Background: Ion selectivity of voltage-gated channels is governed by selectivity filters. Results: Alternative turret region in domain II promotes highly sodium-permeable T-type channels without major changes to gating and kinetic features. Conclusion: T-type channels can generate variable sodium or calcium permeability by gene splicing. Significance: Ion selectivity in T-type channels can be altered using extracellular domains outside the ion selectivity filter.

We report here that unlike what was suggested for many vertebrate neurons, synaptic transmission in Lymnaea stagnalis occurs independent of a physical interaction between presynaptic calcium channels and a functional complement of SNARE proteins. Instead, synaptic transmission in Lymnaea requires the expression of a C-terminal splice variant of the Lymnaea homolog to mammalian N- and P/Q-type calcium channels. We show that the alternately spliced region physically interacts with the scaffolding proteins Mint1 and CASK, and that synaptic transmission is abolished following RNA interference knockdown of CASK or after the injection of peptide sequences designed to disrupt the calcium channel-Mint1 interactions. Our data suggest that Mint1 and CASK may serve to localize the non-L-type channels at the active zone and that synaptic transmission in invertebrate neurons utilizes a mechanism for optimizing calcium entry, which occurs independently of a physical association between calcium channels and SNARE proteins.

NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single gene compared to the calcium and sodium channels which diversified into twenty genes in humans. The single NALCN gene has alternatively-spliced exons at exons 15 or exon 31 that splices in novel selectivity filter residues that resemble calcium channels (EEEE) or sodium channels (EKEE or EEKE). NALCN channels with alternative calcium, (EEEE) and sodium, (EKEE or EEKE) -selective pores are conserved in simple bilaterally symmetrical animals like flatworms to non-chordate deuterostomes. The single NALCN gene is limited as a sodium channel with a lysine (K)-containing pore in vertebrates, but originally NALCN was a calcium-like channel, and evolved to operate as both a calcium channel and sodium channel for different roles in many invertebrates. Expression patterns of NALCN-EKEE in pond snail, Lymnaea stagnalis suggest roles for NALCN in secretion, with an abundant expression in brain, and an up-regulation in secretory organs of sexually-mature adults such as albumen gland and prostate. NALCN-EEEE is equally abundant as NALCN-EKEE in snails, but is greater expressed in heart and other muscle tissue, and 50% less expressed in the brain than NALCN-EKEE. Transfected snail NALCN-EEEE and NALCN-EKEE channel isoforms express in HEK-293T cells. We were not able to distinguish potential NALCN currents from background, non-selective leak conductances in HEK293T cells. Native leak currents without expressing NALCN genes in HEK-293T cells are NMDG+ impermeant and blockable with 10 µM Gd3+ ions and are indistinguishable from the hallmark currents ascribed to mammalian NALCN currents expressed in vitro by Lu et al. in Cell. 2007 Apr 20;129(2):371-83.

The Conus magus peptide toxin -conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both -conotoxin GVIA and MVIIA (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728 -15735), the introduction of five point mutations known to affect GVIA blocking (and located downstream of the EFhand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concentrations (ϳ3 M), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.A number of predatory species such as spiders, scorpions, and fish hunting mollusks have developed venoms to rapidly stun and kill their prey. Their venoms typically contain a mixture of potent peptide toxins that have evolved to specifically bind to voltage-and ligand-gated ion channels (1). Among calcium channel blocking peptides, -conotoxin GVIA (isolated from Conus geographus) and -conotoxin MVIIA (isolated from Conus magus) are perhaps the most widely known. Both of these toxins are thought to specifically block the pore of N-type calcium channels from a variety of species. Block by both toxins is considered to be virtually irreversible at normal membrane potentials (i.e. Refs. 2 and 3), but complete reversibility can be obtained upon strong hyperpolarization (4), or by removal of extracellular divalent carrier ions (5). Our current understanding of the molecular basis of N-type channel block came initially from a study by Ellinor et al. (6) who showed that block of transiently expressed N-type channels by -conotoxin GVIA was dramatically attenuated by sequence substitution in the domain III S5-S6 region of the Ca v 2.2 ␣ 1 subunit. More recently, Feng et al. (7) showed that additional point mutations in an EF-hand consensus motif in this region affected the development of, and recovery from, block by both GVIA and MVIIA, consistent...

