17 research outputs found
K+ Channel Regulator KCR1 Suppresses Heart Rhythm by Modulating the Pacemaker Current If
Hyperpolarization-activated, cyclic nucleotide sensitive (HCN) channels underlie the pacemaker current If, which plays an essential role in spontaneous cardiac activity. HCN channel subunits (HCN1-4) are believed to be modulated by additional regulatory proteins, which still have to be identified. Using biochemistry, molecularbiology and electrophysiology methods we demonstrate a protein-protein interaction between HCN2 and the K+ channel regulator protein 1, named KCR1. In coimmunoprecipitation experiments we show that KCR1 and HCN2 proteins are able to associate. Heterologously expressed HCN2 whole-cell current density was significantly decreased by KCR1. KCR1 profoundly suppressed IHCN2 single-channel activity, indicating a functional interaction between KCR1 and the HCN2 channel subunit. Endogenous KCR1 expression could be detected in adult and neonatal rat ventriculocytes. Adenoviral-mediated overexpression of KCR1 in rat cardiomyocytes (i) reduced If whole-cell currents, (ii) suppressed most single-channel gating parameters, (iii) altered the activation kinetics, (iv) suppressed spontaneous action potential activity, and (v) the beating rate. More importantly, siRNA-based knock-down of endogenous KCR1 increased the native If current size and single-channel activity and accelerated spontaneous beating rate, supporting an inhibitory action of endogenous KCR1 on native If. Our observations demonstrate for the first time that KCR1 modulates IHCN2/If channel gating and indicate that KCR1 serves as a regulator of cardiac automaticity
MODULATION OF TRANSIENT RECEPTOR POTENTIAL VANILLOID 1 (TRPV1) ION CHANNEL BY µ-RECEPTOR-AGONISTS
1\. Titelblatt, Inhalt und Abkürzungen I
2\. Einleitung 5
3\. Zielsetzung 27
4\. Material und Methoden 29
5\. Ergebnisse 58
6\. Diskussion 88
7\. Zusammenfassung 112
8\. Summary 113
9\. Literatur 114
10\. Publikationen, Danksagung 135Opioide spielen eine herausragende Rolle in der Behandlung akuter und
chronischer Schmerzen. Sehr häufig ist die Ursache des Schmerzes auf
entzündungsbedingte Veränderungen im Gewebe zurückzuführen. Die Aktivierung
von peripheren μ-Rezeptoren führt zur Modulation spannungsabhängiger
Calciumkanäle und dadurch zu einer Abnahme der Erregbarkeit afferenter
Neurone. Unklar ist jedoch, inwiefern Opioide den ligandengesteuerten
Ionenkanal transient receptor potential vanilloid 1 (TRPV1) beeinflussen
können. TRPV1 ist entscheidend an der Entstehung von Schmerz und der thermalen
Hyperalgesie unter Entzündungsbedingungen beteiligt und wird insbesondere
durch Entzündungsmediatoren und Proteinkinase-vermittelte Phosphorylierungen
sensibilisiert. Im Rahmen dieser Arbeit wurden die Auswirkungen einer μ
-Rezeptor-Aktivierung auf die Funktion des Ionenkanals TRPV1 in DRG-Neuronen
und in μ-Rezeptor und TRPV1-exprimierenden HEK293-Zellen analysiert. Wir
konnten nachweisen, dass die μ-Rezeptor-Aktivierung zu einer Modulation des
Ionenkanals TRPV1 führt. μ-Rezeptoren und TRPV1 sind in DRG-Neuronen
colokalisiert. Opioide hemmen die capsaicin- und hitzeinduzierte
TRPV1-Aktivität. Die TRPV1-Inhibition wird via inhibierender G-Proteine
vermittelt und ist auf eine Reduktion der cAMP-Konzentration zurückzuführen.
Dieser Effekt ist insbesondere auch unter Entzündungsbedingungen nachweisbar.
Unter Entzündungsbedingungen steigt die TRPV1-Proteinexpression, nicht aber
die Anzahl der mRNA-Transkripte an. Die funktionelle Relevanz der
μ-Rezeptor TRPV1-Interaktion wurde in vivo bestätigt: Die capsaicininduzierte
Hyperalgesie kann in einem Tiermodell durch die lokale Gabe einer nicht
systemisch wirksamen Dosis eines Opioids inhibiert werden. Im Gegensatz zur
opioidvermittelten TRPV1-Inhibition führt der Opioidentzug zu einer
Sensibilisierung der TRPV1-Aktivität. Die TRPV1-Sensibilisierung beim
Opioidentzug ist auf eine erhöhte PKA-Aktivität zurückzuführen, die durch eine
kompensatorische cAMPHochregulation nach Opioidentzug ausgelöst wird. In
unserem Zellsystem sind insbesondere die Adenylylcyclasen AC3 und AC5 an einer
erhöhten Umsetzung von ATP zu cAMP und dem anschließenden Anstieg der PKA-
Aktivität beteiligt. Die μ-Rezeptor-TRPV1-Interaktion stellt einen neuen
molekularen Mechanismus dar, der die analgetischen Eigenschaften der Opioide
beim Entzündungsschmerz charakterisiert.Opioids play an outstanding role in the treatment of acute, postoperative and
tumor-caused pain frequently associated with tissue inflammation. The opioid
mediated activation of peripheral μ-receptors modulates voltage-gated calcium
channels and thus inhibits afferent neuron excitation. However, it is unclear
if opioids affect the ligand-gated ion channel transient receptor potential
vanilloid 1 (TRPV1). TRPV1 is crucially involved in the development of pain
and thermal hypersensitivity associated with tissue inflammation and is
particularly sensitized by inflammatory mediators and protein kinase mediated
phosphorylation processes. In this study, the effects of μ-receptor activation
on TRPV1 ion channel function was analyzed in dorsal root ganglion (DRG)
neurons and in μ-receptor and TRPV1 expressing HEK293-cells. For the first
time we could show that μ-receptor activation leads to a modulation of ion
channel TRPV1. μ-receptors and TRPV1 are co-localized in sensory neurons.
