The L-type (Ca v 1.2) voltage-gated calcium channels play critical roles in membrane excitability, gene expression, and muscle contraction. The generation of splice variants by the alternative splicing of the poreforming Ca v 1.2 ␣ 1 -subunit (␣ 1 1.2) may thereby provide potent means to enrich functional diversity. To date, however, no comprehensive scan of ␣ 1 1.2 splice variation has been performed, particularly in the human context. Here we have undertaken such a screen, exploiting recently developed "transcript scanning" methods to probe the human gene. The degree of variation turns out to be surprisingly large; 19 of the 55 exons comprising the human ␣ 1 1.2 gene were subjected to alternative splicing. Two of these are previously unrecognized exons and two others were not known to be spliced. Comparisons of fetal and adult heart and brain uncovered a large IVS3-S4 variability resulting from combinatorial utilization of exons 31-33. Electrophysiological characterization of such IVS3-S4 variation revealed unmistakable shifts in the voltage dependence of activation, according to an interesting correlation between increased IVS3-S4 linker length and activation at more depolarized potentials. Steady-state inactivation profiles remained unaltered. This systematic portrait of splice variation furnishes a reference library for comprehending combinatorial arrangements of Ca v 1.2 splice exons, especially as they impact development, physiology, and disease.Rapid influx of Ca 2ϩ through the Ca v 1.2 channels initiates physiological responses like gene expression, neurotransmitter release, cardiac or smooth muscle contraction, and regulation of Ca 2ϩ -dependent ion channels (1-4). In these capacities, the functional profile of Ca v 1.2 calcium channels can be customized by combinatorial assembly of the Ca v 1.2 ␣ 1 with several different auxiliary ␤-and ␣ 2 ␦-subunits (5). Even greater flexibility in functional tuning could arise from alternative splicing of the ␣ 1 -subunit genes (6); splicing of the human ␣ 1 1.2 subunit gene is known to generate variants with tissue-specific biases and with distinct pharmacological properties (7). At present, 15 of 53 known exons (Fig. 1) of the human ␣ 1 1.2 gene have been reported to be subjected to alternative splicing (8). However, the full set of possible splice loci and variations and their distributions in heart and brain could far exceed this initial view.Recently, genome-wide analyses suggest that as high as 74% of human genes are alternatively spliced (9). Alternative splicing of pre-mRNA has been implicated in development, physiology, and pathophysiology, and the inclusion or exclusion of exons can be regulated in a tissue-specific or temporal manner (10, 11). Splice variations of human ␣ 1 1.2 subunit confer on the channel isoforms altered properties such as sensitivity to blockade by antagonists, regulation by protein kinase, current density, and activation and inactivation characteristics (12)(13)(14). However, these studies have reported generally on the impact of al...

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 ␣ 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 ␣ 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 ␣ 1 -subunit. The generation of proteomic variations by alternative splicing of the calcium channel Ca v 1.2 ␣ 1 -subunit can potentially provide a flexible mechanism for muscle or neuronal cells to respond to various physiological signals or to diseases.

Scorpion ␣-toxins Lqh␣IT, Lqh-2, and Lqh-3 are representatives of three groups of ␣-toxins that differ in their preference for insects and mammals. These ␣-insect, antimammalian, and ␣-like toxins bind to voltage-gated sodium channels and slow down channel inactivation. Sodium channel mutagenesis studies using various ␣-toxins have shown that they interact with receptor site 3, which is composed mainly of a short stretch of amino-acid residues between S3 and S4 of domain 4. Variation in this region results in marked differences between various subtypes of sodium channels with respect to their sensitivity to the three Lqh toxins. We incorporated the S3-S4 linker of domain 4 from hNa V 1.2/hNa V 1.1, hNa V 1.3, hNa V 1.6, and hNa V 1.7 channels as well as individual point mutations into the rNa V 1.4 skeletal muscle sodium channel. Our data show that the affinity of Lqh-3 and Lqh␣IT to sodium channels is markedly determined by an aspartate residue (Asp1428 in rNa V 1.4); when mutated to glutamate, as is present in Na V 1.1-1.3 channels, Lqh-3-channel interactions are abolished. The interaction of Lqh-2 and Lqh␣IT, however, is strongly reduced when a lysine residue (Lys1432 in rNa V 1.4) is replaced by threonine (as in hNa V 1.7), whereas this substitution is without effect for Lqh-3. The influence of Lys1432 on Lqh-2 and Lqh␣IT strongly depends on the context of the Asp/Glu site at position 1428, giving rise to a wide variety of toxicological phenotypes by means of a combinatorial mixing and matching of only a few residues in receptor site 3.Voltage-gated sodium (Na V ) channels consist of a large (ϳ260 kDa) pore-forming ␣-subunit, composed of four homologous domains (D1-D4), each with six transmembrane segments (S1-S6) and a hairpin-like pore region between S5 and S6. Because of their structural conservation in vertebrates and invertebrates and their pivotal role in cellular excitability, Na V channels are targeted by a large variety of chemically distinct toxins, many of which do not differentiate among channel subtypes (Catterall, 1992;Gordon, 1997). However, some scorpion neurotoxins show specificity for insect or mammalian Na V channels, and others are able to differentiate between Na V subtypes in mammalian neurons . This selectivity is attributed to differences of active sites on the toxins and to variations in receptor binding sites on distinct Na V channels that, when identified, may be used for design of selective drugs.Scorpion toxins affecting Na V channels are 61-to 76-residue polypeptides that comprise two major classes, ␣-and ␤-toxins, according to their mode of action and binding properties to distinct sites (receptor sites-3 and -4, respectively) on Na V channels (Martin-Eauclaire and Couraud, 1995;Gordon et al., 1998). ␣-Toxins inhibit Na V channel inactivation in various excitable preparations, but they show vast differences in preference for insect and mammalian Na V channels. Accordingly, they are divided into classic ␣-toxins, which are highly active in mammalian brain [e.g., Lqh-2 (L...

Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel Na(V)1.4 expressed in mammalian cells was studied by applying the Met-preferring oxidant chloramine-T or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of Met1470 in the putative receptor of the inactivation lid also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues in intracellular linkers connecting the membrane-spanning segments (442, 1139, 1154, 1316, 1469) were of minor importance. The results of mutagenesis, assays of other Na(V) channel isoforms (Na(V)1.2, Na(V)1.5, Na(V)1.7), and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of Na(V) channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion.

Lu S, Das P, Fadool DA, Kaczmarek LK. The Slack sodium-activated potassium channel provides a major outward current in olfactory neurons of Kv1.3Ϫ/Ϫ super-smeller mice.