Alternative splicing of the rat Cav3.3 T-type calcium channel gene produces variants with distinct functional properties11The sequences reported herein have been assigned GenBank accession numbers AY128644–AY128648.

AbstractMolecular diversity in T-type Ca2+ channels is produced by expression of three genes, and alternative splicing of those genes. Prompted by differences noted between rat and human Cav3.3 sequences, we searched for splice variants. We cloned six variants, which are produced by splicing at exon 33 and exon 34. Expression of the variants differed between brain regions. The electrophysiological properties of the variants displayed similar voltage-dependent gating, but differed in their kinetic properties. The functional impact of splicing was inter-related, suggesting an interaction. We conclude that alternative splicing of the Cav3.3 gene produces channels with distinct properties

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

Cloning and expression of the human T-type channel Ca(v)3.3: insights into prepulse facilitation.

The full-length human Ca(v)3.3 (alpha(1I)) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3' untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5 mM Ca(2+) as charge carrier. From a holding potential of -100 mV, step depolarizations elicited inward currents with an apparent threshold of -70 mV, a peak of -30 mV, and reversed at +40 mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Ca(v)3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Ca(v)3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Ca(v)3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100 mV, 200 ms) increased the fraction of channels that gated at low voltages. In contrast, human Ca(v)3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Ca(v)3.3 is similar to high voltage-activated Ca(2+) channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity

Electrophysiological properties of WT, PG6, and PA6 channels and their regulation by β2a and β3.

<p>The values of V₅₀ and k were calculated for each cell, then averaged. R values determined from test pulses to +20 mV. Data shown are mean±SEM from the number of cells shown in parentheses. Statistical significance of the β2a and β3 effects relative to either α₁ alone (+α₂δ1) were determined using ANOVA.</p>†<p>Currents from PG6 were completely inactivated by 350 ms, so the residual current at 25 ms (divided by peak) is reported (R₂₅).</p>§<p>Current density was estimated from the peak of the <i>I–V</i> curve, and statistical significance was determined using Student's <i>t</i>-test.</p><p>*P<0.05, **P<0.01.</p

Model showing possible orientations of β with respect to α₁ assuming a β sheet structure at the site of deletion.

<p>(A) Model showing the orientation of β-subunit in wild-type, and (B) after deletion of 1 amino acid. The β3 core structure was modeled from PDB code 1VYT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003560#pone.0003560-Chen1" target="_blank">[9]</a>. The CFP, Cirulean, was modeled from PDB code 2QYT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003560#pone.0003560-Malo1" target="_blank">[57]</a>. The fragments of CFP were generated using PyMOLWin (Delano Scientific), where CFP-N corresponds to residues 1–158, and CFP-C corresponds to residues 159–238. The approximate size of the α₁2.2 domains and linkers were estimated using the method of Helton and Horne, where the volume occupied by each segment is calculated from the number of amino acids in each segment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003560#pone.0003560-Helton1" target="_blank">[45]</a>. The β3 subunit was scaled using the same method.</p

Introduction of the poly-glycine substitution in the α₁2.2 subunit disrupts (PG6), while poly-alanine substitution (PA6) preserves Ca_vβ regulation.

<p>Panels A–D show data obtained with PG6, while panels E–H show data obtained with PA6. (A, E) Peak current-voltage relationships normalized to cell capacitance for the respective α₁ mutant expressed alone or with β2a or β3. (B, F) Activation represented by the normalized conductance (G/G_max). The residual current after either 25 ms (C) or 350 ms (G) of depolarization divided by the maximum inward current, and plotted against test potential. Representative traces normalized to the peak inward current are shown in the inset. (D, H) Comparison of the β2a and β3 effects on steady-state inactivation estimated using 15 s prepulses to varying potentials. Dotted lines represent steady-state inactivation measured for WT channels in the presence of β3 (α₁2.2+α₂δ1+β3).</p

Estimating P_o of wild-type and Bdel1 channels.

<p>(Aa) Exemplar gating current at reversal potential (∼65 mV) for WT channels expressed with β2a. (Ab) Ionic current trace from the same cell recorded during a depolarizing step to +30 mV from a holding potential of −90 mV. Exemplar gating (Ba) and ionic (Bb) currents for the Bdel1 deletion mutant (also with β2a). Scale bars represent the same units as in panel A. (C) Plot of G_max versus Q_max where each symbol represents an individual cell. Solid line represents the fit to the data with linear regression. The slope, G/Q, is proportional to maximal channel open probability.</p

Deletions in the linker between AID and IS6 (Bdels) affect β regulation.

<p>Panels A–D show data obtained with Bdel1, panels E–H show data obtained with Bdel2, and panels I–L show data obtained with Bdel3. (A, E, I) Peak current-voltage relationships normalized to cell capacitance for Bdels expressed with β2a or β3. In the absence of a β, only Bdel1 produced detectable currents. (B, F,J) Normalized current traces recorded during depolarizing steps to +20 mV from a holding potential of −90 mV. Residual current after either 350 ms (C) or 25 ms (G, K) of depolarization divided by the maximum inward current, and plotted against test potential. (D, H, L) Comparison of the β2a and β3 effects on steady-state inactivation for Bdels estimated using 15 s prepulses to varying potentials. Dotted lines represent β3 effect on steady-state inactivation for WT channel.</p