K+ Channels Channels Research Articles

Loss of anoctamin 1 reveals a subtle role for BK channels in lymphatic muscle action potentials.

Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-activated K+ channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca2+-activated K+ channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT2-Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization.

A Novel Cell Volume Sensor for Real-Time Analysis of Ca2+-Activated K+ Channel.

Potassium channels play a vital role in cell volume regulation. A cell volume sensor was constructed by integrating regulatory volume decrease (RVD) with quartz-crystal microbalance (QCM) for studying potassium channels and their expression. The sensor successfully monitored the K+ channel's activities during RVD by sensitive and noninvasive means. It showed that Ca2+ activated the K+ channel (KCa) and enhanced the RVD level. The inhibition of blockers on K+ channels exhibited an obvious difference in RVD level between normal and cancerous nasopharyngeal cells, suggesting that the KCa channel contributes a dominant role to the RVD function and provides an approach to identify the activation of various K+ channels.

KCa2 and KCa3.1 Channels in the Airways: A New Therapeutic Target.

K+ channels are involved in many critical functions in lung physiology. Recently, the family of Ca2+-activated K+ channels (KCa) has received more attention, and a massive amount of effort has been devoted to developing selective medications targeting these channels. Within the family of KCa channels, three small-conductance Ca2+-activated K+ (KCa2) channel subtypes, together with the intermediate-conductance KCa3.1 channel, are voltage-independent K+ channels, and they mediate Ca2+-induced membrane hyperpolarization. Many KCa2 channel members are involved in crucial roles in physiological and pathological systems throughout the body. In this article, different subtypes of KCa2 and KCa3.1 channels and their functions in respiratory diseases are discussed. Additionally, the pharmacology of the KCa2 and KCa3.1 channels and the link between these channels and respiratory ciliary regulations will be explained in more detail. In the future, specific modulators for small or intermediate Ca2+-activated K+ channels may offer a unique therapeutic opportunity to treat muco-obstructive lung diseases.

Signaling and structures underpinning conducted vasodilation in human and porcine intramyocardial coronary arteries.

BackgroundAdequate blood flow into coronary micro-arteries is essential for myocardial function. Here we assess the mechanisms responsible for amplifying blood flow into myogenically-contracting human and porcine intramyocardial micro-arteries ex vivo using endothelium-dependent and -independent vasodilators.MethodsHuman and porcine atrial and ventricular small intramyocardial coronary arteries (IMCAs) were studied with pressure myography and imaged using confocal microscopy and serial section/3-D reconstruction EM.Results3D rendered ultrastructure images of human right atrial (RA-) IMCAs revealed extensive homo-and hetero-cellular contacts, including to longitudinally-arranged smooth muscle cells (l-SMCs) found between the endothelial cells (ECs) and radially-arranged medial SMCs (r-SMCs). Local and conducted vasodilatation followed focal application of bradykinin in both human and porcine RA-IMCAs, and relied on hyperpolarization of SMCs, but not nitric oxide. Bradykinin initiated asynchronous oscillations in endothelial cell Ca2+ in pressurized RA-IMCAs and, as previously shown in human RA-IMCAs, hyperpolarized porcine arteries. Immunolabelling showed small- and intermediate-conductance Ca2+-activated K+ channels (KCa) present in the endothelium of both species, and concentration-dependent vasodilation to bradykinin followed activation of these KCa channels. Extensive electrical coupling was demonstrated between r-SMCs and l-SMCs, providing an additional pathway to facilitate the well-established myoendothelial coupling. Conducted dilation was still evident in a human RA-IMCA with poor myogenic tone, and heterocellular contacts were visible in the 3D reconstructed artery. Hyperpolarization and conducted vasodilation was also observed to adenosine which, in contrast to bradykinin, was sensitive to combined block of ATP-sensitive (KATP) and inwardly rectifying (KIR) K+ channels.ConclusionsThese data extend our understanding of the mechanisms that coordinate human coronary microvascular blood flow and the mechanistic overlap with porcine IMCAs. The unusual presence of l-SMCs provides an additional pathway for rapid intercellular signaling between cells of the coronary artery wall. Local and conducted vasodilation follow hyperpolarization of the ECs or SMCs, and contact-coupling between l-SMCs and r-SMCs likely facilitates this vasodilation.

