Article Review on mutations in Calcium channels leading to Timothy Syndrome
Introduction
Mutations G406R and G402S in helix IS6 of Cav1.2 are exclusively known to be the leading causes of the multisystem disorder of TS (Timothy Syndrome), which is relatively rare. However, the emergence of new mutations with VSD-II (voltage-sensing domain II) in helix IIS0, particularly the R518C/H, have been described as the cause of cardiac-only TS. Although the article focuses only on cardiac-only TS, the two other Timothy SyndromeSyndrome mutations are relevant to the study. All three mutations are known decelerate VDI (Voltage-dependent inactivation). The end goal is to offer a mechanistic rationale of Timothy syndrome-related mutation in the deceleration of VDIs in calcium channelopathies.
Calcium channels are often expressed as cardiac myocytes, which play a crucial role in cell physiology where entry of calcium ion in such channels is known to trigger various processes which often led to gene mutations. Some of these processes include memory formation, coupling, excitation-transcription, gene transcription, and hormonal secretion (2). Our study focuses on the L-shaped calcium channel of Cav1.2, whose central role revolves around the generation of potential action for excitation-contraction and working myocytes. As such, calcium currents through Cav1.2 channels cause alterations and depolarization of membranes, a product of gene mutations that causes decelerations of CDI and VDI, leading to structural cardiac disorders and arrhythmic cardiac disorders.
Methodology
Before the study’s commencement, the researchers had to adhere to the rules and regulations of mutation identification and research. This involved getting approval from the Ethical Review Boards of Almazov National Medical Research Centre, a Helsinki Declaration attribute. This further included validations under slinger sequencing of all genetic-related disease variants, which would later be classified under American College of Medical Genetics guidelines. Additionally, there had had to be a written consent from the study subject consenting to the clinical study and publication (3).
With the availability of 108 cardiac associated genes disorders for the study, the research methodology commenced by modelling of CAV1.2, presumably iCav1.2-Ia and iCav1.2-II., in an inactive state. Construction of iCav1.2-Ia and iCav1.2-II., templates were built under CAV1.1 channel in a dormant state with the aid of Class ‘Ia’ and II cyro-EM structures (3). The researchers achieved this through the ZMM program, which minimizes energy in generalized coordinates often not covalently bonded. As such, the research maintained the rigidity of bond angles and lengths with the exception of prolines angles and optimized via the MCM method. The Amber force field was used to calculate non-bonded interactions under a shifting function and cut off 9 ‘Å’ distance. The dependent dielectric function was used calculated electrostatic interactions dependent on the environment and distance where ionized interactions were calculated without any cut-offs.
The residues on CAV1.2, which mismatched with the alignment sequence CAV1.1, were optimized using the three consecutive MCM trajectories. The first trajectory maintains side-chain torsions’ flexibility while keeping the rigidity of proline bond angles, backbone torsions, and segment roots orientation and position. The second trajectory aimed at the flexibility of all generalized coordinates by pinpointing constraints that would have caused massive deformations in the first trajectory if they were not relaxed. The third trajectory aimed to ensure the hormonal model’s energy stability, whose computation was conducted without the presence of pins. Decisively, a pin is described as a parabolical energy function, a flat bottom base with a forced constraint of 10 kcal mol. The pin constraint is known to add penalty energy when the model atom deviates from its matching template atom. In the research, each trajectory step was abandoned when the final 2000 energy minimizations failed to reduce each trajectory’s energy composition.
The next methodology step involved transforming the inactive CAV1.2 through steered MCM into open and closed states. Relatively is achieved through two possibilities, where the first one involves moving VSD-II through activated and deactivated conformation states where IS6 and AID (α1-interaction domain) is monitored. However, this method is relatively ineffective as the forced movement of VSD-II as only the P-loop channel is available in its resting structure whereby the S6 helices remain undisturbed. The second method, which is more reliable, moves the S4–S5, S5, and S6 helices into open and closed structures (4). Achievement of this facilitated the modeling and computation of the G406R, G402S, and R518C/H mutants’ residues via the double-shell approach.
