Improved selection of zebrafish CRISPR editing by early next-generation sequencing based genotyping.

Despite numerous prior attempts to improve knock-in (KI) efficiency, the introduction of precise base pair substitutions by the CRISPR-Cas9 technique in zebrafish remains challenging. In our efforts to generate KI zebrafish models of human CACNA1C mutations, we have tested the effect of several CRISPR determinants on KI efficiency across two sites in a single gene and developed a novel method for early selection to ameliorate KI efficiency. We identified optimal KI conditions for Cas9 protein and non-target asymmetric PAM-distal single stranded deoxynucleotide repair templates at both cacna1c sites.

The Selectivity Filter Is Involved in the U-Type Inactivation Process of Kv2.1 and Kv3.1 Channels.

Voltage-gated potassium (Kv) channels display several types of inactivation processes, including N-, C-, and U-types. C-type inactivation is attributed to a nonconductive conformation of the selectivity filter (SF). It has been proposed that the activation gate and the channel's SF are allosterically coupled because the conformational changes of the former affect the structure of the latter and vice versa.

The subfamily-specific interaction between Kv2.1 and Kv6.4 subunits is determined by interactions between the N- and C-termini.

The "silent" voltage-gated potassium (KvS) channel subunit Kv6.4 does not form electrically functional homotetramers at the plasma membrane but assembles with Kv2.1 subunits, generating functional Kv2.

The S4-S5 linker of KCNQ1 channels forms a structural scaffold with the S6 segment controlling gate closure.

In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac potassium current (I(Ks)). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1-S6) with the S1-S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling.

Conserved negative charges in the N-terminal tetramerization domain mediate efficient assembly of Kv2.1 and Kv2.1/Kv6.4 channels.

Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual alpha-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 alpha-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9).

Differential modulation of Kv4 kinetics by KCHIP1 splice variants.

The beta-subunits of the KChIP family modulate properties and expression level of Kv4 channels. We report the cloning of the first splice variant of KChIP1 (KChIP1b) which contains an extra exon, rich in aromatic residues, in the amino terminus. Both splice variants interacted equally well with Kv4.