This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
Structure informs function, and this may be the evolutionary reason why alternative splicing, which is capable of generating different variants of the same protein, arise. But, given the energetic cost of generating different splice variants for testing their capability at a specific task, which incurs cellular functional uncertainty; as well as the exertion of differing physiological effects on cells that may translate into diseased states, what is the evolutionary advantage of this process? Additionally, what are the factors that select a specific variant for a presented task? Using heart tissue samples exposed to hypoxia stress as model system, this research idea entails the illumination of single nucleotide polymorphisms (SNP) of the calcium channel transporter, Cav 1.2 gene in the population through gene sequencing followed by bioinformatic analysis for alternative splice sites. This would be followed by a scan for alternative splice variants through colony polymerase chain reaction using universal primers for Cav 1.2 gene. Confirmation of splice variant identity through Western blot laid the stage for subsequent efforts at cloning and expressing the variant gene in HEK 293 cells lacking endogenous expression of Cav 1.2, for biophysical characterization of calcium conduction through patch clamp electrophysiology. In parallel, structural elucidation efforts necessitate the purification of the calcium channel via hydrophobic interaction or reversed phase liquid chromatography after its heterologous expression in a bacterial host. But, biophysical and biochemical characterization does not speak of the signaling and metabolic pathways laying the path to generation of the splice variant(s). Hence, discovery approaches such as RNA-seq and mass spectrometry proteomics could uncover the molecular mysteries at the transcript and protein level that help guide the selection of specific splice variant in response to hypoxic stress, where HIF is a candidate pathway. Implementing this approach from the retrospective angle of examining diseased human tissue samples provide one important facet for uncovering possible mechanisms driving the generation of a splice variant. However, the complementary prospective approach of identifying the molecular basis and processes for responding to hypoxia in a cell line such as HEK 293 would help provide confirmatory evidence in understanding the key drivers of physiological response to lack of oxygen at the cellular level. Collectively, this research route would illuminate both the nucleotide informational basis of alternative splicing in calcium channel Cav 1.2 as well as identify the molecular mechanisms enabling the selection of specific splice variants useful for conferring, at the cell and tissue level, ability to withstand hypoxic stress without significant negative effects on cell function. Interested readers can expand on the ideas presented.
The author thought about why evolution imbue genes encoding calcium channels with significant alternative splice sites, which have been utilized by the splicesome to generate a plethora of different incarnations of the same transport protein important to the transduction of calcium into and out of cells in tissue requiring contraction for function. More importantly, given increasing knowledge that these alternative splice variants of calcium channels might be responsible for different disease states, a significant gap in understanding exists in profiling the physiological impact of individual splice variants on calcium channel function in different tissues. The author wrote this manuscript preprint to share his ideas with the scientific community, and interested researchers can expand on them in their research.