Membrane Potential Of Muscle Cells Research Articles

Serotonin contracts the rat mesenteric artery by inhibiting 4-aminopyridine-sensitive Kv channels via the 5-HT2A receptor and Src tyrosine kinase

Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca2+-activated K+, inward rectifier K+ and ATP-sensitive K+ channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca2+ channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, α-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.

Angiotensin (1-7) increases the potassium current and the resting potential of arterial myocytes from vascular resistance vessels of normal adult rats: Pathophysiological implications

The influence of angiotensin (Ang) (1-7) on potassium current (Kv) and resting potential of smooth muscle cells isolated from mesenteric artery of Sprague Dawley rats was investigated. Measurements of potassium current were performed using the whole cell configuration of pCLAMP. The results indicated that Ang (1-7) (10−9 M) increased the potassium current by 120% ± 2.6% (P < .05) and the resting potential of smooth muscle cells by 8 ± 2.8 mV (n = 23; P < .05). Ang II (10−9 M) administered to the bath reduced the potassium current by 35% ± 3.6% (n = 23; P < .05) and depolarized the arterial myocytes by 7.8 ± 2.1 mV (n = 25; P < .05). The effect of the heptapeptide on potassium current was inhibited by a Mas receptor inhibitor (A779; 10−8 M) as well as by a protein kinase A (PKA) inhibitor (10−9 M) dialyzed into the cell. Intracellular dialysis of the catalytic subunit of PKA (5 × 10−8 M) enhanced the potassium current by 38% ± 3.4% (n = 14; P < .05) but did not abolish the effect of Ang (1-7). On the other hand, Bis-1 (10−9 M), which is a specific inhibitor of PKC, suppressed the effect of Ang (1-7) on potassium current. In conclusion, Ang (1-7) counteracts the effect of Ang II on potassium current and membrane potential of smooth muscle cells from mesenteric arteries, which are resistance vessels involved in the regulation of peripheral resistance and blood pressure. The activation of the cAMP/PKA cascade is essential for the effect of the heptapeptide. Pathophysiological implications are discussed.

Interstitial cells of Cajal link excitatory and nitrergic inhibitory neurotransmission with slow-wave activity in the intestine

Interstitial cell of Cajal (ICC) form a cellular network that is embedded in the musculature of the gastrointestinal tract. Previous studies provided clear evidence that ICC generate a rhythmic pacemaker current which manifests itself as slow-waves in membrane potential of smooth muscle cells resulting in rhythmic bowel contractions. However, a role of ICC in the transmission of excitatory and inhibitory signals from enteric neurons to smooth muscle cells is highly controversial and remains an outstanding question. This is mainly due to the general lack of models and systems to study ICC function in adult animals at the level of genetics. The aim of this study was to investigate the role of ICC for excitatory and inhibitory nitrergic neurotransmission in the gut.

In vitro motor patterns and electrophysiological changes in patients with colonic diverticular disease

The underlying mechanism responsible for motility changes in colonic diverticular disease (DD) is still unknown. In the present study, our aim was to investigate the structural and in vitro motor changes in the sigmoid colon of patients with DD. Muscle bath, microelectrodes and immunohistochemical techniques were performed with samples obtained from the left and sigmoid colon of patients with DD and compared with those of patients without DD. The amplitude and area under the curve of the spontaneous rhythmic phasic contractions were greatly reduced in patients with DD whereas their frequency and tone remained unaltered. Electrical field stimulation induced a neurally mediated, enhanced ON-contraction (amplitude) in patients with DD and increased the duration of latency of OFF-contractions. The resting membrane potential of smooth muscle cells was hyperpolarized and the amplitude of the inhibitory junction potential was increased in patients with DD. In contrast, no significant histological differences were observed in patients with DD as smooth muscle (circular and longitudinal layers), interstitial cells of Cajal, glial cells and myenteric neurons densities remained unaltered. Sigmoid strips from patients with asymptomatic DD showed an altered motor pattern with reduced spontaneous motility and enhanced neurally mediated colonic responses involving both excitatory and inhibitory motor pathways. No major neural and muscular structural elements were detected at this stage of the disease. These findings could be valuable in understanding the pathophysiology of this prevalent digestive disease.

