin et al. (2023) focused on responsiveness of several K V 7 channel activators across the estrus cycle of the rat.These experiments revealed that during cycle stages associated with high E2, proestrus/estrus, K V 7.2-5 channel activator-mediated relaxations were markedly impaired when compared to diestrus/metestrus, where serum E2 was low [15].Similarly, thromboxane A2 (TXA2) receptor-mediated vasoconstriction was enhanced and β adrenoceptormediated relaxation was impaired during proestrus/diestrus when compared to diestrus/metestrus [15].The sensitivity of these responses to K V 7 inhibition during proestrus/estrus was also diminished [15].An impairment of vascular function during proestrus/estrus was associated with a translocation of K V 7.4 from the plasma membrane.Both K V 7 function and protein membrane abundance were impaired by application of exogenous E2 and the GPER1 agonist, G1, in arteries from females form low serum E2 stages of the estrus cycle, diestrus/metestrus, but not proestrus/metestrus.These ex vivo manipulations were prevented by prior application of the GPER1 antagonist, G36.The findings of Baldwin et al.
(2023) were the first to identify a cyclical estrousregulation of an ion channel in the vasculature and the functional consequences.This work reinforces earlier studies [115][116][117], indicating that K V 7.4 channel abundance in the plasma membrane is labile and at the mercy of the arterial environment.
Whilst the impact of sex hormones on vascular Kv7 channels is a nascent field of research, work in other cell types also indicates an acute estrogenic inhibition of K V 7 function and expression.For example, supraphysiological concentrations of E2 (1 µmol-L −1 ) inhibit K V 7.1:KCNE1 channel derived currents in an heterologous expression system [37] and the E2 receptor antagonist, tamoxifen, increased K V 7.1 function and expression in murine and guinea pig ventricular myocytes [41], suggesting a tonic estrogenic suppression of the channel.E2-induced inhibition of K V 7.1 derived currents in rat distal colic crypt cells occurs in a PKC-δ-dependent manner, independent of the classical estrogen receptors [39], and basolateral K V currents responsible for Cl − secretion are inhibited by E2 via uncoupling of K V 7.1:KCNE3 [40].Moreover, in HT29cl.19Acells, E2 abrogated K V 7.1 membrane expression through PKC-δ-dependent channel endocytosis into early endosomes, which is then recycled to the membrane [38].Taken as a collective, the aforementioned studies provide strong evidence of estrogenic K V 7:KCNE channel regulation in a complex process of channel recycling.Within renal arteries from female rats, VSMC KCNE4 membrane abundance did not fluctuate across the estrus cycle [15].Consequently, the role of KCNE4 in E2 regulation of vascular K V 7.4 remains to be determined, though a process of E2mediated vascular K V 7.4 recycling similar to that described in Rapetti-Mauss et al. (2013) is plausible, in light of the rapid recovery of vascular K V 7.2-5 function between estrus cycle stages [15].
In contrast to E2, short-term supplemental progesterone incubation on ex vivo mesenteric arteries from female rats had no effect on the vasorelaxation elicited by K V 7.2-7.5 channel activators across the estrus cycle [15].These data cannot conclusively rule out a role for progesterone on K V 7 channel function or expression, as K V 7 channel modulator sensitivity and membrane abundance of K V 7.4 was enhanced during metestrus/diestrus, where serum progesterone was highest [15].Similarly, in mouse myometrial tissue, the total copy number for Kcnq1, Kcnq5 and Kcne1 increases during metestrus [42].Future experiments with antiprogestogens, and long-term incubation of arteries (+24hrs) are required to determine the impact of progesterone on vascular K V 7 channel function.
Within male rodents, testosterone increases the relative expression of Kcnq1 in cardiomyocytes [43].This has been postulated to underpin testosterone mediated QT interval shortening [43].The impact of testosterone signaling on vascular K V 7 channels has yet to be investigated, however, testosterone upregulates Kcne4 [44] expression in ventricular myocytes from male mice.Kcne4 transcript is more abundantly expressed in mesenteric arteries from male mice when compared to females, but Kv7.4 protein expression is lower; however, the responses to ML213 were the same [98].These data potentially suggest that females have more K V 7.4 channels but the testosteronemediated upregulation of Kcne4 increases the channel function in the males.Though this remains to be determined.K V 7 channels are being considered as a novel therapeutic target in the treatment of hypertension and other diseases [118].Though the gross impact of "estrus cycling" vascular K V 7 channel function on blood pressure and health has yet to be established, current understanding indicates that the pharmacology required to target the channel conventionally, through activators for example, would change dramatically across the menstrual cycle, thereby excluding these channels as convenient targets in pre-menopausal women.