Distinct patterns of exocytosis elicited by Ca2+, Sr2+ and Ba2+ in bovine chromaffin cells.

Three divalent cations can elicit secretory responses in most neuroendocrine cells, including chromaffin cells. The extent to which secretion is elicited by the cations in intact depolarized cells was Ba2+ > Sr2+ ≥ Ca2+, contrasting with that elicited by these cations in permeabilized cells (Ca2+ > Sr2+ > Ba2+). Current-clamp recordings show that extracellular Sr2+ and Ba2+ cause membrane depolarization and action potentials, which are not blocked by Cd2+ but that can be mimicked by tetra-ethyl-ammonium. When applied intracellularly, only Ba2+ provokes action potentials. Voltage-clamp monitoring of Ca2+-activated K+ channels (KCa) shows that Ba2+ reduces outward currents, which were enhanced by Sr2+. Extracellular Ba2+ increases cytosolic Ca2+ concentrations in Fura-2-loaded intact cells, and it induces long-lasting catecholamine release. Conversely, amperometric recordings of permeabilized cells show that Ca2+ promotes the longest lasting secretion, as Ba2+ only provokes secretion while it is present and Sr2+ induces intermediate-lasting secretion. Intracellular Ba2+ dialysis provokes exocytosis at concentrations 100-fold higher than those of Ca2+, whereas Sr2+ exhibits an intermediate sensitivity. These results are compatible with the following sequence of events: Ba2+ blocks KCa channels from both the outside and inside of the cell, causing membrane depolarization that, in turn, opens voltage-sensitive Ca2+ channels and favors the entry of Ca2+ and Ba2+. Although Ca2+ is less permeable through its own channels, it is more efficient in triggering exocytosis. Strontium possesses both an intermediate permeability and an intermediate ability to induce secretion.

Partial loss of Ca2+‐Activated K+ channel (KCa) in Rat Cerebral Arterial Muscle in Insulin‐dependent diabetes mellitus

Cerebral arteries depolarize and constrict upon pressurization. This is one of the intrinsic mechanisms underlying autoregulation of cerebral blood flow (CBF). The Ca2+‐activated K+ channel (KCa) channel in arterial muscle is an important target through which cerebral arterial response to changes in endogenous or environmental stimulatory factors are sensed. The level of open state probability (NPo) of KCa channels determines the degree of myogenic tone and cytosolic Ca2+ concentration. In the present study we report impaired pressure‐dependent constriction of cerebral arteries and diminished density and NPo of KCa channel currents associated with reduced expression of KCa channel α‐ and β‐subunit proteins in cerebral arteries of genetically insulin‐dependent diabetic rats. In this study we also found that the expression of the CYP 4A ω‐hydroxylase protein and the production of 20‐HETE was significantly increased in the type 1 diabetic cerebral arteries compared to that in the non‐diabetic cerebral arteries. The observed changes in KCa channel function and expression of CYP 4A ω‐hydroxylase and production level of 20‐HETE could represent an important pathologic mechanisms that could underlie abnormalities in cerebral arterial reactivity in insulin‐dependent diabetes mellitus. It is concluded that the diminished KCa channel function, increased production of 20‐HETE and altered pressure‐dependent cerebral arterial constriction in insulin‐dependent diabetes mellitus may lead to impaired autoregulation of CBF that could contribute to increased risk of stroke in this disease condition.Support or Funding InformationNIH RO1 HL0338833This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Beta Adrenergic Induced Pulmonary Arterial Vasodilation Following Long Term Hypoxia in Fetal and Adult Sheep

