Funktionelle Charakterisierung fluoreszenzmarkierter Agonisten an zyklisch-Nukleotid aktivierten Ionenkanälen

Die Nukleotide cAMP und cGMP sind intrazelluläre, sekundäre Botenstoffe und agieren unter anderem als Agonisten an zyklisch-Nukleotid ktivierten (CNG)-Ionenkanälen. Somit besitzen die kleinen Signalmoleküle eine Schlüsselstellung in der Entwicklung von Rezeptorpotentialen im visuellen und olfaktorischen System, indem diese durch Bindung an die Ionenkanäle eine Aktivierung hervorrufen. Dabei ist cAMP der native Ligand der olfaktorischen CNGA2:A4:B1b-Kanäle, jedoch besitzt cGMP eine höhere scheinbare Affinität. Durch die ebenfalls hohe Affinität an den homotetrameren CNGA2-Kanälen wurde in vorherigen Studien ein fluoreszierender Ligand (fcGMP) entwickelt, welcher einen Farbstoff enthält, der mithilfe eines Aminoethylthio-Linkers an Position 8 des Nukleotides verbunden ist. Bei der konfokalen Patch-clamp Fluorometrie (cPCF) wird die Aktivierung synchron zur Bindung gemessen (Biskup et al. 2007). Hierbei wurde eine große Anzahl an neuen Liganden synthetisiert und mittels elektrophysiologischer Methoden die Wirkung auf CNG-Kanäle, welche in Xenopus laevis Oozyten heterolog exprimiert wurden, charakterisiert. Dabei wurden sowohl die scheinbare Affinität als auch die kinetischen Eigenschaften in der inside-out Konfiguration ermittelt. Für die Bestimmung weiterer Kennwerte, wie Bindung, Helligkeit und Fluoreszenzspektren diente die cPCF. Um die publizierten cPCF Experimente (Biskup et al. 2007) auf Einzelmoleküllevel zu beobachten, benötigt es neue Liganden mit verbesserter Affinität und photophysikalischen Eigenschaften. Um die Affinität und die molekulare Helligkeit eines fluoreszierenden Liganden zu optimieren, wurden drei Möglichkeiten verfolgt

Fluorophore-labeled cyclic nucleotides as potent agonists of cyclic nucleotide-regulated ion channels

High‐affinity fluorescent derivatives of cyclic adenosine and guanosine monophosphate are powerful tools to investigate their natural targets. Cyclic nucleotide‐regulated ion channels belong to these targets and are vital for many signal transduction processes, such as vision and olfaction. The relation of ligand binding to activation gating is still challenging and there is a request for fluorescent probes that enable a breaking down to the single molecule level. This inspired us to prepare fluorophore‐labeled cyclic nucleotides, which are composed of a bright dye and a nucleotide derivative with a thiophenol motif at position 8 that has already been shown to enable superior binding affinity. The preparation of these bioconjugates was accomplished via a novel cross‐linking strategy that involves the substitution of the nucleobase with a modified thiophenolate in good yield. Both fluorescent nucleotides are potent activators of different cyclic nucleotide‐regulated ion channels with respect to the natural ligand and previously reported substances. Molecular docking of the probes excluding the fluorophore reveals that the high potency can be attributed to additional hydrophobic and cation‐π interactions between the ligand and the protein. Moreover, the introduced substances bear the potential to investigate related target proteins, such as cAMP‐ and cGMP‐dependent protein kinases, exchange proteins directly activated by cAMP or phosphodiesterases

Fluorophore‐Labeled Cyclic Nucleotides as Potent Agonists of Cyclic Nucleotide‐Regulated Ion Channels

High‐affinity fluorescent derivatives of cyclic adenosine and guanosine monophosphate are powerful tools to investigate their natural targets. Cyclic nucleotide‐regulated ion channels belong to these targets and are vital for many signal transduction processes, such as vision and olfaction. The relation of ligand binding to activation gating is still challenging and there is a request for fluorescent probes that enable a breaking down to the single molecule level. This inspired us to prepare fluorophore‐labeled cyclic nucleotides, which are composed of a bright dye and a nucleotide derivative with a thiophenol motif at position 8 that has already been shown to enable superior binding affinity. The preparation of these bioconjugates was accomplished via a novel cross‐linking strategy that involves the substitution of the nucleobase with a modified thiophenolate in good yield. Both fluorescent nucleotides are potent activators of different cyclic nucleotide‐regulated ion channels with respect to the natural ligand and previously reported substances. Molecular docking of the probes excluding the fluorophore reveals that the high potency can be attributed to additional hydrophobic and cation‐π interactions between the ligand and the protein. Moreover, the introduced substances bear the potential to investigate related target proteins, such as cAMP‐ and cGMP‐dependent protein kinases, exchange proteins directly activated by cAMP or phosphodiesterases

Hydrophobic alkyl chains substituted to the 8-position of cyclic nucleotides enhance activation of CNG and HCN channels by an intricate enthalpy - entropy compensation

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are tetrameric non-specific cation channels in the plasma membrane that are activated by either cAMP or cGMP binding to specific binding domains incorporated in each subunit. Typical apparent affinities of these channels for these cyclic nucleotides range from several hundred nanomolar to tens of micromolar. Here we synthesized and characterized novel cAMP and cGMP derivatives by substituting either hydrophobic alkyl chains or similar-sized more hydrophilic heteroalkyl chains to the 8-position of the purine ring with the aim to obtain full agonists of higher potency. The compounds were tested in homotetrameric CNGA2, heterotetrameric CNGA2:CNGA4:CNGB1b and homotetrameric HCN2 channels. We show that nearly all compounds are full agonists and that longer alkyl chains systematically increase the apparent affinity, at the best more than 30 times. The effects are stronger in CNG than HCN2 channels which, however, are constitutively more sensitive to cAMP. Kinetic analyses reveal that the off-rate is significantly slowed by the hydrophobic alkyl chains. Molecular dynamics simulations and free energy calculations suggest that an intricate enthalpy - entropy compensation underlies the higher apparent affinity of the derivatives with the longer alkyl chains, which is shown to result from a reduced loss of configurational entropy upon binding

Novel Fluorescent Cyclic Nucleotide Derivatives to Study CNG and HCN Channel Function

A highly specific molecular interaction of diffusible ligands with their receptors belongs to the key processes in cellular signaling. Because an appropriate method to monitor the unitary binding events is still missing, most of our present knowledge is based on ensemble signals recorded from a big number of receptors, such as ion currents or fluorescence changes of suitably labeled receptors, and reasoning from these data to the ligand binding. To study the binding process itself, appropriately tagged ligands are required that fully activate the receptors and report the binding at the same time. Herein, we tailored a series of 18 novel fluorescent cyclic nucleotide derivatives by attaching 6 different dyes via different alkyl linkers to the 8-position of the purine ring of cGMP or cAMP. The biological activity was determined in inside-out macropatches containing either homotetrameric (CNGA2), heterotetrameric (CNGA2:CNGA4:CNGB1b), or hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channels. All these novel fluorescent ligands are efficient to activate the channels, and the potency of most of them significantly exceeded that of the natural cyclic nucleotides cGMP or cAMP. Moreover, some of them showed an enhanced brightness when bound to the channels. The best of our derivatives bear great potential to systematically analyze the activation mechanism in CNG and HCN channels, at both the level of ensemble and single-molecule analyses