Within murine mesenteric arteries, no difference in the abundance of K V 1.5 was detected between males and females [136], however whole cell K V currents were greater in mesenteric arteries from female mice [136] and rats [66].Though estrus cycle regulation of vascular K V 1 channels has yet to be investigated, Saito et al. (2009) observed diminished K V 1.5 currents and protein in murine ventricular myocytes harvested from females in estrus when compared to di-estrus [45].Further, supplemental E2 inhibited ventricular K V 1.5 currents and protein expression in ovariectomized mice [45] and tamoxifen increased the relative abundance of K V 1.5 [41].Some evidence indicates that levels of progesterone seen during pregnancy can impair K V 1.3/1.5 transcript expression in native cardiac myocytes and heterologous expression systems [46].Within the vasculature, testosterone deprivation impairs K V current and K V 1.5 expression in aortic VSMCs [49].Similarly, within ventricular myocytes, castration diminished and testosterone replacement enhanced K V 1.5 expression and function [47,48].Evidence therefore exists for estrus cycle dependent regulation of K V 1 channels [45], with E2 impairing and testosterone promoting K V 1 function, though this remains to be investigated within the vasculature.

K V 2 channels
Several studies have identified KCNB-encoded K V 2.1 and K V 2.2 in whole lysates of arteries [137,138].Of note, K V 2.2 is minimally expressed compared to K V 2.1 in rat middle cerebral artery [139] and aorta [140].As K V 2.1 and K V 2.2 form heterotetramers [141], the effects of K V 2.2 can be difficult to delineate.Consequently, the current literature has focused predominantly on K V 2.1 by comparison to K V 2.2.K V 2.1 contributes to K V currents in rat pulmonary [142], mesenteric [143] and aortic [144,145] VSMCs.Functionally, K V 2.1 regulates resting V m in rat middle cerebral artery and opposes myogenic tone in a pressure-sensitive process [146] in heteromultimeric configurations of K V 2.1/K V 9.3 [139].K V 2.1 is also upregulated in response atrial natriuretic peptide-and NO-dependent signaling within the rat aorta [140].
The only study to consider sex-differences in vascular K V 2 channels to date found that mesenteric VSMCs from female C57BL/6J mice expressed greater K V 2.1 protein and current when compared to males [136].Paradoxically, these findings were observed in conjunction with enhanced Ca V 1.2 open probability, greater pressure mediated [Ca 2+ ] i increase and pressure induced tone within arteries from female mice [136].To explain these contrasting findings, O'Dwyer et al. (2020) argue for a novel role for non-conductive K V 2.1 channels in regulating VSMC plasmalemmal Ca V 1.2 channel clustering and function [136].Thus, in their model, a greater ratio of K V 2.1:Ca V 1.2 in arteries from female animals leads to enhanced Ca 2+ influx and constriction [136].
Though the estrus cycle was not considered in the previous study, some evidence exists for the regulation of K V 2 by sex hormones.Supplemental E2 diminishes both native K V 2.1 currents in osteocytes, in a process speculated to be non-genomic [50], and over-expressed K V 2.1 in cultured hippocampal neurons [51].Further, ER inhibition with tamoxifen increases K V 2.1 expression in guinea pig heart [41], ER β signaling impairs K V 2.1/2.2currents in β-pancreatic cells [52] and GPER1 activation inhibits K V 2.2 expression in a PKC-sensitive signaling cascade [53].Though in murine ventricular myocytes, no estrus cycle-sensitive change in K V 2.1 current or protein expression was observed, which calls into question whether Kv2 channels are regulated by estrous/menstrual cycle-dependent changes in E2 levels [45].The role of progesterone in regulating K V 2 is even less clear.Progesterone (1 µmol-L −1 ) diminished K V 2.1 expression in murine cardiac myocytes via mPR, which reportedly underpins prolonged QT intervals and action potential duration [46].Though progesterone (10 µmol-L −1 ) had no effect on K V 2.1-encoded currents in HEK 293 cells [54].The reason for this discrepancy is currently unclear, however, a serum concentration of 1 µmol-L −1 progesterone is not achieved during pregnancy [147].As such, estrus cycle regulation of vascular K V 2 channels remains speculative; however, sex differences in vascular K V 2 expression and function is indicated and merits further investigation.