Long term high altitude exposure is known to cause tissue hypoxia and exposure of fetuses as well as adults to rarefied environments increases morbidity and mortality. In particular, hypoxia can cause developmental abnormalities in the fetal pulmonary vasculature and contribute to pulmonary arterial hypertension in the adult. β2 adrenergic receptor (β‐AR) agonists are a major treatment for asthma, however far less is known about their role in pulmonary arterial function. Even still, previous evidence illustrates that β‐AR stimulation holds promise for the treatment of pulmonary arterial hypertension. Other evidence illustrates that β‐AR stimulation modulates the activity of Ca2+‐activated K+ channels (KCa), which are important mediators of pulmonary vasorelaxation. Dysregulation of KCa may contribute to chronic hypoxia induced pulmonary hypertension and β‐AR signaling may be involved in the process. In these studies we tested the hypothesis that β‐AR ‐ mediated pulmonary arterial vasodilation may be preserved following long term high altitude hypoxia due to dependency on KCa channels. This hypothesis was addressed in isolated pulmonary arteries from adult or fetal sheep that gestated at 700 meters (normoxic) or 3,801 meters for 110+ days (hypoxic). Myography was performed on pulmonary arterial rings to evaluate the vasorelaxation in response to the β‐AR agonist isoproterenol (ISO) and the methylxanthine phosphodiesterase inhibitor, IBMX. ISO‐mediated relaxation was preserved following hypoxia in fetal sheep and relaxation was greater in arteries from adult hypoxic animals relative to fetuses. Inhibition of BKCa with TEA blunted ISO‐mediated vasorelaxation in fetal normoxic and adult hypoxic arteries. IBMX caused a dose dependent relaxation in arteries from all groups and TEA did not influence this effect. Overall, these studies provide evidence that β‐AR stimulated pathways cause pulmonary arterial vasodilation, but with varying reliance on BKCa activation. Targeting β‐AR signaling pathways could therefore be therapeutically significant and functional alterations in the pathways may contribute to the development of disease.Support or Funding InformationThis material is based upon work supported by NIH HD‐069746, P01HD083132, and P20MD006988. MA and BP were Walter E Macpherson Medical Student Summer Research Fellows while AV was an Undergraduate Summer Research Fellow through the Center for Health Disparities and Molecular Mechanisms at Loma Linda University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ca2 +-dependent potassium channels and cannabinoid signaling in the endothelium of apolipoprotein E knockout mice before plaque formation

Endothelial Ca2+-dependent K+ channels (KCa) regulate endothelial function. We also know that stimulation of type 2 cannabinoid (CB2) receptors ameliorates atherosclerosis. However, whether atherosclerosis is accompanied by altered endothelial KCa- and CB2 receptor-dependent signaling is unknown. By utilizing an in situ patch-clamp approach, we directly evaluated the KCa channel function and the CB2 receptor-dependent electrical responses in the endothelium of aortic strips from young ApoE−/− and C57Bl/6 mice. In the ApoE−/− group, the resting membrane potential (−30.1±1.1mV) was less negative (p<0.05) compared to WT (−38.9±1.4mV) and voltage ramps generated an overall KCa current of reduced amplitude. The peak hyperpolarization to 2μM Ach was not different between the groups. However, the sustained component was significantly reduced in ApoE−/− strips. In contrast, the peak hyperpolarization to 0.2μM Ach was increased in the ApoE−/− group, and SKA-31, a direct IKCa/SKCa channel opener, produced a hyperpolarization and whole-cell current of greater amplitude. The BKCa opener NS1619 produced hyperpolarization that was enhanced in ApoE−/− group. N-arachidonoyl glycine, a BKCa opener, produced a hyperpolarization of enhanced amplitude in ApoE−/− arteries. Selective CB2 receptor agonist AM1241 (5μM) had no effect on endothelial membrane potential in WT group; however, in ApoE−/− group, it elicited hyperpolarization that was inhibited by a selective CB2 receptor antagonist AM630. Conclusively, our data point to functional down-regulation of basal IKCa activity in unstimulated endothelium of ApoE−/− mice. Direct and indirect IKCa stimulation resulted in increased recruitment of the channels. In addition, our data point to up-regulation of endothelial BKCa channels and CB2 receptors in ApoE−/− arteries.

