NaV1.2 EFL domain allosterically enhances Ca2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD.

Neuronal voltage-gated sodium channel NaV1.2 C-terminal domain (CTD) binds calmodulin (CaM) constitutively at its IQ motif. A solution structure (6BUT) and other NMR evidence showed that the CaM N domain (CaMN) is structurally independent of the C-domain (CaMC) whether CaM is bound to the NaV1.2IQp (1,901-1,927) or NaV1.2CTD (1,777-1,937) with or without calcium. However, in the CaM + NaV1.2CTD complex, the Ca2+ affinity of CaMN was more favorable than in free CaM, while Ca2+ affinity for CaMC was weaker than in the CaM + NaV1.2IQp complex. The CTD EF-like (EFL) domain allosterically widened the energetic gap between CaM domains. Cardiomyopathy-associated CaM mutants (N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)) all bound the NaV1.2 IQ motif favorably under resting (apo) conditions and bound calcium normally at CaMN sites. However, only N53I and A102V bound calcium at CaMC sites at [Ca2+] < 100 µM. Thus, they are expected to respond like wild-type CaM to Ca2+ spikes in excitable cells.

Photopolymerizable Zwitterionic Polymer Patterns Control Cell Adhesion and Guide Neural Growth.

Developing materials that reduce or eliminate fibrosis encapsulation of neural prosthetic implants could significantly enhance implant fidelity by improving the tissue/electrode array interface. Here, we report on the photografting and patterning of two zwitterionic materials, sulfobetaine methacrylate (SBMA) and carboxybetaine methacrylate (CBMA), for controlling the adhesion and directionality of cells relevant to neural prosthetics. CBMA and SBMA polymers were photopolymerized and grafted on glass surfaces then characterized by X-ray photoelectron spectroscopy, water contact angle, and protein adsorption. Micropatterned surfaces were fabricated with alternating zwitterionic and uncoated bands. Fibroblasts, cells prevalent in fibrotic tissue, almost exclusively migrate and grow on uncoated bands with little to no cells present on zwitterionic bands, especially for CBMA-coated surfaces. Astrocytes and Schwann cells showed similarly low levels of cell adhesion and morphology changes when cultured on zwitterionic surfaces. Additionally, Schwann cells and inner ear spiral ganglion neuron neurites aligned well to zwitterionic patterns.

pClone: Synthetic Biology Tool Makes Promoter Research Accessible to Beginning Biology Students.

The Vision and Change report recommended genuine research experiences for undergraduate biology students. Authentic research improves science education, increases the number of scientifically literate citizens, and encourages students to pursue research. Synthetic biology is well suited for undergraduate research and is a growing area of science. We developed a laboratory module called pClone that empowers students to use advances in molecular cloning methods to discover new promoters for use by synthetic biologists. Our educational goals are consistent with Vision and Change and emphasize core concepts and competencies. pClone is a family of three plasmids that students use to clone a new transcriptional promoter or mutate a canonical promoter and measure promoter activity in Escherichia coli. We also developed the Registry of Functional Promoters, an open-access database of student promoter research results. Using pre- and posttests, we measured significant learning gains among students using pClone in introductory biology and genetics classes. Student posttest scores were significantly better than scores of students who did not use pClone. pClone is an easy and affordable mechanism for large-enrollment labs to meet the high standards of Vision and Change.