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Abstract: Ahnak1 has been implicated in protein kinase A (PKA)-mediated control of cardiac L-type Ca2+ channels (Cav1.2) through its interaction with the Cavβ2 regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavβ2 in the T-tubule system. In previous studies Cavβ2 attachment sites which impacted the channel’s PKA regulation have been located to ahnak1’s proximal C-terminus (ahnak14889–5535, ahnak15462–5535). In this study, we mapped the ahnak1-interacting regions in Cavβ2 and investigated whether Cavβ2 phosphorylation affects its binding behavior. In vitro binding assays with Cavβ2 truncation mutants and ahnak14889–5535 revealed that the core region of Cavβ2 consisting of Src-homology 3 (SH3), HOOK, and guanylate kinase (GK) domains was important for ahnak1 interaction while the C- and N-terminal regions were dispensable. Furthermore, Ser-296 in the GK domain of Cavβ2 was identified as novel PKA phosphorylation site by mass spectrometry. Surface plasmon resonance (SPR) binding analysis showed that Ser-296 phosphorylation did not affect the high affinity interaction (K D ≈35nM) between Cavβ2 and the α1C I–II linker, but affected ahnak1 interaction in a complex manner. SPR experiments with ahnak15462–5535 revealed that PKA phosphorylation of Cavβ2 significantly increased the binding affinity and, in parallel, it reduced the binding capacity. Intriguingly, the phosphorylation mimic substitution Glu-296 fully reproduced both effects, increased the affinity by ≈2.4-fold and reduced the capacity by ≈60%. Our results are indicative for the release of a population of low affinity interaction sites following Cavβ2 phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1´s modulator function on Cav1.2 channel activity. [Copyright &y& Elsevier]

Voltage-gated calcium channels (Cavs) are major Ca 2+ entry pathways in excitable cells. Their β subunits facilitate membrane trafficking of the channel's ion-conducting α1 pore and modulate its gating properties. We report that one β subunit, β3, reduces Ca 2+ release following stimulation of phospholipase C-coupled receptors and inositol 1,4,5-trisphosphate (IP 3 ) formation. This effect requires the SH3-HOOK domain of Cavβ3, includes physical β3/IP 3 receptor interaction, and prevails when agonist-induced IP 3 formation is bypassed by photolysis of caged IP 3 . In agreement with β3 acting as a brake on Ca 2+ release, fibroblast migration is enhanced in vitro, and in vivo, closure of skin wounds is accelerated in the absence of β3. To mediate specific physiological responses and to prevent Ca 2+ toxicity, cytoplasmic Ca 2+ signals must be tightly controlled. The described function of β3, unrelated to its function as a Cav subunit, adds to this tight control., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The anamniote lateral line system, comprising mechanosensory neuromasts and electrosensory ampullary organs, is a useful model for investigating the developmental and evolutionary diversification of different organs and cell types. Zebrafish neuromast development is increasingly well understood, but neither zebrafish nor Xenopus is electroreceptive and our molecular understanding of ampullary organ development is rudimentary. We have used RNA-seq to generate a lateral line-enriched gene-set from late-larval paddlefish ( Polyodon spathula ). Validation of a subset reveals expression in developing ampullary organs of transcription factor genes critical for hair cell development, and genes essential for glutamate release at hair cell ribbon synapses, suggesting close developmental, physiological and evolutionary links between non-teleost electroreceptors and hair cells. We identify an ampullary organ-specific proneural transcription factor, and candidates for the voltage-sensing L-type Ca v channel and rectifying K v channel predicted from skate (cartilaginous fish) ampullary organ electrophysiology. Overall, our results illuminate ampullary organ development, physiology and evolution.