In the double-shell approach, the mutated calcium residue contains a flexible inner shell whose contents contain a 15 ‘Å’ heavy atom. The second outer shell is argued to have a minimum of one heavy atom o 20 ‘Å,’ which doesn’t belong to the flexible shell residue. The outer shell is arguably rigid, which prevents the movement of the flexible shell residues into the residue regions under the fill fledged model (5). Commencement of conformations was processed through the WT channel, which involved models from the rigid and flexible cells. The researchers picked random samples of generalized coordinates from the flexible shell and mutated residues from the 128 gene conformations. The double-shell availability facilitated the minimization of energy and computations due to the limited variables and atoms available for the case study.
Results Discussion
The clinical cases involved a 48 old woman as the study case diagnosed with atrial fibrillation with no history of diastolic cardiac dysfunction, endocrine disorders, obesity, and arterial hypertension. In cariology, the CACNAIC gene is usually associated with carrying clinical conditions often linked to cell excitation and abnormal calcium cycling (18). Mutation of the CACNAIC gene is known to cause structural disorders and cardiac arrhythmic where a wide range of cardiac diseases are never a spectrum of a particular gene mutation but multiple. A broad spectrum of these mutations affects tissues and organs, such as the TS syndrome, caused by alterations of the CAV1.2 homolog. The alterations tend to cause defects in the immune system and severely affect the development of the limbs, heart, and central nervous system. The study focuses on cardiac only TS, which is caused by a mutation of the R518C/H.
The study focuses first on activating the CAV1.2 calcium channel as its composition template of cryo-EM structure Cav1.1, which is usually closed. The prokaryotic sodium channel NavAb via X-ray structures is used as the activation gate to activate CAV1.2 into open and closed pathways. The use of the NavAb channel for the fate way activations of the CAV1.2 is based on the fact that Eukaryotic sodium channels are more related to calcium Eukaryotic than Eukaryotic potassium channels. This is because the open and closed prokaryotic channel channels lack linker S4 and S5 helices and are relatively low resolution. The prokaryotic and eukaryotic channel membranes are somewhat less permeable to large ligands such as batrachotoxin, narrowing the activation gate. Such a structure makes it necessary to have open CAV1.2 as it doesn’t support the modelling of CAV1.2 in a closed state.
However, this modelling approach limits the study as it relies heavily on building Human CAV1.2 model channels in their inactivated and activated open and closed states. This involves utilizing the Prokaryotic sodium channel in closed and open states and cyro-EM structures in their inactive states. This limits the experiment, as the structures of the components constructed contain non-native features as some may lack voltage and lip membrane (20). The non-native elements are brought about by the closed and open state conformation of the CAV1.2 and its combination with CAV1.1. Additionally, the research was limited to study the linker I/II function via the NoVo model’s assistance due to the lack of existence of experimental structural data on the subject. Despite the lack of such vital data, the research study findings were consistent with existing empirical studies on the subject.
Conclusion
The article research focus study is on R518C mutation as the cause of cardiac only TS with the assistance of related TS mutations, G406R and G402S, with the central aim of exploring all possible mechanisms of the atomic-level. The study achieved this by comparing CAV1.2 calcium channels in closed and open state models, with their contact residues (G406, G402, and R518) with their respective Timothy syndrome mutations (G406R, G402S, and R518C). As such, with a focus on R518 contact residue combined with other residues were observed to form strong bonds with the AID at the cytoplasmic face of VSD-II in all the research models. The study assumes that the IS6 would result in VDL when exposed to flexible G402and G406, which will facilitate it to bend, leading to the closure of the activation gate. This will lead to the weakening of Mutation R518C/H, which would displace the AID.
Work Cited
Korkosh, Vyacheslav S., et al. “Atomic Mechanisms of Timothy Syndrome-Associated Mutations in Calcium Channel Cav1. 2.” Frontiers in physiology 10 (2019): 1-20.