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Here we describe features of the first non-mammalian T-type calcium channel (LCa v 3) expressed in vitro. This molluscan channel possesses combined biophysical properties that are reminiscent of all mammalian T-type channels. It exhibits T-type features such as "transient" kinetics, but the "tiny" label, usually associated with Ba 2؉ conductance, is hard to reconcile with the "bigness" of this channel in many respects. LCa v 3 is 25% larger than any voltage-gated ion channel expressed to date. It codes for a massive, 322-kDa protein that conducts large macroscopic currents in vitro. LCa v 3 is also the most abundant Ca 2؉ channel transcript in the snail nervous system. A window current at typical resting potentials appears to be at least as large as that reported for mammalian channels. This distant gene provides a unique perspective to analyze the structural, functional, drug binding, and evolutionary aspects of T-type channels.T-type calcium channels open in response to slight depolarizations in the low voltage range. Paradoxically, they are also recruited after membrane hyperpolarization as occurs during rebound burst firing (1). A window current of T-type channels is a feature that permits Ca 2ϩ entry at rest (2) and contributes to differentiation and growth promoting functions in both excitable and non-excitable cells (3). T-type channels are also a leading pharmaceutical drug target and are implicated in a wide range of conditions such as epilepsy, pain, hypertension, cancer, and mental disorders (4).T-type Ca 2ϩ currents were first measured in starfish eggs using a two-electrode voltage clamp (5). Currents conducted by "Channel I" were evoked by small depolarizations (low voltageactivated), visible as a small hump in a current amplitude versus test potential plot, appearing inconsequential beside the Channel II currents elicited by larger depolarizations (high voltageactivated). Ca 2ϩ channel types would be discriminated further by Tsien and co-workers (6) on the basis of properties where Ba 2ϩ is the charge carrier. High voltage-activated L-type channels have a large unitary Ba 2ϩ conductance with long-lasting openings, N-type (or non-L-type) channels are typically associated with neurons of intermediate unitary conductance, and the low voltage-activated, T-type channels produce transient currents that are of tiny unitary conductance in Ba 2ϩ and close slowly upon membrane repolarization, producing a slowly deactivating tail current (6).T-type channels remain as the least understood among the Ca 2ϩ channel families. Although most of the 10 mammalian Ca 2ϩ channel genes were characterized in the late 1980s, an additional decade was required for a description of the three T-type genes, Ca v 3.1 (␣1G), Ca v 3.2 (␣1H), and Ca v 3.3 (␣1I) (7). Progress in understanding T-type channel functions continues to be hampered by the lack of highly selective blockers that discriminate between Ca v 3 channel types or separate Ca v 3 channels from related L-type (Ca v 1) and non-L-type (Ca v 2) Ca 2ϩ channels, which usu...

NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.

Here we report the first assessment of the expression and modulation of an invertebrate ␣ 1 subunit homolog of mammalian presynaptic Ca v 2 calcium channels (Ntype and P/Q-type) in mammalian cells. Our data show that molluscan channel (LCa v 2a) isolated from Lymnaea stagnalis is effectively membrane-targeted and electrophysiologically recordable in tsA-201 cells only when the first 44 amino acids of LCa v 2a are substituted for the corresponding region of rat Ca v 2.1. When coexpressed with rat accessory subunits, the biophysical properties of LCa v 2a-5rbA resemble those of mammalian N-type calcium channels with respect to activation and inactivation, lack of pronounced calcium dependent inactivation, preferential permeation of barium ions, and cadmium block. Consistent with reports of native Lymnaea calcium currents, the LCa v 2a-5rbA channel is insensitive to micromolar concentrations of -conotoxin GVIA and is not affected by nifedipine, thus confirming that it is not of the L-type. Interestingly, the LCa v 2a-5rbA channel is almost completely and irreversibly inhibited by guanosine 5-3-O-(thio)triphosphate but not regulated by syntaxin1, suggesting that invertebrate presynaptic calcium channels are differently modulated from their vertebrate counterparts.