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studies are providing insights into how these stimuli are transduced into signals that regulate VGIC gene transcription (Mori et al., 1993; Dolmetsch et al., 2001; Tao et al., 2002; Chen et al., 2003). Less is known about the promoter regions that control VGIC transcription.

A series of interesting studies using cleavage-arrested blastomeres of the tunicate Halocynthia roretzi indicated that neural induction leads to the appearance of voltage-gated sodium current in the cell that adopts a neural fate (Takahashi and Yoshii,

1981; Takahashi and Okamura, 1998; for review, see Okamura et al., 1993; Figure 7). More recent studies have characterized the effects at a molecular level and demonstrated that neural induction leads to the expression of a specific voltage-gated sodium channel isotype, TuNa1 (Okamura et al., 1997). Transcription of TuNa1 is activated during neural induction by interactions between specific blasto-meres of the animal and vegetal poles during a critical period of time (Okado and Takahashi, 1988, 1990a, 1990b; Okamura et al., 1994).

Inappropriate cell contacts lead to differentiation of alternate cell types and the expression of different ion channels (Okado and Takahashi, 1990b). Similarly, a specific potassium channel transcript begins expression at the time of neural induction in embryonic spinal neurons of the frog Xenopus laevis (Ribera, 1990).

While it is clear that cell-cell interactions during development can activate transcription of specific ion channel genes, the underlying mechanisms are poorly understood. Moreover, depending upon its identity, a neuron will express a specific repertoire of ion channels. It is possible to accelerate the expression of one type of channel by premature expression of another (Linsdell and Moody, 1994). Future work needs to address how transcription of the subset of ion channel genes expressed in any individual neuron is coordinated. For example, co-regulation of transient potassium and hyper-polarization-activated inward currents has been observed in lobster stomatogastric ganglion neurons (Maclean et al., 2003).

In comparison to the vast number of ion channel genes that have been identified by molecular cloning, little is known about their DNA regulatory elements or the transcription factors that regulate transcription. The best-studied transcription factor for an ion channel gene is REST, or repressor element silencing transcription factor (also known as neuron-restrictive silencing factor or NRSF, Kraner et al., 1992; Mori et al., 1992; Schoenherr and Anderson, 1995a, 1995b). REST specifically controls expression of the voltage-gated sodium channel a-subunit Nav1.2 (Chong et al., 1995; Schade and Brown, 2000; Dallman et al., 2004). REST binds to RE-1, a DNA element found in the regulatory region of many neuronal vertebrate genes (Kraner et al., 1992; Schoenherr and Anderson, 1995b). Most non-neuro-nal tissues express REST. Overexpression of REST recombinant protein in neuronal cells prevents Nav1.2 expression (Huang et al., 1999; Nadeau and Lester, 2002). Conversely, expression of a dominant negative REST in non-neuronal cells results in Nav1.2 transcription (Chong et al., 1995). Thus, REST functions in a negative pathway and suppresses expression of Nav1.2 in non-neuronal cells.

Future work also needs to address what role tran-scriptional mechanisms play in the developmental upregulation of ion channels. For example, Xenopus spinal neurons display a threefold increase in potassium current density that underlies the developmental shortening of the AP duration (O'Dowd et al., 1988; Lockery and Spitzer, 1992). A critical period of RNA synthesis is required for

Young Mature

Young Mature

for 2 days 1 day recovery

Figure 8 AP development requires a critical period of transcription. Blockade of transcription with an inhibitor of the RNA synthesis (DRB, 5,6-dichlorobenzimidazole 1-3-D-ribofurano-side) during a critical period of development prevents maturation of the AP and a voltage-gated potassium current. Reproduced from Ribera, A. B. and Spitzer, N. C. 1989. A critical period of transcription required for differentiation of the action potential of spinal neurons. Neuron 2, 1055-1062, with permission from Elsevier.

for 2 days 1 day recovery

Figure 8 AP development requires a critical period of transcription. Blockade of transcription with an inhibitor of the RNA synthesis (DRB, 5,6-dichlorobenzimidazole 1-3-D-ribofurano-side) during a critical period of development prevents maturation of the AP and a voltage-gated potassium current. Reproduced from Ribera, A. B. and Spitzer, N. C. 1989. A critical period of transcription required for differentiation of the action potential of spinal neurons. Neuron 2, 1055-1062, with permission from Elsevier.

maturation of IKv (Ribera and Spitzer, 1989; Figure 8). Transient inhibition of RNA synthesis during a 9 h period prevents the normal threefold increase in density of IKv, even when a 48 h recovery period is allowed. Conversely, increasing the levels of potassium channel RNAs (e.g., Kv1.1 or Kv2.2) leads to premature maturation of IKv (Jones and Ribera, 1994; Blaine et al., 2004). These findings are consistent with the notion that the critical RNA synthesized during the 9 h period codes for a potassium channel subunit.

