I–II Loop Structural Determinants in the Gating and Surface Expression of Low Voltage-Activated Calcium Channels

The intracellular loops that interlink the four transmembrane domains of Ca2+- and Na+-channels (Cav, Nav) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I–II loop) in high voltage-activated (HVA) Ca2+ channels possesses the binding site for Cavβ subunits and plays significant roles in channel function, including trafficking the α1 subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I–II loop of Cav1/Cav2 HVA channels and Cav3 LVA/T-type channels, evidence for a regulatory role of the I–II loop in T-channel function has recently emerged for Cav3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Cav3 channels, we have performed a structure-function analysis of the I–II loop in Cav3.1 and Cav3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Cav3.1 and Cav3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Cav3 channels. In contrast to findings in Cav3.2, deletion of the central region of the I–II loop in Cav3.1 and Cav3.3 yielded a modest increase (+30%) and a reduction (−30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Cav3 function by highlighting the unique role played by the intracellular I–II loop in Cav3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Cav3.3

Diet-Induced Obesity Impairs Endothelium-Derived Hyperpolarization via Altered Potassium Channel Signaling Mechanisms

BACKGROUND: The vascular endothelium plays a critical role in the control of blood flow. Altered endothelium-mediated vasodilator and vasoconstrictor mechanisms underlie key aspects of cardiovascular disease, including those in obesity. Whilst the mechanism of nitric oxide (NO)-mediated vasodilation has been extensively studied in obesity, little is known about the impact of obesity on vasodilation to the endothelium-derived hyperpolarization (EDH) mechanism; which predominates in smaller resistance vessels and is characterized in this study. METHODOLOGY/PRINCIPAL FINDINGS: Membrane potential, vessel diameter and luminal pressure were recorded in 4(th) order mesenteric arteries with pressure-induced myogenic tone, in control and diet-induced obese rats. Obesity, reflecting that of human dietary etiology, was induced with a cafeteria-style diet (∼30 kJ, fat) over 16-20 weeks. Age and sexed matched controls received standard chow (∼12 kJ, fat). Channel protein distribution, expression and vessel morphology were determined using immunohistochemistry, Western blotting and ultrastructural techniques. In control and obese rat vessels, acetylcholine-mediated EDH was abolished by small and intermediate conductance calcium-activated potassium channel (SK(Ca)/IK(Ca)) inhibition; with such activity being impaired in obesity. SK(Ca)-IK(Ca) activation with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) and 1-ethyl-2-benzimidazolinone (1-EBIO), respectively, hyperpolarized and relaxed vessels from control and obese rats. IK(Ca)-mediated EDH contribution was increased in obesity, and associated with altered IK(Ca) distribution and elevated expression. In contrast, the SK(Ca)-dependent-EDH component was reduced in obesity. Inward-rectifying potassium channel (K(ir)) and Na(+)/K(+)-ATPase inhibition by barium/ouabain, respectively, attenuated and abolished EDH in arteries from control and obese rats, respectively; reflecting differential K(ir) expression and distribution. Although changes in medial properties occurred, obesity had no effect on myoendothelial gap junction density. CONCLUSION/SIGNIFICANCE: In obese rats, vasodilation to EDH is impaired due to changes in the underlying potassium channel signaling mechanisms. Whilst myoendothelial gap junction density is unchanged in arteries of obese compared to control, increased IK(Ca) and Na(+)/K(+)-ATPase, and decreased K(ir) underlie changes in the EDH mechanism

Openers of calcium-activated potassium channels and endothelium-dependent hyperpolarizations in the guinea pig carotid artery

This study was designed to determine whether putative openers of calcium-activated potassium channels of small and/or intermediate conductance (SKCa and IKCa) induce vascular smooth muscle hyperpolarizations and to identify the underlying mechanisms. The membrane potential of guinea pig carotid artery smooth muscle cells was recorded with intracellular microelectrodes in the presence of N ω-nitro-l- arginine and indomethacin. Acetylcholine and NS-309 produced endothelium-dependent hyperpolarizations. The effects of acetylcholine were partially and significantly inhibited by apamin. The combinations of charybdotoxin plus apamin and TRAM-34 plus apamin markedly and significantly reduced these hyperpolarizations. 1-ethyl-2-benzimidazolinone (1-EBIO) induced hyperpolarizations that were unaffected by TRAM-34 but partially inhibited by charybdotoxin, apamin, TRAM-34 plus apamin, and charybdotoxin plus apamin. Riluzole produced only marginal hyperpolarizations. Therefore, in the guinea pig carotid artery, endothelium-dependent hyperpolarization to acetylcholine involves the activation of both SKCa and IKCa, with a predominant role for the former channel. 1-EBIO is a non-selective and weak opener of SKCa, while riluzole is virtually ineffective. By contrast, NS-309 is a reasonably potent and selective opener of both SKCa and IKCa, and this compound mimics the endothelium-dependent hyperpolarizations to acetylcholine. © 2008 Springer-Verlag.link_to_subscribed_fulltex

C-type natriuretic peptide and endothelium-dependent hyperpolarization in the guinea-pig carotid artery

Background and purpose: C-type natriuretic peptide (CNP) has been proposed to make a fundamental contribution in arterial endothelium-dependent hyperpolarization to acetylcholine. The present study was designed to address this hypothesis in the guinea-pig carotid artery. Experimental approach: The membrane potential of vascular smooth muscle cells was recorded in isolated arteries with intracellular microelectrodes. Key results: Acetylcholine induced endothelium-dependent hyperpolarizations in the presence or absence of N G-nitro-L-arginine, indomethacin and/or thiorphan, inhibitors of NO-synthases, cyclooxygenases or neutral endopeptidase, respectively. Acetycholine hyperpolarized smooth muscle cells in resting arteries and produced repolarizations in phenylephrine-stimulated arteries. CNP produced hyperpolarizations with variable amplitude. They were observed only in the presence of inhibitors of NO-synthases and cyclooxygenases and were endothelium-independent, maintained in phenylephrine-depolarized carotid arteries, and not affected by the additional presence of thiorphan. In arteries with endothelium, the hyperpolarizations produced by CNP were always significantly smaller than those induced by acetylcholine. Upon repeated administration, a significant tachyphylaxis of the hyperpolarizing effect of CNP was observed, while consecutive administration of acetycholine produced sustained responses. The hyperpolarizations evoked by acetylcholine were abolished by the combination of apamin plus charybdotoxin, but unaffected by glibenclamide or tertiapin. In contrast, CNP-induced hyperpolarizations were abolished by glibenclamide and unaffected by the combination of apamin plus charybdotoxin. Conclusions and implications: In the isolated carotid artery of the guinea-pig, CNP activates K ATP and is a weak hyperpolarizing agent. In this artery, the contribution of CNP to EDHF-mediated responses is unlikely. © 2008 Nature Publishing Group All rights reserved.link_to_subscribed_fulltex

Low-Threshold Exocytosis Induced by cAMP-Recruited Ca(V)3.2 (α(1H)) Channels in Rat Chromaffin Cells

We have studied the functional role of Ca(V)3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). Ca(V)3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (−50, −40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 μM Ni(2+), which selectively blocks T-type currents in RCCs. This suggests that the “low-threshold exocytosis” induced by cAMP is due to increased Ca(2+) influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca(2+) entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca(2+) dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited Ca(V)3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca(2+) efficacy similar to that of already existing high-threshold Ca(2+) channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca(2+) current component recruited by cAMP is selectively associated to the α(1H) (Ca(V)3.2) channel isoform