K V 11.1 channels
Isoforms of KCNH2 encode K V 11.1, commonly known as ether-a-go-go (ERG)-related channels, including ERG1a and ERG1b, contribute to the fast-component of the delayed rectifying current (QT interval) of the cardiac action potential [148,149].Within the vasculature, currents sensitive to the ERG1 blocker, E-4031, were demonstrated in murine portal vein [150,151], and K V 11 protein and transcript were identified within isolated murine aortic, carotid and femoral arterial smooth muscle cells [152], though no ERG currents were detectable.Modulation of K V 11.1 did however alter VSMC proliferation [152].Additionally, K V 11.1 inhibitor dofetilide had no functional effect in quiescent vessels, including rat aorta, mesenteric and intralobar arteries [153].The current consensus is that K V 11.1 functionally contributes to repolarization kinetics of spontaneously contractile smooth muscle, including portal vein [62,151,152], esophageal [154], jejunum [155], myometrial [62], and bladder [156], as well as playing a role in VSMC proliferation.
No papers to date have investigated the role of K V 11.1 within the vasculature of the female; however, a host of literature has investigated the role of sex hormones in regulating cardiac ERG channels.Ablation of E2 production by aromatase knockout in a mouse model impaired QT interval sensitivity to E-4031 [55].Furthermore, E2 drives K V 11.1 forward trafficking in cardiomyocytes by enhanced interaction with HSP90, thereby increasing ventricular repolarization [57].However, the E2 receptor inhibitor tamoxifen increased K V 11.1 protein expression [41] and physiological concentrations of E2 reduce K V 11.1 currents in guinea pig hearts [56] and HEK cells [58].Additionally, mutagenesis [56], pharmacological [58], and computational [59] studies indicate that E2 binds to the K V 11.1 pore in a manner similar to K V 11.1 inhibitor dofetilide [59].
In contrast to E2, the role of progesterone and testosterone derivatives in regulating K V 11.1 is clearer.Physiological levels of progesterone seen during the follicular phase upregulate cardiac K V 11.1 currents in guinea pig ventricular myocytes [60].Though progesterone, as seen during pregnancy, impairs K V 11.1 forward trafficking in rat neonatal cardiac myocytes [61].Similarly, the considerable impact of K V 11.1 blockers on myometrial contractility is lost in pregnant mice and humans as they progress through pregnancy [62,63].This effect is associated with an increase in KCNE2 proteins as labor develops [62,63], reinforcing the crucial role for auxiliary subunit proteins in K + channel regulation and as the site of hormone based regulation.Similarly to progesterone, testosterone improves native K V 11.1 function in HEK cells [64] and rabbit ventricular myocytes in a non-genomic post-transcriptional mechanism which enhances K V 11.1 current density and voltage sensitivity [65].
These findings implicate a cyclical regulation of E2 suppression and progesterone promotion of K V 11.1 function across the estrus cycle, either through channel block or through auxiliary subunit regulation, respectively.As no ERG currents have been demonstrated in K V 11.1 expressing quiescent systemic vascular beds [152], the functional consequence for sex hormones in mediating sex-differences in K V 11.1 regulation may be slight.Though in light of the role of ERG in mediating VSMC proliferation, there is a potential role for androgenic regulation of K V 11.1 in sex-differences in vessel size [157], angiogenesis [158], vascularization [159], and angiogenesis [160].