Expression of a Diverse Array of Ca2+-Activated K+ Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney.

The voltage- and Ca2+-activated, large conductance K+ channel (BK, maxi-K) is expressed in the collecting duct system of kidney where it underlies flow- and Ca2+-dependent K+ excretion. To determine if other Ca2+-activated K+ channels (KCa) may participate in this process, mouse kidney and the K+-secreting mouse cortical collecting duct (CCD) cell line, mCCDcl1, were assessed for TRPV4 and KCa channel expression and cross-talk. qPCR mRNA analysis and immunocytochemical staining demonstrated TRPV4 and KCa expression in mCCDcl1 cells and kidney connecting tubule (CNT) and CCD. Three subfamilies of KCa channels were revealed: the high Ca2+-binding affinity small-conductance SK channels, SK1and SK3, the intermediate conductance channel, IK1, and the low Ca2+-binding affinity, BK channel (BKα subunit). Apparent expression levels varied in CNT/CCD where analysis of CCD principal cells (PC) and intercalated cells (IC) demonstrated differential staining: SK1:PC<IC, and SK3:PC>IC, IK1:PC>IC, BKα:PC = IC, and TRPV4:PC>IC. Patch clamp analysis and fluorescence Ca2+ imaging of mCCDcl1 cells demonstrated potent TRPV4-mediated Ca2+ entry and strong functional cross-talk between TRPV4 and KCa channels. TRPV4-mediated Ca2+ influx activated each KCa channel, as evidenced by selective inhibition of KCa channels, with each active KCa channel enhancing Ca2+ entry (due to membrane hyperpolarization). Transepithelial electrical resistance (TEER) analysis of confluent mCCDcl1 cells grown on permeable supports further demonstrated this cross-talk where TRPV4 activation induce a decrease in TEER which was partially restored upon selective inhibition of each KCa channel. It is concluded that SK1/SK3 and IK1 are highly expressed along with BKα in CNT and CCD and are closely coupled to TRPV4 activation as observed in mCCDcl1 cells. The data support a model in CNT/CCD segments where strong cross talk between TRPV4-mediated Ca2+ influx and each KCa channel leads to enhance Ca2+ entry which will support activation of the low Ca2+-binding affinity BK channel to promote BK-mediated K+ secretion.

Calcium-Activated Potassium Channels: Potential Target for Cardiovascular Diseases.

Ca(2+)-activated K(+) channels (KCa) are classified into three subtypes: big conductance (BKCa), intermediate conductance (IKCa), and small conductance (SKCa) KCa channels. The three types of KCa channels have distinct physiological or pathological functions in cardiovascular system. BKCa channels are mainly expressed in vascular smooth muscle cells (VSMCs) and inner mitochondrial membrane of cardiomyocytes, activation of BKCa channels in these locations results in vasodilation and cardioprotection against cardiac ischemia. IKCa channels are expressed in VSMCs, endothelial cells, and cardiac fibroblasts and involved in vascular smooth muscle proliferation, migration, vessel dilation, and cardiac fibrosis. SKCa channels are widely expressed in nervous and cardiovascular system, and activation of SKCa channels mainly contributes membrane hyperpolarization. In this chapter, we summarize the physiological and pathological roles of the three types of KCa channels in cardiovascular system and put forward the possibility of KCa channels as potential target for cardiovascular diseases.

Rhythmic variations in BKCa channel activity, [Ca2+]i level and PKC‐delta expression in rat cerebral arterial muscle cells (1070.6)

