What can naturally occurring mutations tell us about Cav1.x channel function?

AbstractVoltage-gated Ca2+ channels allow for Ca2+-dependent intracellular signaling by directly mediating Ca2+ ion influx, by physical coupling to intracellular Ca2+ release channels or functional coupling to other ion channels such as Ca2+ activated potassium channels. L-type Ca2+ channels that comprise the family of Cav1 channels are expressed in many electrically excitable tissues and are characterized by their unique sensitivity to dihydropyridines. In this issue, we summarize genetic defects in L-type Ca2+ channels and analyze their role in human diseases (Ca2+ channelopathies); e.g. mutations in Cav1.2 α1 cause Timothy and Brugada syndrome, mutations in Cav1.3 α1 are linked to sinoatrial node dysfunction and deafness while mutations in Cav1.4 α1 are associated with X-linked retinal disorders such as an incomplete form of congenital stationary night blindness. Herein, we also put the mutations underlying the channel's dysfunction into the structural context of the pore-forming α1 subunit. This analysis highlights the importance of combining functional data with structural analysis to gain a deeper understanding for the disease pathophysiology as well as for physiological channel function. This article is part of a Special Issue entitled: Calcium channels

Symmetric Allosteric Mechanism of Hexameric Escherichia coli Arginine Repressor Exploits Competition between L-Arginine Ligands and Resident Arginine Residues

An elegantly simple and probably ancient molecular mechanism of allostery is described for the Escherichia coli arginine repressor ArgR, the master feedback regulator of transcription in L-arginine metabolism. Molecular dynamics simulations with ArgRC, the hexameric domain that binds L-arginine with negative cooperativity, reveal that conserved arginine and aspartate residues in each ligand-binding pocket promote rotational oscillation of apoArgRC trimers by engagement and release of hydrogen-bonded salt bridges. Binding of exogenous L-arginine displaces resident arginine residues and arrests oscillation, shifting the equilibrium quaternary ensemble and promoting motions that maintain the configurational entropy of the system. A single L-arg ligand is necessary and sufficient to arrest oscillation, and enables formation of a cooperative hydrogen-bond network at the subunit interface. The results are used to construct a free-energy reaction coordinate that accounts for the negative cooperativity and distinctive thermodynamic signature of L-arginine binding detected by calorimetry. The symmetry of the hexamer is maintained as each ligand binds, despite the conceptual asymmetry of partially-liganded states. The results thus offer the first opportunity to describe in structural and thermodynamic terms the symmetric relaxed state predicted by the concerted allostery model of Monod, Wyman, and Changeux, revealing that this state is achieved by exploiting the dynamics of the assembly and the distributed nature of its cohesive free energy. The ArgR example reveals that symmetry can be maintained even when binding sites fill sequentially due to negative cooperativity, which was not anticipated by the Monod, Wyman, and Changeux model. The molecular mechanism identified here neither specifies nor requires a pathway for transmission of the allosteric signal through the protein, and it suggests the possibility that binding of free amino acids was an early innovation in the evolution of allostery

Cholesterol Slows down the Lateral Mobility of an Oxidized Phospholipid in a Supported Lipid Bilayer

We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect become dramatic when immobile obstacles were inserted into the bilayer. which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane

Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding

UID/Multi/04378/2019 Grant no. BB/P010660/1 grant number BB/M011216/1Saturation-transfer difference (STD) NMR spectroscopy is a fast and versatile method which can be applied for drug-screening purposes, allowing the determination of essential ligand binding affinities (KD). Although widely employed to study soluble proteins, its use remains negligible for membrane proteins. Here the use of STD NMR for KD determination is demonstrated for two competing substrates with very different binding affinities (low nanomolar to millimolar) for an integral membrane transport protein in both detergent-solubilised micelles and reconstituted proteoliposomes. GltPh, a homotrimeric aspartate transporter from Pyrococcus horikoshii, is an archaeal homolog of mammalian membrane transport proteins—known as excitatory amino acid transporters (EAATs). They are found within the central nervous system and are responsible for fast uptake of the neurotransmitter glutamate, essential for neuronal function. Differences in both KD’s and cooperativity are observed between detergent micelles and proteoliposomes, the physiological implications of which are discussed.publishersversionpublishe