1.13.8.2 Post-Transcriptional

Post-transcriptional mechanisms, such as alternative splicing, editing, mRNA stability, and localization, influence ion channel function. Neural activity can activate post-transcriptional effects, as is the case for stabilization of transcripts coding for specific calcium channel isoforms (Schorge et al., 1999). Moreover, recent studies indicate that post-transcriptional control of ion channel function occurs throughout development of the nervous system.

1.13.8.2.1 Alternative splicing Alternative splicing influences translational efficiency as well as protein stability, transport, and localization (Black, 2003; Stamm et al., 2005). Alternative exon usage constitutes a major mechanism to increase functional diversity of VGICs. Almost all aspects of VGIC function can be affected by alternative splicing: channel activation and inactivation, gating, kinetic properties, and sensitivity to blockers and modulators (Iverson et al., 1997; Chemin et al., 2001; Decher et al., 2001; Tian et al., 2001a, 2001b). The effects range from subtle ones to complete loss of function.

Alternative exon usage can change dynamically in response to diverse stimuli, including growth factors, pH, and neural activity. Of particular interest, development influences alternative exon usage (Kaufer et al., 1998; Oh and Waxman, 1998; Stamm et al., 2005). For example, multiple sites for alternative splicing usage have been identified in voltage-gated potassium and sodium channels. In Drosophila, the major class of sodium channel is encoded by a single gene known as para. Alternative splicing of para generates several different channel isoforms. The isoforms display temporally and spatially distinct expression patterns (Thackeray and Ganetzky, 1994; Lee et al., 2002). Alternative splicing also regulates sodium channels in the cockroach and results in mRNA variants coding for proteins with different activation, inactivation, and gating characteristics (Song et al., 2004). Further, several of the isoforms display tissue-specific distribution and developmental specificity. Similar results have been obtained by the study of Drosophila genes coding for voltage-gated (i.e., Shaker) or calcium-dependent (i.e., Slo) potassium channels (Atkinson et al., 1991; Adelman et al., 1992; Butler et al., 1993).

Ion channel gene families that are represented by a single gene in invertebrates often have undergone duplications during evolution and exist as multigene families in vertebrates (i.e., Shaker gene vs. Kv1 gene family; para gene vs. Nav1 gene family). Despite the increased functional diversity created by gene duplication, alternative splicing still operates to create molecularly diverse channel transcripts. For example, the mammalian sodium channel a-subunit genes, Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.9, are alternatively spliced (Sarao et al., 1991; Gustafson et al., 1993; Plummer et al., 1997; Lu and Brown, 1998; Jeong et al., 2000). Moreover, expression of specific splice variants is developmentally regulated. In Xenopus, alternative spicing of the slo (xSlo) gene generates variants that differ in their tissue and developmental expression patterns (Kukuljan et al., 2003; Figure 9). Additionally, the variants code for channels that differ with respect to both voltage and calcium sensitivities.

Nav1.1, Nav1.2, and Nav1.6 genes display devel-opmentally regulated patterns of alternative splicing (Sarao et al., 1991; Plummer et al., 1997; Alessandri-Haber et al., 2002). Interestingly, the coding regions of neonatal variants of Nav1.2 and Nav1.6 genes xSlo15 LLLTLPCLTALPVFAV

xSlo56 PKMSIRKRLIQACCIGCSEIDCSCMSGTLRNNMGTLEQAF PISPVTVNDFSTSLRGF

xSlo59 LIYSKMSIRKRLIQACCIGCSEIDCSCMSGTLRNNMGTLE QAFPISPVTVNDFSTSLRGF

xSlo99 PAKEEHRLSIHRLSIHSQAAKASYSVTSSKLCTEQQEPVP LVNNRKGSLFLPCDSSLLHLQLLSSSGTGHHTSIKLQRAL SLPGKYRYHPNQPILIQKQF

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