Objectives: The objective of the present study is to investigate preence of rhythmic variation in KCa single‐channel activity, intracellular Ca2+ ([Ca2+]i ) level and PKC‐δ protein expression in rat cerebral arterial muscle cells.Methods. Although the underlying signaling mechanisms have not been elucidated yet, a circadian variation in the occurrence of cerebrovascular events and higher stroke incidence during morning hours has been reported. In the present study we investigated and compared changes in activities of a 238 pS Ca2+‐activated K+ channel (KCa) current, levels of intracellular Ca2+ ([Ca2+]i ) and PKC‐δ expression in freshly isolated rat cerebral arterial muscle cells during the early afternoon (2:00 PM) and in the early morning (2:00 AM) using the cell‐attached mode of the Patch Clamp technique, the ratiometric fluorescent dye Fura‐2AM to measure [Ca2+]i. and western blotting, respectively.Results. We found that the isolated cerebral arterial muscle cells exhibited altered NPo of a 238 pS KCa channel current (0.0038 ± 0.0004 at 2:00 PM and 0.0086 ± 0.0003 at 2:00 AM (n=5. P &lt; 0.05) when recorded at a patch potential of +40 mV using symmetrical KCl solution, changed [Ca2+]i levels (172 ± 21 nM at 2:00 AM and 343 ± 76 nM at 2:00 PM, n= 3 trials, p &lt; 0.05) and reduced expression of PKC‐δ protein at 2:00 AM compared to that determined at 2:00 PM of the day.Conclusions. Given that activities of KCa channel currents, [Ca2+]i level and PKC‐δ activity are requisite for the initiation and development of cerebral arterial myogenic tone, the observed rhythmic changes in these endogenous signaling events may be regarded as possible contributing factors for altered vascular reactivity and increased incidence of cerebral stroke at different times of the day.

20-Hydroxyeicosatetraenoic Acid Contributes to the Inhibition of K+ Channel Activity and Vasoconstrictor Response to Angiotensin II in Rat Renal Microvessels

The present study examined whether 20-hydroxyeicosatetraenoic acid (HETE) contributes to the vasoconstrictor effect of angiotensin II (ANG II) in renal microvessels by preventing activation of the large conductance Ca2+-activated K+ channel (KCa) in vascular smooth muscle (VSM) cells. ANG II increased the production of 20-HETE in rat renal microvessels. This response was attenuated by the 20-HETE synthesis inhibitors, 17-ODYA and HET0016, a phospholipase A2 inhibitor AACOF3, and the AT1 receptor blocker, Losartan, but not by the AT2 receptor blocker, PD123319. ANG II (10-11 to 10-6 M) dose-dependently decreased the diameter of renal microvessels by 41 ± 5%. This effect was blocked by 17-ODYA. ANG II (10-7 M) did not alter KCa channel activity recorded from cell-attached patches on renal VSM cells under control conditions. However, it did reduce the NPo of the KCa channel by 93.4 ± 3.1% after the channels were activated by increasing intracellular calcium levels with ionomycin. The inhibitory effect of ANG II on KCa channel activity in the presence of ionomycin was attenuated by 17-ODYA, AACOF3, and the phospholipase C (PLC) inhibitor U-73122. ANG II induced a peak followed by a steady-state increase in intracellular calcium concentration in renal VSM cells. 17-ODYA (10-5 M) had no effect on the peak response, but it blocked the steady-state increase. These results indicate that ANG II stimulates the formation of 20-HETE in rat renal microvessels via the AT1 receptor activation and that 20-HETE contributes to the vasoconstrictor response to ANG II by blocking activation of KCa channel and facilitating calcium entry.

Oxygen-sensitive reduction in Ca2+-activated K+ channel open probability in turtle cerebrocortex