Regulation of Cerebral Artery Smooth Muscle Membrane Potential by Ca2+‐Activated Cation Channels

Arterial tone is dependent on the depolarizing and hyperpolarizing currents regulating membrane potential and governing the influx of Ca²⁺ needed for smooth muscle contraction. Several ion channels have been proposed to contribute to membrane depolarization, but the underlying molecular mechanisms are not fully understood. In this review, we will discuss the historical and physiological significance of the Ca²⁺-activated cation channel, TRPM4, in regulating membrane potential of cerebral artery smooth muscle cells. As a member of the recently described transient receptor potential super family of ion channels, TRPM4 possesses the biophysical properties and upstream cellular signaling and regulatory pathways that establish it as a major physiological player in smooth muscle membrane depolarization.

Ionic and Molecular Mechanisms of β-Amyloid-Induced Depolarization in Mouse Skeletal Muscle Fibers

Excessive production and accumulation of β-amyloid peptide (βAP) underlies the pathogenesis of Alzheimer’s disease. Many studies have demonstrated that βAP has a wide spectrum of toxic effects on neuron functions, while the mechanism of action of βAP on other types of excitable cells, particularly skeletal muscle fibers, has received virtually no study. We report here electrophysiological experiments on mouse diaphragm muscle providing the first demonstration that βAP (fragment 25–35, 10−6 M) impairs the processes generating the resting membrane potential in muscle fibers, leading to marked depolarization of fibers via two mechanisms: 1) inhibition of Na+,K+-ATPase, leading to disappearance of the contribution of this pump to forming the resting membrane potential; 2) increases in the cation permeability of membranes due to the formation in them of “amyloid” channels blocked by Zn2+ ions. These data significantly extend our understanding of the mechanisms by which motor impairments and skeletal muscle pathology develop in Alzheimer’s disease, inclusion body myositis, and other diseases associated with βAP accumulation.

Nitric oxide generated by the compound RuBPY promotes the vascular smooth cell membrane hyperpolarization

The cis-[Ru(bpy)2(py)NO2](PF6) specie (RuBPY) has been used as nitric oxide (NO) delivery agent. It is an NO reservoir and it is thermodynamically stable in aqueous solution. This study aimed to evaluate the NO specie generated by RuBPY as compared to NO released from sodium nitroprusside (SNP) and to study the cellular mechanisms specially focusing the activation of soluble guanylyl-cyclase (sGC), K+ channels and the cell membrane hyperpolarization, which are the main targets for NO-inducing vascular relaxation.NO generated by RuBPY and the vascular smooth muscle cell (VSMC) membrane potential were measured by confocal microscopy. The cellular mechanisms of aorta relaxation were investigated using K+ channel blockers and sGC inhibitor. NO released from RuBPY was higher than NO released from SNP. RuBPY released only radicalar NO0 and SNP released both NO− and NO0. The concentration–effect curves for RuBPY-induced relaxation was shifted to the right by inhibition of sGC with ODQ and by the non-selective blockade of K+ channels with TEA. The simultaneous combination of ODQ and TEA abolished the vasorelaxation induced by RuBPY. The membrane potential measured by the sensitive dye 4-Di-ANNEPS demonstrated that RuBPY induces cell membrane hyperpolarization. Taken together, our results indicate that the large amount of NO0 specie generated by RuBPY induces vasorelaxation due to activation of sGC, K+ channels sensitive to TEA, and cell membrane hyperpolarization. These results indicate that NO0 generated from RuBPY can also directly activate the K+ channels in an independent way of sGC.