Opioids restrain capsaicin and heat induced TRPV1 activity, mediated by
μ-receptor activation and inhibitory G proteins. This effect was also
confirmed under inflammatory conditions. TRPV1 protein expression increases
under inflammatory conditions while the number of mRNA transcripts does not
change. The functional relevance of the interaction between μ-receptors and
TRPV1 interaction could be confirmed in vivo: Behavioural experiments show,
that capsaicininduced thermal hyperalgesia is attenuated by local
administration of a not systemically effective dose of an opioid. In opposite
to opioid mediated TRPV1 inhibition, TRPV1 is sensitized to capsaicin after
longer opioid administration followed by opioid withdrawal. The opioid
mediated TRPV1 sensitization is to be due to an increased protein kinase A
(PKA) activity, which is triggered by a compensatory cAMP upregulation after
opioid withdrawal. In our cell system, mainly adenylylcyclases AC3 and AC5
contribute to the improved conversion from ATP to cAMP and the following
increase in PKA activity. The interaction between μ-receptors and TRPV1
represents a new molecular mechanism, which characterizes the analgesic
characteristics of opioids during inflammatory pain
mu-opioid receptor activation modulates transient receptor potential vanilloid 1 (TRPV1) currents in sensory neurons in a model of inflammatory pain
Current therapy for inflammatory pain includes the peripheral application of opioid receptor agonists. Activation of opioid receptors modulates voltage-gated ion channels, but it is unclear whether opioids can also influence ligand-gated ion channels [e.g., the transient receptor potential vanilloid type 1 (TRPV1)]. TRPV1 channels are involved in the development of thermal hypersensitivity associated with tissue inflammation. In this study, we investigated \u3bc-opioid receptor and TRPV1 expression in primary afferent neurons in the dorsal root ganglion (DRG) in complete Freund's adjuvant (CFA)-induced paw inflammation. In addition, the present study examined whether the activity of TRPV1 in DRG neurons can be inhibited by \u3bc-opioid receptor (\u3bc-receptor) ligands and whether this inhibition is increased after CFA inflammation. Immunohistochemistry demonstrated colocalization of TRPV1 and \u3bc-receptors in DRG neurons. CFA-induced inflammation increased significantly the number of TRPV1- and \u3bc-receptor-positive DRG neurons, as well as TRPV1 binding sites. In whole-cell patch clamp studies, opioids significantly decreased capsaicin-induced TRPV1 currents in a naloxone- and pertussis toxin-sensitive manner. The inhibitory effect of morphine on TRPV1 was abolished by forskolin and 8-bromo-cAMP. During inflammation, an increase in TRPV1 is apparently rivaled by an increase of \u3bc-receptors. However, in single dissociated DRG neurons, the inhibitory effects of morphine are not different between animals with and without CFA inflammation. In in vivo experiments, we found that locally applied morphine reduced capsaicin-induced thermal allodynia. In summary, our results indicate that \u3bc-receptor activation can inhibit the activity of TRPV1 via Gi/o proteins and the cAMP pathway. These observations demonstrate an important new mechanism underlying the analgesic efficacy of peripherally acting \u3bc-receptor ligands in inflammatory pain. Copyright \ua9 2007 The American Society for Pharmacology and Experimental Therapeutics
Correction: K+ Channel Regulator KCR1 Suppresses Heart Rhythm by Modulating the Pacemaker Current If
KCR1 suppresses spontaneous action potential activity.
<p>(A) Representative original recordings of spontaneous action potentials of control neonatal rat ventricular cardiomyocytes. (B) KCR1 infection suppressed spontaneous beating activity in neonatal cells. Action potential artificially induced by a depolarizing pulse in a quiescent neonatal cardiocyte. (C) KCR1<sup>siRNA</sup> infection accelerated spontaneous beating activity in neonatal cells. (D) Overexpression and knock-down of endogenous KCR1 resulted in a significant (<sub>*</sub>, p<0.001) decrease and increase of the beating rate, respectively. For data see text.</p
Single-channel parameters of native I<sub>f</sub> in adult and neonatal cardiomyocytes modulated by KCR1 and KCR1<sup>siRNA</sup>
<p>Modulation of single-channel parameters of native I<sub>f</sub> (control) by KCR1 overexpression (KCR1-infected) and knock-down of endogenous KCR1 (KCR1<sup>siRNA</sup>-infected) in adult and neonatal cardiomyocytes. Holding potential −35 mV, test potential −90 mV. I<sub>peak</sub> was measured from ensemble average currents. For closed time and latency analysis, only experiments containing just one detected open level were used for calculation. Numbers of experiments given in parentheses indicate number of experiments with only one channel in the patch. Pooled data are presented as mean±SEM.</p>*<p>p<0.05 vs. control;</p>#<p>p<0.05 vs. KCR1-infected.</p