Orientation of palmitoylated CaVβ2a relative to CaV2.2 is critical for slow pathway modulation of N-type Ca2+ current by tachykinin receptor activation

The Gq-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca2+ channel (CaV2.2 or N channel) activity at two distinct sites by a pathway involving a lipid metabolite, most likely arachidonic acid (AA). In another study published in this issue (Heneghan et al. 2009. J. Gen Physiol. doi:10.1085/jgp.200910203), we found that the form of modulation observed depends on which CaVβ is coexpressed with CaV2.2. When palmitoylated CaVβ2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when CaVβ3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of CaVβ2a may occupy an inhibitory site on CaV2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of CaVβ2a relative to CaV2.2 may displace the palmitoyl groups and prevent them from antagonizing AA's actions, thereby allowing inhibition even in the presence of CaVβ2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the α interacting domain (AID) of CaV2.2's I–II linker. CaVβs bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple CaVβ's movements to CaV2.2 gating. Although Bdel1/β2a currents exhibited more variable enhancement by SP, Bdel2/β2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from CaV2.2 coexpressed with CaVβ3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/β2a currents, suggesting that when palmitoylated CaVβ2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that CaVβ2a's palmitoyl groups directly interact with an inhibitory site on CaV2.2 to block N-current inhibition by SP

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

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

Molecular Pharmacology of Human Cav3.2 T-Type Ca2+ Channels: Block by Antihypertensives, Antiarrhythmics, and Their Analogs

Antihypertensive drugs of the “calcium channel blocker” or “calcium antagonist” class have been used to establish the physiological role of L-type Ca2+ channels in vascular smooth muscle. In contrast, there has been limited progress on the pharmacology T-type Ca2+ channels. T-type channels play a role in cardiac pacemaking, aldosterone secretion, and renal hemodynamics, leading to the hypothesis that mixed T- and L-type blockers may have therapeutic advantages over selective L-type blockers. The goal of this study was to identify compounds that block the Cav3.2 T-type channel with high affinity, focusing on two classes of compounds: phenylalkylamines (e.g., mibefradil) and dihydropyridines (e.g., efonidipine). Compounds were tested using a validated Ca2+ influx assay into a cell line expressing recombinant Cav3.2 channels. This study identified four clinically approved antihypertensive drugs (efonidipine, felodipine, isradipine, and nitrendipine) as potent T-channel blockers (IC50 < 3 μM). In contrast, other widely prescribed dihydropyridines, such as amlodipine and nifedipine, were 10-fold less potent, making them a more appropriate choice in research studies on the role of L-type currents. In summary, the present results support the notion that many available antihypertensive drugs block a substantial fraction of T-current at therapeutically relevant concentrations, contributing to their mechanism of action

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

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

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