Abstract
Voltage-gated calcium channels play a major role in many important processes including muscle contraction, neurotransmission, excitation-transcription coupling, and hormone secretion. To date, 10 calcium channel alpha(1)-subunits have been reported, of which four code for L-type calcium channels. In our previous work, we uncovered by transcript-scanning the presence of 19 alternatively spliced exons in the L-type Ca(v)1.2 alpha(1)-subunit. Here, we report the smooth muscle-selective expression of alternatively spliced exon 9(*) in Ca(v)1.2 channels found on arterial smooth muscle. Specific polyclonal antibody against exon 9(*) localized the intense expression of 9(*)-containing Ca(v)1.2 channels on the smooth muscle wall of arteries, but the expression on cardiac muscle was low. Whole-cell patch clamp recordings of the 9(*)-containing Ca(v)1.2 channels in HEK293 cells demonstrated -9 and -11-mV hyperpolarized shift in voltage-dependent activation and current-voltage relationships, respectively. The steady-state inactivation property and sensitivity to blockade by nifedipine of the +/-exon 9(*) splice variants were, however, not significantly different. Such cell-selective expression of an alternatively spliced exon strongly indicates the customization and fine tuning of calcium channel functions through alternative splicing of the pore-forming alpha(1)-subunit. The generation of proteomic variations by alternative splicing of the calcium channel Ca(v)1.2 alpha(1)-subunit can potentially provide a flexible mechanism for muscle or neuronal cells to respond to various physiological signals or to diseases.
Highlights
Despite the fundamental importance of the hyperpolarized activation threshold of smooth muscle L-type channels, the molecular basis for this specialization remains elusive. This unknown represents a critical deficit in our understanding of vascular biology, especially given that the underlying structural elements could represent valuable molecular targets for novel therapeutics to modulate vascular tone
We demonstrated the high expression of exon 9* in the smooth muscle layer of arteries
Acknowledgments—We thank Drs David Yue and Jinsong Bian for critically reading the manuscript and for invaluable suggestions and comments
Summary
Generation of ␣1C77-9* Expression Construct—We performed RTPCR of human heart cDNA (Quickclone; catalog no. 7121-1; Clontech) to amplify the region spanning exons 6 –11 of the ␣11.2 subunit. To determine nonspecific cross-reactivity with ␣11.3 exon 9* protein, a PCR fragment containing exons 9, 9*, 10, 12, 13, and 14 of ␣11.3 subunit isolated from a rat cochlea cDNA library was inserted into pET22b vector using the EcoRI and SalI restriction enzyme sites. Determination of Tissue Specificity of pAb9* Antibody by Western Blot—Tissues from rat aorta, brain, and heart were homogenized in lysis buffer containing 50 mM Tris, pH 8, 1 mM EDTA, and 150 mM NaCl. Due to the small quantity of rat aorta, six or seven aortas were pooled together for protein extraction. After washing with 0.1% Triton/phosphate-buffered saline, goat anti-rabbit fluorescein isothiocyanate-conjugated or goat antimouse Texas Red-conjugated secondary antibody was added to the samples. The steady-state inactivation curves were obtained from experiments by stepping from a holding potential of Ϫ90 mV to a family of 15-s-long prepulses, followed by a 104-ms test pulse to ϩ10 mV. Statistical significance of differences between means was calculated with Student’s t test
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