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. 2004 Feb 18;24(7):1707-18.
doi: 10.1523/JNEUROSCI.4846-03.2004.

The CACNA1F gene encodes an L-type calcium channel with unique biophysical properties and tissue distribution

John E McRory  1 , Jawed Hamid, Clinton J Doering, Esperanza Garcia, Robin Parker, Kevin Hamming, Lina Chen, Michael Hildebrand, Aaron M Beedle, Laura Feldcamp, Gerald W Zamponi, Terrance P Snutch
Affiliations

The CACNA1F gene encodes an L-type calcium channel with unique biophysical properties and tissue distribution

John E McRory et al. J Neurosci. .

Abstract

Glutamate release from rod photoreceptors is dependent on a sustained calcium influx through L-type calcium channels. Missense mutations in the CACNA1F gene in patients with incomplete X-linked congenital stationary night blindness implicate the Ca(v)1.4 calcium channel subtype. Here, we describe the functional and pharmacological properties of transiently expressed human Ca(v)1.4 calcium channels. Ca(v)1.4 is shown to encode a dihydropyridine-sensitive calcium channel with unusually slow inactivation kinetics that are not affected by either calcium ions or by coexpression of ancillary calcium channel beta subunits. Additionally, the channel supports a large window current and activates near -40 mV in 2 mM external calcium, making Ca(v)1.4 ideally suited for tonic calcium influx at typical photoreceptor resting potentials. Introduction of base pair changes associated with four incomplete X-linked congenital night blindness mutations showed that only the G369D alteration affected channel activation properties. Immunohistochemical analyses show that, in contrast with previous reports, Ca(v)1.4 is widely distributed outside the retina, including in the immune system, thus suggesting a broader role in human physiology.