11,12-EET Stimulates the Association of BK Channel α and β1 Subunits in Mitochondria to Induce Pulmonary Vasoconstriction

In the systemic circulation, 11,12-epoxyeicosatrienoic acid (11,12-EET) elicits nitric oxide (NO)- and prostacyclin-independent vascular relaxation, partially through the activation of large conductance Ca2+-activated potassium (BK) channels. However, in the lung 11,12-EET contributes to hypoxia-induced pulmonary vasoconstriction. Since pulmonary artery smooth muscle cells also express BK channels, we assessed the consequences of BKβ1 subunit deletion on pulmonary responsiveness to 11,12-EET as well as to acute hypoxia. In buffer-perfused mouse lungs, hypoxia increased pulmonary artery pressure and this was significantly enhanced in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors. Under these conditions the elevation of tissue EET levels using an inhibitor of the soluble epoxide hydrolase (sEH-I), further increased the hypoxic contraction. Direct administration of 11,12-EET also increased pulmonary artery pressure, and both the sEH-I and 11,12-EET effects were prevented by iberiotoxin and absent in BKβ1 −/− mice. In pulmonary artery smooth muscle cells treated with NOS and COX inhibitors and loaded with the potentiometric dye, di-8-ANEPPS, 11,12-EET induced depolarization while the BK channel opener NS1619 elicited hyperpolarization indicating there was no effect of the EET on classical plasma membrane BK channels. In pulmonary artery smooth muscle cells a subpopulation of BK channels is localized in mitochondria. In these cells, 11,12-EET elicited an iberiotoxin-sensitive loss of mitochondrial membrane potential (JC-1 fluorescence) leading to plasma membrane depolarization, an effect not observed in BKβ1 −/− cells. Mechanistically, stimulation with 11,12-EET time-dependently induced the association of the BK α and β1 subunits. Our data indicate that in the absence of NO and prostacyclin 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the α and β1 subunits of mitochondrial BK channels. The 11,12-EET-induced activation of BK channels results in loss of the mitochondrial membrane potential and depolarization of the pulmonary artery smooth muscle cells.

Effects of β-hydroxybutyrate and different calcium and potassium concentrations on the membrane potential and motility of abomasal smooth muscle cells in cattle

The left displacement of the abomasum (LDA) is a common disease in periparturient dairy cows. Plasma β-hydroxybutyrate (BHBA) levels above the reference range are regarded as risk factors for the occurrence of LDA. Additionally, hypokalemia and hypocalcemia have been observed in LDA cows. The aim of the present study was to characterize the membrane potential and the slow waves in abomasal smooth muscle cells by a microelectrode technique and to determine possible effects of BHBA and of various calcium and potassium concentrations on the membrane potential. Subsequently, the results obtained by the microelectrode technique were combined with in vitro motility experiments of abomasal smooth muscles. Strips of the abomasal circular smooth muscles were prepared and incubated in different buffer solutions. For the microelectrode technique, healthy bulls and cows that underwent surgery for LDA were sampled. These measurements showed a frequency of the slow waves between 3.5 and 10.9 per minute (for amplitudes ≥3mV) and between 0.6 and 4.5 per minute (for amplitudes ≥5mV). The frequency of contractions (1.8 to 3.1 per minute) were in the same order as the frequency of the slow waves with amplitudes ≥5mV. Blocking potassium conductance with barium chloride induced a depolarization of the basal membrane potential (from −43±2.9 to −37±4.1mV; mean ± standard error of the mean) without affecting the frequency or the height of the slow waves. The reduction in the potassium concentration from 5.4 to 2mmol/L resulted in a nominal decrease in the activity of contractions (from 22.2 to 18.6mN/min). The subsequent addition of 1mmol of KCl/L induced a nominal increase in contraction activity (from 18.6 to 25.7mN/min). An effect of BHBA (5mmol/L) could not be demonstrated, neither on the electric nor on the motility parameters. A simulated hypocalcemia (1.2mmol/L total, 0.9mmol/L ionized Ca) did not change slow waves and motility. In conclusion, changes in membrane potential ≥5mV correlated with contractions of abomasal muscles. Hypokalemic conditions may reduce abomasal contraction activity via an effect on the membrane potential. An exclusive increase in BHBA or a slight hypocalcemia (0.9mmol/L ionized Ca) had no effects on the motility of healthy abomasal smooth muscle cells. A prolonged or severe exposition of muscles to a combination of low Ca and high BHBA concentrations might nevertheless be able to affect abomasal motility.

