Orientation of the Calcium Channel β Relative to the α12.2 Subunit Is Critical for Its Regulation of Channel Activity

BACKGROUND: The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure. METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity

I–II Loop Structural Determinants in the Gating and Surface Expression of Low Voltage-Activated Calcium Channels

The intracellular loops that interlink the four transmembrane domains of Ca2+- and Na+-channels (Cav, Nav) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I–II loop) in high voltage-activated (HVA) Ca2+ channels possesses the binding site for Cavβ subunits and plays significant roles in channel function, including trafficking the α1 subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I–II loop of Cav1/Cav2 HVA channels and Cav3 LVA/T-type channels, evidence for a regulatory role of the I–II loop in T-channel function has recently emerged for Cav3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Cav3 channels, we have performed a structure-function analysis of the I–II loop in Cav3.1 and Cav3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Cav3.1 and Cav3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Cav3 channels. In contrast to findings in Cav3.2, deletion of the central region of the I–II loop in Cav3.1 and Cav3.3 yielded a modest increase (+30%) and a reduction (−30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Cav3 function by highlighting the unique role played by the intracellular I–II loop in Cav3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Cav3.3

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

State-dependent Ras signaling and AMPA receptor trafficking

Synaptic trafficking of AMPA-Rs, controlled by small GTPase Ras signaling, plays a key role in synaptic plasticity. However, how Ras signals synaptic AMPA-R trafficking is unknown. Here we show that low levels of Ras activity stimulate extracellular signal-regulated kinase kinase (MEK)–p42/44 MAPK (extracellular signal-regulated kinase [ERK]) signaling, whereas high levels of Ras activity stimulate additional Pi3 kinase (Pi3K)–protein kinase B (PKB) signaling, each accounting for ∼50% of the potentiation during long-term potentiation (LTP). Spontaneous neural activity stimulates the Ras–MEK–ERK pathway that drives GluR2L into synapses. In the presence of neuromodulator agonists, neural activity also stimulates the Ras–Pi3K–PKB pathway that drives GluR1 into synapses. Neuromodulator release increases with increases of vigilance. Correspondingly, Ras–MEK–ERK activity in sleeping animals is sufficient to deliver GluR2L into synapses, while additional increased Ras–Pi3K–PKB activity in awake animals delivers GluR1 into synapses. Thus, state-dependent Ras signaling, which specifies downstream MEK–ERK and Pi3K–PKB pathways, differentially control GluR2L- and GluR1-dependent synaptic plasticity

Alternative splicing within the I-II loop controls surface expression of T-type Ca(v)3.1 calcium channels

Molecular diversity of T-type/Cav3 Ca2+ channels is created by expression of three genes and alternative splicing of those genes. Prompted by the important role of the I-II linker in gating and surface expression of Cav3 channels, we describe here the properties of a novel variant that partially deletes this loop. The variant is abundantly expressed in rat brain, even exceeding transcripts with the complete exon 8. Electrophysiological analysis of the Δ8b variant revealed enhanced current density compared to Cav3.1a, but similar gating. Luminometry experiments revealed an increase in the expression of Δ8b channels at the plasma membrane. We conclude that alternative splicing of Cav3 channels regulates surface expression and may underlie disease states in which T-channel current density is increased

Estimating the effect of the deletions on the probability of channel opening.

<p><i>Aa</i>, Schematic of the P/-8 voltage protocol. <i>Ab</i>, Representative current at full scale, which is expanded in panel <i>Ac</i>. <i>B,C</i> Representative gating current traces recorded during depolarizing voltage steps from −100 to ∼ +50 mV (reversal potential): WT Ca_v3.1 (<i>Ba</i>); GD1–2 (<i>Bb</i>); GD3–5 (<i>Bc</i>); WT Ca_v3.3 (<i>Ca</i>); ID1–2 (<i>Cb</i>); and ID3–5 (<i>Cc</i>). Vertical scale bar is same size for all six traces (0.1 nA), while the horizontal scale bar is 1 ms in <i>B</i> and 2 ms in <i>C</i>. Data were acquired at 20 kHz, filtered at 10 kHz, and represent the average of 20 runs. G_max vs. Q_max for WT Ca_v3.1 and GD1–2 (<i>D</i>), or WT Ca_v3.3 and ID1–2 (<i>E</i>). The slope of the linear regression fit provides an estimate of <i>P_o,</i> and in both cases the slope of the line fitting the D1–2 mutants was 2-fold higher than for WT (Ca_v3.1, 0.26±0.03, n = 9; GD1–2, 0.55±0.06, n = 6, P<0.001; Ca_v3.3, 0.12±0.01, n = 9; and ID1–2, 0.26±0.02, n = 6, P<0.05). The difference between Ca_v3.1 and Ca_v3.3 is also statistically significant (P<0.001, one-way ANOVA followed by Tukey's multiple comparison test, Prism).</p

Effect of Ca_v3.1 I–II loop deletions on the voltage dependence of channel activation.

<p><i>A, B,</i> Normalized current traces recorded during depolarizing voltage steps from −80 to +30 mV (holding potential, −100 mV, except for GD1–2 mutant, which due to shifted inactivation was −110 mV) in WT Ca_v3.1 (<i>Aa</i>), GD1–2 (<i>Ab</i>), GD3–5 (<i>Ac</i>), WT Ca_v3.3 (<i>Ba</i>), ID1–2 (<i>Bb</i>) and ID3–5 (<i>Bc</i>). Thick gray lines represent the current at −50 mV, demonstrating the negative shift in voltage dependence of activation observed in the deletion mutants. Currents were normalized to the maximum peak current in that cell. Time calibration bar scale applies to all three sets of traces in each case. Peak current-voltage plots for either Ca_v3.1 and its deletions (<i>C</i>) or Ca_v3.3 and its deletions (<i>D</i>). Peak currents were normalized to the cell size as estimated by capacitance. Normalized current-voltage plots for either Ca_v3.1 and its deletions (<i>E</i>) or Ca_v3.3 and its deletions (<i>F</i>). Same symbol definition as in panels <i>C</i> and <i>D</i>. Smooth curves in <i>C–F</i> represent fits to the average data using a Boltzmann–Ohm equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002976#pone.0002976-AriasOlgun1" target="_blank">[4]</a>.</p

Electrophysiological properties of Ca_v3.1, Ca_v3.3, and their deletion mutants.

<p>The <i>G_max</i> and <i>V_0.5</i> of activation were determined from the <i>I-V</i> protocol, and therefore have the same number of cells (n) in each measurement. The <i>G/Q</i> ratio was calculated for each individual cell, and then averaged. Statistical significance is denoted with asterisks, where three asterisks indicates P<0.001, two for P<0.01, and one for P<0.05.</p