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Figures

Figure 1.
Figure 1.
Proposed transmembrane topology of the calcium channel α1 subunit illustrating the approximate loci of amino acid differences in our cDNA construct relative to the previously available GenBank sequence (accession number AF15290). Lys-58 (N terminus) region, Glu-381 (domain I-II linker), Arg-1282 (IVS3-S4), and Ile-1306 (IVS4) in the original GenBank sequence are substituted in our cDNA by Arg, Gly, His, and Val residues, respectively. The presence of these residues was confirmed in five independent PCR and sequencing reactions. The # symbol indicates the location of an 11 amino acid insertion attributable to sliding of an intron-exon boundary at the end of exon 9 (domain I-II linker). The asterisk indicates a deletion of seven amino acids in the domain IV S3-S4 region attributable to skipping of exon 32. Locations of missense mutations associated with night blindness (G369D, G674D, A928D, W1459stop) are indicated by dotted lines.
Figure 2.
Figure 2.
HEK cell transfected with human Cav1.4 cDNA/pcDNA 3-zeo and stained using the Cav1.4 antibody. Top left, An image captured with a confocal microscope demonstrating expression of the Cav1.4 channel in the cell membrane. Top right, The same section, but visualized using bright-field illumination. Bottom, Overlay of above images demonstrating localization of the Cav1.4 calcium channel to the outer membrane.
Figure 3.
Figure 3.
Biophysical properties of the human Cav1.4 calcium channel. A, Family of whole-cell currents recorded from the human Cav1.4 calcium channel coexpressed with rat β2a and α2-δ1 subunits in tsA-201 cells. The cell was held at -100 mV and depolarized to various test potentials at 10 sec intervals. B, Ensemble of whole-cell current-voltage relationships from 75 different experiments. In each case, the holding potential was -100 mV. The solid line reflects a fit with the Boltzman relationship. Parameters obtained from this fit were: G = 5.7 nS, S = 6.68 mV, Erev = 49.0 mV, and Va = -0.95 mV. C, Ensemble of 14 inactivation curves obtained at a test potential of +20 mV. The solid line is a fit with the Boltzmann relationship. The parameters obtained from the fit were: Vh = -11.98 mV and z = 2.26. The dashed line represents the activation curve plotted using parameters obtained in B. Note the large window current. D, Activity of Cav1.4 in 2 mm Ca2+ and low (0.5 mm) EGTA internal recording solution containing 4 mm ATP. The light trace represents an average of I-V relationships obtained from five separate recordings, each normalized to unity at peak. Because the individual I-V relationships did not peak at exactly the same potential, the peak of the displayed I-V curves and its Boltzmann fit (solid line) do not extend to -1. The fitting parameters were: Va = -11.0 mV, Erev = 38.1 mV, G = 0.028 nS, and S = 8.65 mV. E, Comparison of the biophysical parameters of the human Cav1.4 calcium channel coexpressed with rat α2-δ1 and various rat β subunits (20 mm Ba). Note that the β subunits mediate only minor changes in the half-activation (Va) and half-inactivation potentials (Vh) and in the time constants for inactivation (determined from monoexponential fits to raw current records such as the one shown in D).
Figure 4.
Figure 4.
Pharmacological properties of human Cav1.4 calcium channels. A, Sample traces at +20 mV for currents obtained in 20 mm external ion in the presence of low (0.5 mm) EGTA internal recording solution. Note that even under low buffering conditions, the current appears to be insensitive to inactivation even in the presence of high calcium. Inset, Average fraction of remaining currents (R800) after an 800 msec depolarization to +20 mV. In each case, R800 values were obtained in barium and calcium from the same cell. A total of seven cells are included in the analysis. Error bars denote SEs. B, Dose dependence of nifedipine block of Cav1.4 channels (coexpressed with rat β2a and rat α2-δ1) at a test potential of +20 mV (n = 10). The solid line reflects a fit with the Michaelis-Menton equation (IC50 = 900 nm; n = 0.88). Inset, Comparison of the blocking effects of 300 nm nifedipine at holding potentials of -100 and -50 mV. Note the slight increase in the amount of block at more depolarized holding potentials. C, Ensemble of whole-cell current-voltage relationships from nine different paired experiments before and after the application of 3 μm BayK8644. In each case, the holding potential was -100 mV. Note the increase in maximum slope conductance and leftward shift in the peak of the current-voltage relationship after application of BayK 8644. The parameters obtained from the Boltzman fits were: control: Va = -0.7 mV, Erev = 46.4 mV, G = 2.53 nS, and S = 8.04; BayK: Va = -9.6 mV, Erev = 54.5 mV, G = 9.7 nS, and S = 4.82 mV. D, Summary of Cav1.4 (plus rat β2a plus rat α2-δ1) calcium channel pharmacology. The cells were bathed in 20 mm external barium, the holding potential was -100 mV, and the test potential was +20 mV. Note the small degree of inhibition mediated by 3 μm ω-conotoxin GVIA (17.4 ± 0.9%; n = 8). Nifedipine (3 μm) and cadmium (10 μm) both potently inhibit channel activity [73.4 ± 3.1% (n = 9) and 91.2 ± 2.0% (n = 7) block, respectively]. BayK 8644 (3 μm) significantly upregulates current activity (406 ± 50%; n = 9).
Figure 5.
Figure 5.
Expression of the Cav1.4 calcium channel in human retina. A, Tissue section of human retina stained with H and E demonstrating the constituents of the retina. ONL, Outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer. B, Tissue section of retina immunostained with Cav1.4 antibody (staining indicated by brown precipitate). Immunoreactivity of cells and synapses is observed in the OPL, INL, IPL, and NFL. C, Tissue section of retina and retinoblastoma tumor stained with preabsorbed Cav1.4 antibody (negative control). All images were captured at 400× magnification.
Figure 6.
Figure 6.
Expression the Cav1.4 calcium channel in human retinoblastoma (Rb). A tissue section of human Rb tumor (400×) stained with H and E (A) and with Cav1.4 antibody (B) (staining indicated brown precipitate) demonstrating immunoreactivity of tumor cells is shown. Areas of tumor cell necrosis (Nec) and endothelial cells lining the blood vessel (BV) do not stain. C, Section of Rb (200×) stained with preabsorbed Cav1.4 antibody (negative control).
Figure 7.
Figure 7.
Tissue distribution of the Cav1.4 calcium channel in human and rat. A, RT-PCR detected Cav1.4 calcium channel transcripts in RNA from human adrenal gland, bone marrow, skeletal muscle, spinal cord, spleen, and thymus. One microgram of total human RNA was used in each RT-PCR reaction together with oligonucleotides specific to the Cav1.4 channel. PCR products were electrophoresed through a 1.8% agarose gel, blotted onto a nylon membrane, and probed with a [γ-32P] ATP radiolabeled oligonucleotide specific to Cav1.4. B, Western blot with a Cav1.4-specific antibody detects protein expression in rat retina, spleen, spinal cord, bone marrow, and thymus. Ten milligrams of total protein were separated through a 5% SDS-PAGE acrylamide gel, blotted to nitrocellulose membrane, and then probed with the Cav1.4-specific antibody. The results shown are representative of identical experiments using tissue from three separate rats.
Figure 8.
Figure 8.
Cav1.4 calcium channel expression in human lymphoid tissue. A, Tissue section of human lymph node immunostained with Cav1.4 antibody demonstrating immunoreactivity of plasma cells (PC) in a germinal center. B, Tissue section of chronically inflamed human uterine cervix immunostained with Cav1.4 antibody demonstrating immunoreactivity of PCs as well as mast cells (MC), in the cervical stroma. C, Section of cervix stained with Cav1.4 antibody incubated with the blocking peptide (negative control). All images were captured at 400× magnification.
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