Abstract 166: Interaction Between LDL and Sympathetic Adrenoreceptors

Introduction: LDL is a significant risk factor for cardiovascular disease and is known to impair flow-dependent vasodilatation. Furthermore, it has been demonstrated that LDL can even directly cause vasoconstricton. Recent studies suggested that lower LDL levels, achieved through administration of statins, are correlated with lower blood pressure, independent from pleiotropic statin effects. Our objective was to investigate whether LDL-induced impairment in flow-dilatation was due to an interaction of LDL with sympathetic adrenoreceptors. Materials and Methods: Flow-dependent isometric tension, intracellularly recorded membrane potential and cAMP-cGMP were measured in segments of 22 coronaries from heart transplantations. Results: As compared to Krebs solution, LDL affected flow-dilatation and caused a relative contraction [Δ(T 3 -T 100 ): Krebs 0.459 g; LDL 0.278 g]. Complete blockade of both α- (phentolamine 10 -7 mol/L) and β-receptors (propranolol 10 -7 mol/L) resulted in a ∼50% reduction of flow-dilatation impairment induced by LDL. Blockage of either α- (1) or β-receptors (2) showed smaller effects which added up to the measured effect under blockade of both receptors (4). Similar effects were apparent in the recorded membrane potential of the vascular smooth muscle cells. Discussion: We conclude that the LDL-induced impairment in flow-dependent dilatation is partially due to the interaction between lipoprotein and both α- and β-adrenoreceptors. Since blockade of each receptor resulted in a relative dilatation, we assume that the LDL effect is caused by a stimulation of α-adrenoreceptors and an inhibition of β-adrenoreceptors.

Impaired propulsive motility in the distal but not proximal colon of BK channel β1‐subunit knockout mice

Large-conductance Ca(2+) -activated K(+) (BK) channels regulate smooth muscle tone. The BK channel β1-subunit increases Ca(2+) sensitivity of the α-subunit in smooth muscle. We studied β1-subunit knockout (KO) mice to determine if gastrointestinal (GI) motility was altered. Colonic and intestinal longitudinal muscle reactivity to bethanechol and colonic migrating motor complexes (CMMCs) were measured in vitro. Gastric emptying and small intestinal transit were measured in vivo. Colonic motility was assessed in vivo by measuring fecal output and glass bead expulsion time. Myoelectric activity of distal colon smooth muscle was measured in vitro using intracellular microelectrodes. Bethanechol-induced contractions were larger in the distal colon of β1-subunit KO compared to wild type (WT) mice; there were no differences in bethanechol reactivity in the duodenum, ileum, or proximal colon of WT vsβ1-subunit KO mice. There were more retrogradely propagated CMMCs in the distal colon of β1-subunit KO compared to WT mice. Gastrointestinal transit was unaffected by β1-subunit KO. Fecal output was decreased and glass bead expulsion times were increased in β1-subunit KO mice. Membrane potential of distal colon smooth muscle cells from β1-subunit KO mice was depolarized with higher action potential frequency compared to WT mice. Paxilline (BK channel blocker) depolarized smooth muscle cells and increased action potential frequency in WT distal colon. BK channels play a prominent role in smooth muscle function only in the distal colon of mice. Defects in smooth muscle BK channel function disrupt colonic motility causing constipation.

