Alternative Splicing at N Terminus and Domain I Modulates CaV1.2 Inactivation and Surface Expression

The Ca_V1.2 L-type calcium channel is a key conduit for Ca²⁺ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its α_1C pore-forming subunit is known to undergo extensive alternative splicing to produce many Ca_V1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human Ca_V1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with Ca_Vβ_2a, displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V_1/2inact of Ca_V1.2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V_1/2act. However, the measured effects were β-subunit-dependent when comparing Ca_Vβ_2a with Ca_Vβ₃ coexpression. Notably, calcium-dependent inactivation mediated by local Ca²⁺-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing Ca_V1.2 as compared to exon-1a-containing Ca_V1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with Ca_Vβ_2a or Ca_Vβ₃ subunit. This finding correlated well with a higher channel surface expression for the exon 1-Ca_V1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human Ca_V1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.     

The Oligomerization Landscape of Histones

In eukaryotes, DNA is packaged within nucleosomes. The DNA of each nucleosome is typically centered around an octameric histone protein core: one central tetramer plus two separate dimers. Studying the assembly mechanisms of histones is essential for understanding the dynamics of entire nucleosomes and higher-order DNA packaging. Here, we investigate canonical histone assembly and that of the centromere-specific histone variant, centromere protein A (CENP-A), using molecular dynamics simulations. We quantitatively characterize their thermodynamical and dynamical features, showing that two H3/H4 dimers form a structurally floppy, weakly bound complex, the latter exhibiting large instability around the central interface manifested via a swiveling motion of two halves. This finding is consistent with the recently observed DNA handedness flipping of the tetrasome. In contrast, the variant CENP-A encodes distinctive stability to its tetramer with a rigid but twisted interface compared to the crystal structure, implying diverse structural possibilities of the histone variant. Interestingly, the observed tetramer dynamics alter significantly and appear to reach a new balance when H2A/H2B dimers are present. Furthermore, we found that the preferred structure for the (CENP-A/H4)₂ tetramer is incongruent with the octameric structure, explaining many of the unusual dynamical behaviors of the CENP-A nucleosome. In all, these data reveal key mechanistic insights and structural details for the assembly of canonical and variant histone tetramers and octamers, providing theoretical quantifications and physical interpretations for longstanding and recent experimental observations. Based on these findings, we propose different chaperone-assisted binding and nucleosome assembly mechanisms for the canonical and CENP-A histone oligomers.     

Polymodal Nociception in Drosophila Requires Alternative Splicing of TrpA1

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Simulating Genetic Circuits in Bacterial Populations with Growth Heterogeneity

We computationally study genetic circuits in bacterial populations with heterogeneities in the growth rate. To that end, we present a stochastic simulation method for gene circuits in populations of cells and propose an efficient implementation that we call the “Next Family Method”. Within this approach, we implement different population setups, specifically Chemostat-type growth and growth in an ideal Mother Machine and show that the population structure and its statistics are different for the different setups whenever there is growth heterogeneity. Such dependence on the population setup is demonstrated, in the case of bistable systems with different growth rates in the stable states, to have distinctive signatures on quantities including the distributions of protein concentration and growth rates, and hysteresis curves. Applying this method to a bistable antibiotic resistance circuit, we find that as a result of the different statistics in different population setups, the estimated minimal inhibitory concentration of the antibiotic becomes dependent on the population setup in which it is measured.     

Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization

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