Ion-pair interactions between voltage-sensing domain IV and pore domain I regulate CaV1.1 gating

Abstract

The voltage-gated calcium channel Ca V 1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of Ca V 1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire Ca V 1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for Ca V 1.1 and Ca V 1.2 L-type calcium channels. Moreover, in Ca V 1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patchclamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-Ca V 1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V 1/2 ) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-Ca V 1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V 1/2 , whereas no further shift of V 1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter-and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in Ca V 1.1.

The Role of Force Fields and Water Models in Protein Folding and Unfolding Dynamics

“…Proteins are essential macromolecules throughout the kingdom of life. Due to their high variability in sequence and structure they can fold into a panoply of different structures and fulfill various different roles and functions, e.g., in metabolism, , signal transduction, , immunity, , muscle contraction, , or drug delivery. , Protein-based therapeutics, also known as biologics, are an increasingly important class of drugs, which has been highlighted in various previous studies. − Hence, understanding protein folding, unfolding, and misfolding is an important ongoing field of research. − It is well-known that most proteins can adopt a distinct fold, which is evolutionarily well conserved, even more than their sequence . However, it still remains elusive why a specific sequence adopts a certain fold. , The accurate prediction of protein folding and unfolding dynamics is crucial for numerous fields of research, including drug design, , enzyme engineering, and understanding the molecular basis of diseases. , After all, certain conformational rearrangements, such as misfolds, are expected to be involved in numerous diseases, like Alzheimer’s disease, Huntington disease, Parkinson’s disease, or diabetes type 2. − Additionally, refolding events resulting from point mutations have been shown to elucidate changes in specificity or even polyreactivity, providing new applications in drug design and engineering. − …”

“…Proteins are essential macromolecules throughout the kingdom of life. Due to their high variability in sequence and structure they can fold into a panoply of different structures and fulfill various different roles and functions, e.g., in metabolism, 1 , 2 signal transduction, 3 , 4 immunity, 5 , 6 muscle contraction, 7 , 8 or drug delivery. 9 , 10 Protein-based therapeutics, also known as biologics, are an increasingly important class of drugs, which has been highlighted in various previous studies.…”