Influence of Hyperpolarization Caused by High Salt Diet on β-Adrenoceptor–Mediated Responses in Rat Pulmonary Artery

The effect of high salt diet on β-adrenoceptor-mediated response was assessed in rat-isolated pulmonary arteries. Isoprenaline-induced relaxations were not different in tissues from rats on either, high salt or regular diets. However, acidic buffer (pH 6.4) alone and in combination with Ba2+ or Nω-nitro-L-arginine methyl ester (L-NAME) significantly attenuated isoprenaline-induced relaxations in tissues from rats on high salt compared with those on a regular diet. Also, Ba2+ and ouabain together produced a significantly greater inhibition of isoprenaline-induced relaxation in tissues from rats on high salt when compared with those on the regular diet. The resting membrane potential of smooth muscle cells of pulmonary arteries of rats on high salt diet compared with regular diet was more negative (ie, hyperpolarized) than that on the regular diet. This hyperpolarization was reversed on exposure of blood vessels to acidic buffer and/or Ba2+ and ouabain combined but not L-NAME. Treatment with isoprenaline did not cause further hyperpolarization of smooth muscle cells of arteries from rats on the high salt diet. Taken together, the electrical changes due to the high salt diet can be attributed to the opening of K+ channels (2 pore acid-sensitive K+ channels and K(ir2.1)) and the activation of Na+/K+-ATPase. Furthermore, hyperpolarization observed after consumption of high salt diet seems to preserve β-adrenoceptor-mediated vasorelaxation.

Characteristics of the actions by which 5‐hydroxytryptamine affects electrical and mechanical activities in rabbit jugular vein graft

The vasomodulating actions of 5-HT in vein grafts, and the underlying mechanisms, remain to be fully clarified. Here, we characterized the actions by which 5-HT affects electrical and mechanical activities in rabbit autologous jugular vein grafts. Smooth muscle cell (SMC) membrane potential and isometric tension were measured in vein grafts 4 weeks after implantation into carotid arteries. Changes in the expression of 5-HT receptor subtypes and in myosin heavy chain isoforms (SM1, SM2 and SMemb) were examined by immunohistochemistry and Western blot analysis. The walls of grafted veins displayed massive increases in the number of SM1- and SM2-positive SMCs. 5-HT induced a large depolarization and contraction that were each reduced by both 5-HT(2A) - and 5-HT(1B/1D) -receptor antagonists. The 5-HT-induced contraction was not modified by a 5-HT₇ -receptor antagonist. The 5-HT₇ -receptor-selective agonist AS 19 did not induce relaxation during the contraction to prostaglandin F(2α) . Immunohistochemical and Western blot analyses revealed that immunoreactive responses against 5-HT(2A) and 5-HT(1B/1D) receptors were increased in the vein graft. 5-HT is able to induce a large contraction in rabbit autologous jugular vein grafts through (i) an increased number of differentiated contractile SMCs; (ii) an increased number of SMCs expressing contractile 5-HT(2A) - and 5-HT(1B/1D) receptors; and (iii) a down-regulation of the function of the relaxant SMC 5-HT₇ receptors. These changes in the vein graft may help it to resist the higher pressure present on the arterial side of the circulation.

Quick and effective hyperpolarization of the membrane potential in intact smooth muscle cells of blood vessels by synchronization modulation electric field

Blood vessel dilation starts from activation of the Na/K pumps and inward rectifier K channels in the vessel smooth muscle cells, which hyperpolarizes the cell membrane potential and closes the Ca channels. As a result, the intracellular Ca concentration reduces, and the smooth muscle cells relax and the blood vessel dilates. Activation of the Na/K pumps and the membrane potential hyperpolarization plays a critical role in blood vessel functions. Previously, we developed a new technique, synchronization modulation, to control the pump functions by electrically entraining the pump molecules. We have applied the synchronization modulation electric field noninvasively to various intact cells and demonstrated the field-induced membrane potential hyperpolarization. We further applied the electric field to blood vessels and investigated the field induced functional changes of the vessels. In this paper, we report the results in a study of the membrane potential change in the smooth muscle cells of mesenteric blood vessels in response to the oscillating electric field. We found that the synchronization modulation electric field can effectively hyperpolarize the muscle membrane potential quickly in seconds under physiological conditions.

