Assessment of mutations in KCNN2 and ZNF135 to patient neurological symptoms.

Exome sequencing from a patient with neurological and developmental symptoms revealed two mutations in separate genes. One was a homozygous transition mutation that results in an in-frame, premature translational stop codon in the ZNF135 gene predicted to encode a transcriptional repressor. Another mutation was heterozygous, a single nucleotide duplication in the KCNN2 gene that encodes a Ca-activated K channel, SK2, and leads to a translational frame shift and a premature stop codon. Heterologous expression studies, brain slice recordings, and coordination tests from a transgenic mouse line carrying the SK2 mutation suggest that it does not contribute to the patient's symptoms. ZNF135 is expressed in human brain and it is likely that the homozygous mutation underlies the human phenotype.

A plasmid-based expression system to study protein-protein interactions at the Golgi in vivo.

There is still an unmet need for simple methods to verify, visualize, and confirm protein-protein interactions in vivo. Here we describe a plasmid-based system to study such interactions. The system is based on the transmembrane domain (TMD) of the EF-hand Ca(2+) sensor protein calneuron-2. We show that fusion of 28 amino acids that include the TMD of calneuron-2 to proteins of interest results in prominent localization on the cytoplasmic side of the Golgi. The recruitment of binding partners to the protein of interest fused to this sequence can then be easily visualized by fluorescent tags.

IK1 channels do not contribute to the slow afterhyperpolarization in pyramidal neurons.

In pyramidal neurons such as hippocampal area CA1 and basolateral amygdala, a slow afterhyperpolarization (sAHP) follows a burst of action potentials, which is a powerful regulator of neuronal excitability. The sAHP amplitude increases with aging and may underlie age related memory decline. The sAHP is due to a Ca(2+)-dependent, voltage-independent K(+) conductance, the molecular identity of which has remained elusive until a recent report suggested the Ca(2+)-activated K(+) channel, IK1 (KCNN4) as the sAHP channel in CA1 pyramidal neurons. The signature pharmacology of IK1, blockade by TRAM-34, was reported for the sAHP and underlying current. We have examined the sAHP and find no evidence that TRAM-34 affects either the current underling the sAHP or excitability of CA1 or basolateral amygdala pyramidal neurons. In addition, CA1 pyramidal neurons from IK1 null mice exhibit a characteristic sAHP current. Our results indicate that IK1 channels do not mediate the sAHP in pyramidal neurons.

Molecular dynamics of the neuronal EF-hand Ca2+-sensor Caldendrin.

Caldendrin, L- and S-CaBP1 are CaM-like Ca2+-sensors with different N-termini that arise from alternative splicing of the Caldendrin/CaBP1 gene and that appear to play an important role in neuronal Ca2+-signaling. In this paper we show that Caldendrin is abundantly present in brain while the shorter splice isoforms L- and S-CaBP1 are not detectable at the protein level. Caldendrin binds both Ca2+ and Mg2+ with a global Kd in the low µM range. Interestingly, the Mg2+-binding affinity is clearly higher than in S-CaBP1, suggesting that the extended N-terminus might influence Mg2+-binding of the first EF-hand. Further evidence for intra- and intermolecular interactions of Caldendrin came from gel-filtration, surface plasmon resonance, dynamic light scattering and FRET assays. Surprisingly, Caldendrin exhibits very little change in surface hydrophobicity and secondary as well as tertiary structure upon Ca2+-binding to Mg2+-saturated protein. Complex inter- and intramolecular interactions that are regulated by Ca2+-binding, high Mg2+- and low Ca2+-binding affinity, a rigid first EF-hand domain and little conformational change upon titration with Ca2+ of Mg2+-liganted protein suggest different modes of binding to target interactions as compared to classical neuronal Ca2+-sensors.

Ca(2+) sensor proteins in dendritic spines: a race for Ca(2+).

Dendritic spines are believed to be micro-compartments of Ca(2+) regulation. In a recent study, it was suggested that the ubiquitous and evolutionarily conserved Ca(2+) sensor, calmodulin (CaM), is the first to intercept Ca(2+) entering the spine and might be responsible for the fast decay of Ca(2+) transients in spines. Neuronal calcium sensor (NCS) and neuronal calcium-binding protein (nCaBP) families consist of Ca(2+) sensors with largely unknown synaptic functions despite an increasing number of interaction partners. Particularly how these sensors operate in spines in the presence of CaM has not been discussed in detail before. The limited Ca(2+) resources and the existence of common targets create a highly competitive environment where Ca(2+) sensors compete with each other for Ca(2+) and target binding. In this review, we take a simple numerical approach to put forth possible scenarios and their impact on signaling via Ca(2+) sensors of the NCS and nCaBP families. We also discuss the ways in which spine geometry and properties of ion channels, their kinetics and distribution, alter the spatio-temporal aspects of Ca(2+) transients in dendritic spines, whose interplay with Ca(2+) sensors in turn influences the race for Ca(2+).

Post-translational membrane insertion of tail-anchored transmembrane EF-hand Ca2+ sensor calneurons requires the TRC40/Asna1 protein chaperone.

Calneuron-1 and -2 are neuronal EF-hand-type calcium sensor proteins that are prominently targeted to trans-Golgi network membranes and impose a calcium threshold at the Golgi for phosphatidylinositol 4-OH kinase IIIß activation and the regulated local synthesis of phospholipids that are crucial for TGN-to-plasma membrane trafficking. In this study, we show that calneurons are nonclassical type II tail-anchored proteins that are post-translationally inserted into the endoplasmic reticulum membrane via an association of a 23-amino acid-long transmembrane domain (TMD) with the TRC40/Asna1 chaperone complex. Following trafficking to the Golgi, calneurons are probably retained in the TGN because of the length of the TMD and phosphatidylinositol 4-phosphate lipid binding. Both calneurons rapidly self-associate in vitro and in vivo via their TMD and EF-hand containing the N terminus. Although dimerization and potentially multimerization precludes TRC40/Asna1 binding and thereby membrane insertion, we found no evidence for a cytosolic pool of calneurons and could demonstrate that self-association of calneurons is restricted to membrane-inserted protein. The dimerization properties and the fact that they, unlike every other EF-hand calmodulin-like Ca(2+) sensor, are always associated with membranes of the secretory pathway, including vesicles and plasma membrane, suggests a high degree of spatial segregation for physiological target interactions.