In response to low ambient oxygen levels the western painted turtle brain undergoes a large depression in metabolic rate which includes a decrease in neuronal action potential frequency. This involves the arrest of N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) currents and paradoxically an increase in γ-aminobutyric acid receptor (GABAR) currents in turtle cortical neurons. In a search for other oxygen-sensitive channels we discovered a Ca2+-activated K+ channel (KCa) that exhibited a decrease in open time in response to anoxia. Single-channel recordings of KCa activity were obtained in cell-attached and excised inside-out patch configurations from neurons in cortical brain sheets bathed in either normoxic or anoxic artificial cerebrospinal fluid (aCSF). The channel has a slope conductance of 223pS, is activated in response to membrane depolarization, and is controlled in a reversible manner by free [Ca2+] at the intracellular membrane surface. In the excised patch configuration anoxia had no effect on KCa channel open probability (Popen); however, in cell-attached mode, there was a reversible fivefold reduction in Popen (from 0.5±0.05 to 0.1±0.03) in response to 30-min anoxia. The inclusion of the potent protein kinase C (PKC) inhibitor chelerythrine prevented the anoxia-mediated decrease in Popen while drip application of a phorbol ester PKC activator decreased Popen during normoxia (from normoxic 0.4±0.05 to phorbol-12-myristate-13-acetate (PMA) 0.1±0.02). Anoxia results in a slight depolarization of turtle pyramidal neurons (∼8mV) and an increase in cytosolic [Ca2+]; therefore, KCa arrest is likely important to prevent Ca2+ activation during anoxia and to reduce the energetic cost of maintaining ion gradients. We conclude that turtle pyramidal cell Ca2+-activated K+ channels are oxygen-sensitive channels regulated by cytosolic factors and are likely the reptilian analog of the mammalian large conductance Ca2+-activated K+ channels (BK channels).

Ca2+-activated K+ channels of small and intermediate conductance control eNOS activation through NAD(P)H oxidase

Ca2+-activated K+ channels (KCa) and NO play a central role in the endothelium-dependent control of vasomotor tone. We evaluated the interaction of KCa with NO production in isolated arterial mesenteric beds of the rat. In phenylephrine-contracted mesenteries, acetylcholine (ACh)-induced vasodilation was reduced by NO synthase (NOS) inhibition with Nω-nitro-l-arginine (L-NA), but in the presence of tetraethylammonium, L-NA did not further affect the response. In KCl-contracted mesenteries, the relaxation elicited by 100nM ACh or 1μM ionomycin was abolished by L-NA, tetraethylammonium, or simultaneous blockade of small-conductance KCa (SKCa) channels with apamin and intermediate-conductance KCa (IKCa) channels with triarylmethane-34 (TRAM-34). Apamin–TRAM-34 treatment also abolished 100nM ACh-activated NO production, which was associated with an increase in superoxide formation. Endothelial cell Ca2+ buffering with BAPTA elicited a similar increment in superoxide. Apamin–TRAM-34 treatment increased endothelial NOS phosphorylation at threonine 495 (P-eNOSThr495). Blockade of NAD(P)H oxidase with apocynin or superoxide dismutation with PEG-SOD prevented the increment in superoxide and changes in P-eNOSThr495 observed during apamin and TRAM-34 application. Our results indicate that blockade of SKCa and IKCa activates NAD(P)H oxidase-dependent superoxide formation, which leads to inhibition of NO release through P-eNOSThr495. These findings disclose a novel mechanism involved in the control of NO production.

Profound Alterations in the Intrinsic Excitability of Cerebellar Purkinje Neurons Following Neurotoxin 3-Acetylpyridine (3-AP)-Induced Ataxia in Rat: New Insights Into the Role of Small Conductance K+ Channels

Alterations in the intrinsic properties of Purkinje cells (PCs) may contribute to the abnormal motor performance observed in ataxic rats. To investigate whether such changes in the intrinsic neuronal excitability could be attributed to the role of Ca(2+)-activated K(+) channels (K(Ca)), whole cell current clamp recordings were made from PCs in cerebellar slices of control and ataxic rats. 3-AP induced profound alterations in the intrinsic properties of PCs, as evidenced by a significant increase in both the membrane input resistance and the initial discharge frequency, along with the disruption of the firing regularity. In control PCs, the blockade of small conductance K(Ca) channels by UCL1684 resulted in a significant increase in the membrane input resistance, action potential (AP) half-width, time to peak of the AP and initial discharge frequency. SK channel blockade also significantly decreased the neuronal discharge regularity, the peak amplitude of the AP, the amplitude of the afterhyperpolarization and the spike frequency adaptation ratio. In contrast, in ataxic rats, both the firing regularity and the initial firing frequency were significantly increased by the blockade of SK channels. In conclusion, ataxia may arise from alterations in the functional contribution of SK channels, to the intrinsic properties of PCs.

