Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2

The Cacna1fnob2 mouse is reported to be a naturally occurring null mutation for the Cav1.4 calcium channel gene and the phenotype of this mouse is not identical to that of the targeted gene knockout model. We found two mRNA species in the Cacna1fnob2 mouse: approximately 90% of the mRNA represents a transcript with an in-frame stop codon within exon 2 of CACNA1F, while approximately 10% of the mRNA represents a transcript in which alternative splicing within the ETn element has removed the stop codon. This latter mRNA codes for full length Cav1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Cav1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Cav1.4wt or Cav1.4nob2 cDNA revealed that the alternatively spliced protein does not differ from wild type with respect to activation and inactivation characteristics; however, while the wild type N-terminus interacted with filamin proteins in a biochemical pull-down experiment, the alternatively spliced N-terminus did not. The Cacna1fnob2 mouse electroretinogram displayed reduced b-wave and oscillatory potential amplitudes, and the retina was morphologically disorganized, with substantial reduction in thickness of the outer plexiform layer and sprouting of bipolar cell dendrites ectopically into the outer nuclear layer. Nevertheless, the spatial contrast sensitivity (optokinetic response) of Cacna1fnob2 mice was generally similar to that of wild type mice. These results suggest the Cacna1fnob2 mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Cav1.4 protein, albeit at reduced levels, and the functional Cav1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1fnob2 mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts

Ca(v)1.4 Encodes a Calcium Channel with Low Open Probability and Unitary Conductance

When transiently expressed in tsA-201 cells, Ca(v)1.4 calcium channels support only modest whole-cell currents with unusually slow voltage-dependent inactivation kinetics. To examine the basis for this unique behavior we used cell-attached patch single-channel recordings using 100 mM external barium as the charge carrier to determine the single-channel properties of Ca(v)1.4 and to compare them to those of the Ca(v)1.2. Ca(v)1.4 channel openings occurred infrequently and were of brief duration. Moreover, openings occurred throughout the duration of the test depolarization, indicating that the slow inactivation kinetics observed at the whole-cell level are caused by sustained channel activity. Ca(v)1.4 and Ca(v)1.2 channels displayed similar latencies to first opening. Because of the rare occurrence of events, the probability of opening could not be precisely determined but was estimated to be <0.015 over a voltage range of −20 to +20 mV. The single-channel conductance of Ca(v)1.4 channels was ∼4 pS compared with ∼20 pS for Ca(v)1.2 under the same experimental conditions. Additionally, in the absence of divalent cations, Ca(v)1.4 channels pass cesium ions with a single-channel conductance of ∼21 pS. Although Ca(v)1.2 opening events were best described kinetically with two open time constants, Ca(v)1.4 open times were best described by a single time constant. BayK8644 slightly enhanced the single-channel conductance in addition to increasing the open time constant for Ca(v)1.4 channels by ∼45% without, however, causing the appearance of an additional slower gating mode. Overall, our data indicate that single Ca(v)1.4 channels support only minute amounts of calcium entry, suggesting that large numbers of these channels are needed to allow for significant whole-cell current activity, and providing a mechanism to reduce noise in the visual system

RT-PCR analysis of <i>Cacna1f^wt</i> and <i>Cacna1f^nob2</i> mice.

<p>A. Schematic representation of the location of PCR primers used. Primers RR44, 45, and 46 were used for RT-PCR reactions; primers RR50, 51, 52, and 53 were used for genomic PCR reactions. B. Agarose gel depicting RT-PCR reaction products for mRNA isolated from <i>Cacna1f^wt</i> and <i>Cacna1f^nob2</i> mice. Regardless of the primer pair used, only a single band is detected using mRNA from <i>Cacna1f^wt</i> mice. Using mRNA from <i>Cacna1f^nob2</i> mice, however, two bands are visible (see arrows). The relative intensities of the fluorescence signals indicate that the larger-M_r band accounts for ∼90%, and the smaller-M_r band for ∼10%, of the total mRNA.</p

Biophysical properties of Ca_v1.4^wt and Ca_v1.4^nob2 channels, coexpressed with β_2a and α₂–δ₁ subunits in tSA-201 cells.

<p>A. Representative current waveforms for Ca_v1.4^wt (left) and Ca_v1.4^nob2 (right) recorded with 20 mM Ba²⁺ external saline. Horizontal scale bars denote 25 ms, and vertical scale bars 25 pA. B. Average activation (filled symbols) and inactivation (hollow) symbols for Ca_v1.4^wt (squares) and Ca_v1.4^nob2 (circles) recorded with 20 mM Ba²⁺ external saline. Average activation parameters from 11 Ca_v1.4^wt cells and 13 Ca_v1.4^nob2 cells are: V_{act, wt} = −3±4 mV, V_{act, nob2} = −1±4 mV (n = 13); G_{max, wt} = 4±3 nS and G_{max, nob2} = 3±1 nS; S_wt = 9±1 mV and S_nob2 = 8.2±0.8 mV. These values are stastically identical, and are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone-0002538-t003" target="_blank">Table 3</a>. Average inactivation parameters from these cells are V_{inact, wt} = −18±11 mV and V_{inact, nob2} = −22±10 mV, with a large fraction of non-inactivating current for both channels. These values statistically identical and are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone-0002538-t003" target="_blank">Table 3</a>. C. Average half-inactivation potentials for channels recorded with 2 mM Ca²⁺ as charge carrier. Currents were substantially smaller than with 20 mM Ba²⁺, but were distinguishable from background noise, and were obtained using a ramp protocol identical to that previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone.0002538-Doering2" target="_blank">[31]</a>, obtained by ramping voltage from −100 mV to +100 mV over 500 ms. Values were V_{act, wt} = −17±8 mV (average peak current size −9±4 pA) and V_{act, nob2} = −17±6 mV (average peak current size −9±3 pA). The shift observed with switching from 20 mM Ba²⁺ to 2 mM Ca²⁺ as external charge carrier is similar to that we have previously reported for the human Ca_v1.4 channels <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone.0002538-McRory1" target="_blank">[16]</a>.</p

Western blot of spleen samples from <i>Cacna1f^wt</i>, <i>Cacna1f^nob2</i>, and <i>Cacna1f^G305X</i> mice probed with a Ca_v1.4-specific antibody directed against the C-terminus of the channel.

<p>Full-length protein is visible in all lanes except for <i>Cacna1f^G305X</i>.</p