TASK1 (K2P3.1) K+ channel inhibition by endothelin‐1 is mediated through Rho kinase‐dependent phosphorylation

TASK1 (K(2P)3.1) two-pore-domain K(+) channels contribute substantially to the resting membrane potential in human pulmonary artery smooth muscle cells (hPASMC), modulating vascular tone and diameter. The endothelin-1 (ET-1) pathway mediates vasoconstriction and is an established target of pulmonary arterial hypertension (PAH) therapy. ET-1-mediated inhibition of TASK1 currents in hPASMC is implicated in the pathophysiology of PAH. This study was designed to elucidate molecular mechanisms underlying inhibition of TASK1 channels by ET-1. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record TASK1 currents from hPASMC and Xenopus oocytes. ET-1 inhibited TASK1-mediated I(KN) currents in hPASMC, an effect attenuated by Rho kinase inhibition with Y-27632. In Xenopus oocytes, TASK1 current reduction by ET-1 was mediated by endothelin receptors ET(A) (IC(50) = 0.08 nM) and ET(B) (IC(50) = 0.23 nM) via Rho kinase signalling. TASK1 channels contain two putative Rho kinase phosphorylation sites, Ser(336) and Ser(393) . Mutation of Ser(393) rendered TASK1 channels insensitive to ET(A) - or ET(B)-mediated current inhibition. In contrast, removal of Ser(336) selectively attenuated ET(A) -dependent TASK1 regulation without affecting the ET(B) pathway. ET-1 regulated vascular TASK1 currents through ET(A) and ET(B) receptors mediated by downstream activation of Rho kinase and direct channel phosphorylation. The Rho kinase pathway in PASMC may provide a more specific therapeutic target in pulmonary arterial hypertension treatment.

Methionine Enhances the Contractile Activity of Human Colon Circular Smooth Muscle In Vitro

Effective drug to manage constipation has been unsatisfactory. We sought to determine whether methionine has effect on the human colon. Human colon tissues were obtained from the specimens of colon resection. Microelectrode recording was performed and contractile activity of muscle strips and the propagation of the contractions in the colon segment were measured. At 10 µM, methionine depolarized the resting membrane potential (RMP) of circular muscle (CM) cells. In the CM strip, methionine increased the amplitude and area under the curve (AUC) of contractions. In the whole segment of colon, methionine increased the amplitude and AUC of the high amplitude contractions in the CM. These effects on contraction were maximal at 10 µM and were not observed in longitudinal muscles in both the strip and the colon segment. Methionine reversed the effects of pretreatment with sodium nitroprusside, tetrodotoxin and Nw-oxide-L-arginine, resulting in depolarization of the RMP, and increased amplitude and AUC of contractions in the muscle strip. Methionine treatment affected the wave pattern of the colon segment by evoking small sized amplitude contractions superimposed on preexisting wave patterns. Our results indicate that a compound mimicking methionine may provide prokinetic functions in the human colon.

Molecular Modelling and Experimental Analysis of the ATP Sensitivity of Cloned Smooth Muscle KATP Channels

