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

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

Location of deletions in the I–II loop.

<p>(<i>A</i>), Schematic representation of the I–II loop connecting repeat IS6 to repeat IIS1 in Ca_v3.1 and Ca_v3.3. Deleted regions are shown as open boxes. (<i>B</i>), Alignment of the I–II loop of human Ca_v3 channels. Arrows indicate where deletions begin and end. Dashes represent gaps in the alignment. Amino acids are color-coded by their physical properties as follows: red, positively-charged; green, negatively-charged; blue, polar; and yellow, hydrophobic.</p