Small and intermediate conductance Ca2+‐activated K+channels regulate spontaneous contraction of mouse portal vein myocytes

Ca2+‐activated K+ channels (KCa) are key components of vascular myocytes EC coupling through their control of smooth muscle cell membrane potential. Whilst BKCa is expressed ubiquitously in smooth muscle cells, only proliferative VSMCs express KCa3.1 channels. However, mouse portal vein (mPV) is an atypical blood vessel showing spontaneous and rhythmic contractions. Since KCa channels are involved in the regulation of excitability of other cell types like neurons, the contribution of KCa2.x and KCa3.1 channels to the control of mPV excitability was investigated. Expression of KCa2.x and KCa3.1 was detected in mPV by PCR and immunohistochemistry. In single cell patch clamp studies, the RMP of isolated mPV myocytes was hyperpolarized (≈15 mV) by exposure to NS309 (10 μM), a KCa2.x and KCa3.1 channel activator. Inhibition of KCa2.x channels with UCL1689 (3 μM) reversed the effect of NS309 whilst inhibition of KCa3.1 channels (TRAM‐34; 10 μM) had no effect. NS309 significantly decreased the amplitude and increased the frequency of spontaneous contractions of intact mPV. These effects were reversed by application of UCL1689, suggesting that the NS309‐induced changes in spontaneous mPV contractions is carried out by KCa2.x channels. This study suggest that KCa2.x and KCa3.1 channels are expressed in mouse portal vein myocytes and play an important role in regulation of spontaneous contractions.Supported by FICM

The endothelium at the brink of calamity in storage disease: more than just overloaded with junk?

This editorial refers to ‘Globotriaosylceramide leads to KCa3.1 channel dysfunction: a new insight into endothelial dysfunction in Fabry disease’ by S. Park et al ., pp. 290–299, this issue. The endothelium crucially controls the contractile status of vascular smooth muscle and thereby vascular diameter. This is achieved by the release of dilating endothelial autacoids such as nitric oxide (NO), prostaglandins, and a third factor that elicits relaxation through smooth muscle hyperpolarization and is therefore known as ‘endothelium-derived hyperpolarizing factor’ (EDHF). Although the importance of EDHF is well accepted, its chemical nature is still elusive, and a wide range of agents, such as K+ ions, eicosanoids, H2O2, H2S, or C-type natriuretic peptide, have been suggested to serve as EDHF. Despite this diversity, it is firmly established that endothelial hyperpolarization is critical to initiate EDHF-type dilation. It is either achieved by releasing one of the above-mentioned molecules or by a direct current transfer from endothelium to smooth muscle via the gap junctions that interconnect these cells.1 The initial endothelial hyperpolarization results from opening of Ca2+-activated K+-channels (KCa), which have been identified as KCa3.1 and/or KCa2.3 (also named IKCa and SKCa, respectively) by pharmacological blockers as well as by genetic approaches. Both endothelial KCa channels are activated by Ca2+ and are voltage independent, which is an important biophysical feature that prevents their inactivation by the ensuing hyperpolarization, thus …

Key role of Ca2+‐activated K+ channels of small and intermediate conductance in the NO signaling of resistance vessels

Ca2+‐activated K+ channels (KCa) and nitric oxide (NO) play a central role in the control of vasomotor tone in resistance arteries. To evaluate the contribution of KCa channels to NO‐mediated dilation, we analyzed the vasomotor responses and NO production (chemiluminiscence) induced by the endothelium‐dependent vasodilator, acetylcholine (ACh), or the Ca2+ ionophore, ionomycin, in isolated arterial mesenteric bed of rat. ACh (0.01–10μM) elicited a concentration‐dependent vasodilatation. In phenylephrine‐contracted mesenteries, NO synthase (NOS) inhibition with Nω‐nitro‐L‐arginine (L‐NA) reduced the response to ACh. Blockade of KCa channels with tetraethylammonium (TEA) or inward rectifier K+ channels with Ba2+ blunted the response to ACh. L‐NA inhibited the ACh‐induced relaxation in presence of Ba2+, but not TEA. In addition, TEA blocked the NO production induced by ACh. In KCl‐contracted vessels, the relaxation‐elicited by ACh or 1μM ionomycin was abolished by L‐NA and inhibited by TEA or simultaneous blockade of KCa channels of small (SKCa) and intermediate (IKCa) conductance with apamin and TRAM‐34, respectively. Apamin and TRAM‐34 also abolished the NO production activated by 100nM ACh. These results indicate that functional SKCa and IKCa channels are required for Ca2+‐mediated eNOS activation. Membrane hyperpolarization is not involved in the regulation of NO production by these channels.VRAID 15/2009

