Characterization of CaV1.2 exon 33 heterozygous knockout mice and negative correlation between Rbfox1 and CaV1.2 exon 33 expressions in human heart failure.

Recently, we reported that homozygous deletion of alternative exon 33 of CaV1.2 calcium channel in the mouse resulted in ventricular arrhythmias arising from increased CaV1.2Δ33 ICaL current density in the cardiomyocytes. We wondered whether heterozygous deletion of exon 33 might produce cardiac phenotype in a dose-dependent manner, and whether the expression levels of RNA splicing factors known to regulate alternative splicing of exon 33 might change in human heart failure. Unexpectedly, we found that exon 33+/- cardiomyocytes showed similar CaV1.2 channel properties as wild-type cardiomyocyte, even though CaV1.2Δ33 channels exhibit a gain-in-function. In human hearts, we found that the mRNA level of splicing factor Rbfox1, but not Rbfox2, was downregulated in dilated cardiomyopathy, and CACNA1C mRNA level was dramatically decreased in the both of dilated and ischemic cardiomyopathy. These data imply Rbfox1 may be involved in the development of cardiomyopathies via regulating the alternative splicing of CaV1.2 exon 33. (149 words).

Exclusion of alternative exon 33 of CaV1.2 calcium channels in heart is proarrhythmogenic.

Alternative splicing changes the CaV1.2 calcium channel electrophysiological property, but the in vivo significance of such altered channel function is lacking. Structure-function studies of heterologously expressed CaV1.2 channels could not recapitulate channel function in the native milieu of the cardiomyocyte. To address this gap in knowledge, we investigated the role of alternative exon 33 of the CaV1.2 calcium channel in heart function. Exclusion of exon 33 in CaV1.2 channels has been reported to shift the activation potential -10.4 mV to the hyperpolarized direction, and increased expression of CaV1.2Δ33 channels was observed in rat myocardial infarcted hearts. However, how a change in CaV1.2 channel electrophysiological property, due to alternative splicing, might affect cardiac function in vivo is unknown. To address these questions, we generated mCacna1c exon 33-/--null mice. These mice contained CaV1.2Δ33 channels with a gain-of-function that included conduction of larger currents that reflects a shift in voltage dependence and a modest increase in single-channel open probability. This altered channel property underscored the development of ventricular arrhythmia, which is reflected in significantly more deaths of exon 33-/- mice from ß-adrenergic stimulation. In vivo telemetric recordings also confirmed increased frequencies in premature ventricular contractions, tachycardia, and lengthened QT interval. Taken together, the significant decrease or absence of exon 33-containing CaV1.2 channels is potentially proarrhythmic in the heart. Of clinical relevance, human ischemic and dilated cardiomyopathy hearts showed increased inclusion of exon 33. However, the possible role that inclusion of exon 33 in CaV1.2 channels may play in the pathogenesis of human heart failure remains unclear.

Alternative splicing generates a novel truncated Cav1.2 channel in neonatal rat heart.

L-type Cav1.2 Ca(2+) channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Cav1.2 Ca(2+) channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Cav1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Cav1.233L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Cav1.2 channels, Cav1.233L channels reduced the current density and altered the electrophysiological properties of the wild type Cav1.2 channels. Interestingly, the truncated 3.5-domain Cav1.233L channels also yielded a dominant negative effect on Cav1.3 channels, but not on Cav3.2 channels, suggesting that Cavß subunits is required for Cav1.233L regulation. A biochemical study provided evidence that Cav1.233L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Cav1.233L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Cav1.2 channels. Moreover, the human Cav1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Cav1.2 channel. An electrophysiological study showed that human Cav1.233L channel is a functional channel but conducts Ca(2+) ions at a much lower level.

Molecular alteration of Ca(v)1.2 calcium channel in chronic myocardial infarction.

Ca(v)1.2 channels are important for excitation-contraction coupling of cardiac muscles. Alternative splicing of Ca(v)1.2 channels could produce extensive phenotypic variations of channel properties. In a rat model of chronic myocardial infarction, we investigated whether Ca(v)1.2 channels may alter the use of alternatively spliced exons to generate functional variants. A myocardial infarction model on rat was generated by ligating the left anterior descending artery. Eight weeks after ligation, we found that in the scar region, the expression of a number of alternatively spliced exons were changed. The proportions of exon 9* inclusion and exon 33 deletion were detected to increase and localize at the surviving cardiac muscle cells with reverse transcriptase polymerase chain reaction, laser capture microdissection, and immunostaining. The wild-type Delta9*/33 (deletion of exon 9* and inclusion of exon 33) channel was reduced greatly in the scar region and several other isoforms increased. Importantly, a novel 9*/Delta33 (inclusion of exon 9* and deletion of exon 33) channel was generated in the scar region. Electrophysiological studies showed that the channels found in scar region exhibited hyperpolarized shifts in both the activation and inactivation potentials when expressed in HEK293 cells. The changes of Ca(v)1.2 channels may play a role either in maintenance of muscle excitability and contractility or contribute to arrhythmogenesis.