BK Ca channels
KCNMA1 encodes for the pore-forming α subunit K Ca 1.1, which in turn gives rise to homotetrameric channels within the membrane termed BK Ca .Unique amongst K Ca , BK Ca are both Ca 2+ and voltage activated [161][162][163].Within the vasculature, BK Ca expression is predominantly restricted to VSMCs, though some expression may be present within the endothelium [164,165].Vascular BK Ca channels primarily couple with the K Ca β1 auxiliary subunit, which improves [Ca 2+ ] i and voltage sensitivity when compared to K Ca 1.1 alone [166][167][168].In a vascular bed specific manner, inhibitors of BK Ca channels increase basal and pressure-induced tone [11,[169][170][171][172][173].These descriptions provide only a brief insight into the role of BK Ca channels within the vasculature, for more detail see [174].
BK Ca channels have been identified in male and female human mesenteric [175] and coronary [176] arteries, though data was not separated dependent on sex.Functional BK Ca channels have also been demonstrated in omental and myometrial arteries from women [177,178].Within rodents, BK Ca -derived currents are greater within middle cerebral artery VSMCs isolated from adult female rats when compared to age-matched males, though this phenotype dissipates with age, due to a decrease in K Ca β1 expression [66].Though the estrus cycle was not factored into this study, ovariectomy diminished BK Ca channel expression and enhanced pressuremediated increases in vessel tone [66] and BK Ca expression and vessel function was ameliorated by supplemental E2 dietary pellets [66].
Mechanistic insight into E2-mediated changes in BK Ca function indicate that E2 (0.02-5 µmol-L −1 )-GPER1 signaling upregulated vascular BK Ca currents in human [68,69] and porcine [70] coronary artery VSMCs, via PKC.Further, ER αderived PI3K signaling increased BK Ca 1.1 transcript expression in cultured human coronary and aortic VSMCs [71] and E2 (0.3 nmol-L −1 ) treatment increased BK Ca currents in ovine uterine arteries via K Ca β1 modulation [72][73][74].The importance of the discrepancies in the receptor responsible for E2-mediated increases in vascular BK Ca activity is debatable, as E2 has similar affinities for both ER α/β and GPER1 [179].These data strongly support an upregulation of BK Ca channels in VSMCs by E2, although it should be noted that outside VSMCs, including in cultured human endothelial cells [67], BK Ca channel expression and function is negatively regulated by E2 [180].
Investigations regarding the effect of progesterone on vascular BK Ca are sparse.In heterologous expression systems, progesterone (10 µmol-L −1 ) inhibits BK Ca derived currents [75], in a process speculated to be due to an increase in intracellular pH, and a decrease in cytosolic Ca 2+ /cAMP.Though progesterone (0.1 µmol-L −1 ) has no effect on native BK Ca currents in uterine artery VSMCs from sheep [72].
Conversely, testosterone can enhance BK Camediated relaxations through activation of the NO-PKG cascade in porcine coronary [76,77] and rat mesenteric arteries [78].As both serum testosterone and E2 are raised significantly during proestrus [17], current literature would indicate an increase in vascular responsiveness during proestrus.In contrast to this hypothesis, we observed no change in relaxation response to the BK Ca channel activator NS11021 in mesenteric nor renal arteries harvested from female animals across the estrus cycle [15].The reason for this discrepancy remains to be determined and requires further investigation.

K ATP channels
ATP-sensitive K + channels (K ATP ) are a member of the inwardly rectifying K + channel (K IR ) subfamily and are derived from K IR 6.x proteins.K ATP however express poor inward rectification by comparison to other K IR channels [181,182].Expression of K IR 6.x alone does not form functional K + channels, but requires the co-expression of sulphonylurea receptors (SURs) [183][184][185][186][187], including SUR1, SUR2A and SUR2B [188].K ATP are now recognized as intracellular sensors that convey changes in metabolic states to electrical signals across the membrane.K ATP inhibitors contract coronary [189,190] and renal arteries [191] in normotensive animals, implicating K ATP in the regulation of basal arterial tone.K ATP is the target of myriad receptor-mediated signaling cascades, for more detail, see [174].