ATP-sensitive K+ (KATP) channels, comprised of pore-forming Kir6.x and regulatory SURx subunits, play important roles in many cellular functions. Vascular (Kir6.1/SUR2B) and non-vascular (Kir6.2/SUR2B) smooth muscle KATP channels regulate the resting membrane potential in smooth muscle cells and modulate the muscle tone. In spite of the fact that Kir6.1 and Kir6.2 share ∼70% sequence identity, their sensitivity to ATP inhibition differs by more than 30-fold with IC50 values of 3.1±1.3mM (n=7) and 101±22μM (n=17), respectively. We investigated the molecular mechanisms that underlie this differential response. All (17) Kir6.2 residues putatively involved in binding of ATP are conserved in Kir6.1 with the exception of K39 and K185. We constructed a homology model of Kir6.2 and carried out computational docking of ATP to determine the conformation of bound ATP in its putative binding site. The model was validated by assessing the binding free-energy difference of ATP for wild-type (Kir6.2/SUR2B) and a range of Kir6.2 ATP-binding site mutants for which the affinity for ATP was previously experimentally determined. The model predicted that substitution of K39 and K185 on Kir6.2 with the equivalent residues on Kir6.1 (K39S/K185R) did not affect the ATP affinity. Patch-clamp experiments confirmed that there was no difference in the ATP sensitivity between these channels. Furthermore, the open probability (Po) of Kir6.1/SUR2B, Kir6.2/SUR2B and Kir6.2-K39S-K185R/SUR2B were identical (0.41±0.05 (n=7), 0.38±0.06 (n=7) and 0.45±0.02 (n=8), respectively), indicating that their different ATP sensitivity is not caused by a change in channel gating. In conclusion, the difference in ATP sensitivity between Kir6.1 and Kir6.2 is not caused by a difference in the ATP binding site meaning other regions of the channel must be involved.This work was supported by BBSRC (BB/H000259/1) and by CASPUR (gpu11-905)

Role of vascular potassium channels in the regulation of renal hemodynamics

K(+) conductance is a major determinant of membrane potential (V(m)) in vascular smooth muscle (VSMC) and endothelial cells (EC). The vascular tone is controlled by V(m) through the action of voltage-operated Ca(2+) channels (VOCC) in VSMC. Increased K(+) conductance leads to hyperpolarization and vasodilation, while inactivation of K(+) channels causes depolarization and vasoconstriction. K(+) channels in EC indirectly participate in the control of vascular tone by several mechanisms, e.g., release of nitric oxide and endothelium-derived hyperpolarizing factor. In the kidney, a change in the activity of one or more classes of K(+) channels will lead to a change in hemodynamic resistance and therefore of renal blood flow and glomerular filtration pressure. Through these effects, the activity of renal vascular K(+) channels influences renal salt and water excretion, fluid homeostasis, and ultimately blood pressure. Four main classes of K(+) channels [calcium activated (K(Ca)), inward rectifier (K(ir)), voltage activated (K(V)), and ATP sensitive (K(ATP))] are found in the renal vasculature. Several in vitro experiments have suggested a role for individual classes of K(+) channels in the regulation of renal vascular function. Results from in vivo experiments are sparse. We discuss the role of the different classes of renal vascular K(+) channels and their possible role in the integrated function of the renal microvasculature. Since several pathological conditions, among them hypertension, are associated with alterations in K(+) channel function, the role of renal vascular K(+) channels in the control of salt and water excretion deserves attention.

Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle.

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca(2+) mobilization and Ca(2+) sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca(2+)]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca(2+)]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.

Models of neurovascular coupling via potassium and EET signalling

Functional hyperemia is an important metabolic autoregulation mechanism by which increased neuronal activity is matched by a rapid and regional increase in blood supply. This mechanism is facilitated by a process known as “neurovascular coupling”—the orchestrated communication system involving neurons, astrocytes and arterioles. Important steps in this process are the production of EETs in the astrocyte and the release of potassium, via two potassium channels (BK and KIR), into the perivascular space. We provide a model which successfully accounts for several observations seen in experiment. The model is capable of simulating the approximate 15% arteriolar dilation caused by a 60-s neuronal activation (modelled as a release of potassium and glutamate into the synaptic cleft). This model also successfully emulates the paradoxical experimental finding that vasoconstriction follows vasodilation when the astrocytic calcium concentration (or perivascular potassium concentration) is increased further. We suggest that the interaction of the changing smooth muscle cell membrane potential and the changing potassium-dependent resting potential of the KIR channel are responsible for this effect. Finally, we demonstrate that a well-controlled mechanism of potassium buffering is potentially important for successful neurovascular coupling.