Differential splicing patterns of L-type calcium channel Cav1.2 subunit in hearts of Spontaneously Hypertensive Rats and Wistar Kyoto Rats.

Cav1.2 L-type calcium channels are essential in heart and smooth muscle contraction. Rat Cav1.2 gene contains 11 alternatively spliced exons (1a, 1, 8a, 8, 9*, 21, 22, 31, 32, 32-6nt and 33) which can be assorted to generate a large number of functionally distinct splice variants. Until now, it is unknown whether the utilization of these alternatively spliced exons is altered in the hypertrophied hearts of hypertensive rats. By comparing the assortments of these 11 exons in full-length Cav1.2 transcripts derived from Spontaneously Hypertensive Rats (SHRs) and Wistar Kyoto Rats (WKYs) hearts, we found that the inclusion of Cav1.2 alternative exons was significantly different between the two rats both at individual loci and in combinatorial arrangements. Functional characterizations of three Cav1.2 channel splice variants that were identified to be significantly altered in SHR hypertrophied cardiomyocytes demonstrated distinct whole-cell electrophysiological properties when expressed in HEK 293 cells. Interestingly, aberrant splice variants which included or excluded both mutually exclusive exons 21/22 or exons 31/32 were found to be increased in hypertensive rats. Two aberrant splice variants that included both exons 21 and 22 were found to be unable to conduct currents even though they expressed proteins with the predicted molecular mass. Characterization of one of the aberrant splice variants showed that it exerted a dominant negative effect on the functional Cav1.2 channels when co-expressed in HEK293 cells. The altered combinatorial splicing profiles of Cav1.2 transcripts identified in SHR hearts provide a different and new perspective in understanding the possible role of molecular remodeling of Cav1.2 channels in cardiac hypertrophy as a consequence of hypertension.

A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine.

Native smooth muscle L-type Ca(v)1.2 calcium channels have been shown to support a fraction of Ca(2+) currents with a window current that is close to resting potential. The smooth muscle L-type Ca(2+) channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca(v)1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Ca(v)1.2 channels. Here, we report a smooth muscle L-type Ca(v)1.2 calcium channel splice variant, Ca(v)1.2SM (1/8/9(*)/32/Delta33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca(v)1.2b clone (1/8/9(*)/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Ca(v)1.2CM clone (1a/8a/Delta9(*)/32/33) that is different from the established Ca(v)1.2a (1a/8a/Delta9(*)/31/33). Importantly, Ca(v)1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca(v)1.2b and cardiac Ca(v)1.2CM channels when currents were recorded in either 5 mM Ba(2+) or 1.8 mM Ca(2+) external solutions. This is the first time that a smooth muscle Ca(v)1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.

Splicing for alternative structures of Cav1.2 Ca2+ channels in cardiac and smooth muscles.

An estimate of up to 60% of genes are subjected to alternative splicing, and 15% of human genetic diseases are associated with mutation of the splice sites [Krawczak M, Reiss J, and Cooper DN. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 1992; 90: 41-54; Cooper TA, and Mattox W. The regulation of splice-site selection, and its role in human disease. Am J Hum Genet 1997; 61: 259-66; Modrek B and Lee CJ. Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss. Nat Genet 2003; 34: 177-80; Modrek B, Resch A, Grasso C, and Lee C. Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res 2001; 29: 2850-9; Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature 2001; 409: 860-921] . The molecular diversity of alternatively spliced transcripts provides templates for a myriad of protein structures that are potentially crucial to sustaining the complexity of human physiology. The extensive alternative splicing of the alpha(1)1.2-subunit of the L-type Ca(v)1.2 channel, producing splice variants with distinct electrophysiological and pharmacological properties, would impact directly on the function of the cardiovascular system. Cell-selective expression of Ca(v)1.2 channels containing a specific alternatively spliced exon increases the functional variations for specific cellular activities in response to changing physiological signals. However, the regulation or control of the alpha(1)1.2-subunit alternative splicing machinery is unknown, and the role of numerous splice variants expressed in a cell is a mystery. A systematic and concerted effort is required to determine all the possible combinations of alternatively spliced exons in alpha(1)1.2-subunits in smooth and cardiac muscles. This will provide useful information to monitor changes on the usage of the entire suite of alternatively spliced exons to help relate altered Ca(v)1.2 channel function to physiology and disease.

The Candida albicans antiporter gene CNH1 has a role in Na+ and H+ transport, salt tolerance, and morphogenesis.

The isolation and functional characterization of a Candida albicans Na+/H+ antiporter gene, CNH1, is reported here. The gene encodes a protein of 840 amino acids that exhibits high levels of similarity in sequence, size, and structural and functional domains to a group of known Na+/H+ antiporters of fungi. The CNH1 gene is able to functionally complement the salt-sensitivity of a Saccharomyces cerevisiae ena1 nha1 mutant, and mutations of two conserved aspartate residues to asparagines in the putative Na+-binding site abolish this activity. Deletion of CNH1 results in retardation of growth and a highly elongated morphology in a significant fraction of cells under conditions that normally support yeast growth. These results indicate that CNH1 has a role in Na+ and H+ transport, salt-tolerance, and morphogenesis.