Vascular KCa-channels as therapeutic targets in hypertension and restenosis disease

Importance of the field: Cardiovascular disease is a leading cause of death in modern societies. Hyperpolarizing Ca2+-activated K+ channels (KCa) are important membrane proteins in the control of arterial tone and pathological vascular remodelling and thus could serve as new drug targets.Areas covered in this review: We summarize recent advances in the field of vascular KCa and their roles in cardiovascular pathologies such as hypertension and restenosis disease and draw attention to novel small-molecule channel modulators and their possible therapeutic utility. This review focuses on literature from the last four to five years.What the reader will gain: Pharmacological opening of endothelial KCa3.1/KCa2.3 channels stimulates endothelium-derived-hyperpolarizing-factor-mediated arteriolar dilation and lowers blood pressure. Inhibition of smooth muscle KCa3.1 channels has beneficial effects in restenosis disease and atherosclerosis. We consider the therapeutic potential of KCa3.1/KCa2.3 openers as novel endothelium-specific antihypertensive drugs as well as of KCa3.1-blockers for the treatment of pathological vascular remodelling and discuss advantages and disadvantages of the pharmacotherapeutic approaches.Take home message: Pharmacological manipulation of vascular KCa channels by novel small-molecule modulators offers new venues for alternative treatments of hypertension, restenosis and atherosclerosis. Additional efforts are required to optimize these compounds and to validate them as cardiovascular-protective drugs.

Single-nucleotide polymorphisms in vascular Ca2+-activated K+-channel genes and cardiovascular disease

In the cardiovascular system, Ca2+-activated K+-channels (KCa) are considered crucial mediators in the control of vascular tone and blood pressure by modulating the membrane potential and shaping Ca2+-dependent contraction. Vascular smooth muscle cells express the BKCa channel which fine-tunes contractility by providing a negative feedback on Ca2+-elevations. BKCa channel's ion-conducting alpha-subunit is encoded by the KCa1.1 gene, and the accessory and Ca2+-sensitivity modulating beta1-subunit is encoded by the KCNMB1 gene. Vascular endothelial cells express the calmodulin-gated KCa channels IKCa (encoded by the KCa3.1 gene) and SKCa (encoded by the KCa2.3 gene). These two channels mediate endothelial hyperpolarization and initiate the endothelium-derived hyperpolarizing factor-dilator response. Considering these essential roles of KCa in arterial function, mutations in KCa genes have been suspected to contribute to cardiovascular disease in humans. So far, DNA sequence analysis in the population and patient cohorts has identified single-nucleotide polymorphisms (SNPs) in the BKCa beta1-subunit gene as well as in the alpha-subunit gene (KCa1.1). Some of these SNPs produce amino acid exchanges and evoke alterations of channel functions ("gain-of-function" as well as "loss-of-function"). Moreover, the epidemiological studies showed that the presence of the E65K polymorphism in, e.g., BKCa beta1-subunit gene (producing a "gain-of-function") lowers the prevalence for severe hypertension and myocardial infarction. Other SNPs in the BKCa alpha-subunit gene and also in the KCa3.1 gene expressed in the endothelium have been suggested to increase the risk of cardiovascular disease. These findings from sequence analysis of human KCa genes, and epidemiological studies thus provide evidence that genetic variations and mutations in KCa channel genes contribute to human cardiovascular disease.