Functional K ATP channels are an essential components of human chorionic plate, umbilical and myometrial artery reactivity [178,[192][193][194][195]. K ATP channels have been demonstrated in middle cerebral, meningeal, superficial temporal and cutaneous arteries within male and female humans [196][197][198][199][200]. Reports of sexual dimorphisms in vascular K ATP function are contradicting.For example, there are no apparent sex-dependent differences in sensitivity to K ATP channel modulators in rodent cerebral [201,202] nor mesenteric [203] arteries from normotensive rats, though a later study found impaired K ATP activator mediated relaxation within arteries from male hypertensive rats when compared to females [203].Similarly, inhibition of K ATP had greater impact on skeletal muscle tissue oxygenation in female rats when compared to males [204], reportedly as a result of increased VSMC K ATP activity in female rats [204].
As regards to the role of sex hormones in regulating K ATP , E2 protects against ischemic reperfusion injury within myocardium [79] and neurons [85] by enhanced mitochondrial-K ATP (mKATP) and K IR 6.2/SUR1 function, respectively.mKATP channels when activated prevent apoptosis, enhance respiration and reduce matrix Ca 2+ accumulation [205].E2-mediated upregulation of K ATP function is reported as both a genomic and non-genomic process, for example, E2 upregulates SUR expression within myocardial and neuronal cells [79,84,85] and also gives rise to a PKA-PKC-mediated increases in K ATP activity in neuronal cells [86,87].Similarly within humans, following angioplasty, E2 protects against myocardial ischemia by increasing myocardial K ATP activity and through enhanced coronary artery vasodilation [83], however, increased coronary artery vasodilation in response to E2 was insensitive to K ATP inhibition [83].In contrast to neuronal and myocardial cells, E2 rapidly inhibited K ATP function within murine β-pancreatic cells via ERβ [80,81] in a cGMP-PKG-sensitive signaling cascade [82].Consequently, the role of E2 in regulating vascular K ATP expression or activity remains speculative, and may be either positive or negative.
A comprehensive study on the effect of progesterone on K ATP is yet to be undertaken.Within our own study, we observed an estrus cycledependent shift in sensitivity to relaxations elicited by a K ATP activator within mesenteric, but not renal, arteries from female rats within proestrus/ estrus, when compared to arteries from female rats within diestrus/metestrus [15].When comparing serum sex hormone concentration between these groups we observed raised E2 in the former and raised progesterone in the later [15].Consequently, it is unclear if mesenteric vascular K ATP channel function is impaired by E2 or enhanced by progesterone [15]; however, pinacidil sensitivity was comparable between arteries from females of a low-serum estradiol stage of the estrus cycle and arteries from male animals (data not published), indicative of E2 inhibition of vascular K ATP function.Further, the cause of the vascular bed specific estrus cycle regulation of K ATP channel function was also unclear.
Finally, conflicting reports indicate testosterone as either a K ATP -dependent or K ATP -independent vasodilator [206].For example, testosterone mediates K ATP sensitive vasorelaxation in canine coronary [88], rat aortic [90] and human corporal [91] arteries, either via release of endothelial derived relaxant factors [88,90] or via direct interaction with the channel [91].Conversely, the K ATP inhibitor Glibenclamide had no effect on testosterone-mediated relaxation in rat aorta [89].The reason for the discrepancy between Honda et al. (1999) and Ding and Stallone (2001), may be accounted for by differences in rat models.Both studies employ similar concentrations of both testosterone and Glibenclamide yielding conflicting results, warranting further investigation.

Conclusion
This brief review demonstrates a range of extravascular and vascular studies, which detail sexual dimorphisms in ion channel function.These differences are further complicated by genomic and non-genomic sex hormone regulation of ion channels.However, fluctuations in sex hormone levels across the estrus cycle is rarely taken into consideration and studies commonly favor supraphysiological concentrations of sex hormones to investigate their role.Our lack of understanding of female biology from a basic preclinical science perspective has culminated in a greater prevalence in adverse drug reactions within women when compared with age-matched men [207].This review highlights the need to revisit basic principles of ion channels within the vasculature of the female, with keen consideration of the estrus cycle.

Disclosure statement
No potential conflict of interest was reported by the authors.

Table 1 .
The effect of sex hormones on vascular and non-vascular K + channels.*= hormone mediator speculated.