Cav3 T-type channels: regulators for gating, membrane expression, and cation selectivity.

Cav3 T-type channels are low-voltage-gated channels with rapid kinetics that are classified among the calcium-selective Cav1 and Cav2 type channels. Here, we outline the fundamental and unique regulators of T-type channels. An ubiquitous and proximally located "gating brake" works in concert with the voltage-sensor domain and S6 alpha-helical segment from domain II to set the canonical low-threshold and transient gating features of T-type channels. Gene splicing of optional exon 25c (and/or exon 26) in the short III-IV linker provides a developmental switch between modes of activity, such as activating in response to membrane depolarization, to channels requiring hyperpolarization input before being available to activate. Downstream of the gating brake in the I-II linker is a key region for regulating channel expression where alternative splicing patterns correlate with functional diversity of spike patterns, pacemaking rate (especially in the heart), stage of development, and animal size. A small but persistent window conductance depolarizes cells and boosts excitability at rest. T-type channels possess an ion selectivity that can resemble not only the calcium ion exclusive Cav1 and Cav2 channels but also the sodium ion selectivity of Nav1 sodium channels too. Alternative splicing in the extracellular turret of domain II generates highly sodium-permeable channels, which contribute to low-threshold sodium spikes. Cav3 channels are more ubiquitous among multicellular animals and more widespread in tissues than the more brain centric Nav1 sodium channels in invertebrates. Highly sodium-permeant Cav3 channels can functionally replace Nav1 channels in species where they are lacking, such as in Caenorhabditis elegans.

Modulation of jellyfish potassium channels by external potassium ions.

The amplitude of an A-like potassium current (I(Kfast)) in identified cultured motor neurons isolated from the jellyfish Polyorchis penicillatus was found to be strongly modulated by extracellular potassium ([K(+)](out)). When expressed in Xenopus oocytes, two jellyfish Shaker-like genes, jShak1 and jShak2, coding for potassium channels, exhibited similar modulation by [K(+)](out) over a range of concentrations from 0 to 100 mM. jShak2-encoded channels also showed a decreased rate of inactivation and an increased rate of recovery from inactivation at high [K(+)](out). Using site-directed mutagenesis we show that inactivation of jShak2 can be ascribed to an unusual combination of a weak "implicit" N-type inactivation mechanism and a strong, fast, potassium-sensitive C-type mechanism. Interaction between the two forms of inactivation is responsible for the potassium dependence of cumulative inactivation. Inactivation of jShak1 was determined primarily by a strong "ball and chain" mechanism similar to fruit fly Shaker channels. Experiments using fast perfusion of outside-out patches with jShak2 channels were used to establish that the effects of [K(+)](out) on the peak current amplitude and inactivation were due to processes occurring at either different sites located at the external channel mouth with different retention times for potassium ions, or at the same site(s) where retention time is determined by state-dependent conformations of the channel protein. The possible physiological implications of potassium sensitivity of high-threshold potassium A-like currents is discussed.

Residues in a jellyfish shaker-like channel involved in modulation by external potassium.

The jellyfish gene, jShak2, coded for a potassium channel that showed increased conductance and a decreased inactivation rate as [K(+)](out) was increased. The relative modulatory effectiveness of K(+), Rb(+), Cs(+), and Na(+) indicated that a weak-field-strength site is present. Cysteine substituted mutants (L369C and F370C) of an N-terminal truncated construct, (jShak2Delta2-38) which only showed C-type inactivation, were used to establish the position and nature of this site(s). In comparison with jShak2Delta2-38 and F370C, L369C showed a greater relative increase in peak current when [K(+)](out) was increased from 1 to 100 mM because the affinity of this site was reduced at low [K(+)](out). Increasing [K(+)](out) had little effect on the rate of inactivation of L369C; however, the appearance of a second, hyperbolic component to the inactivation curve for F370C indicated that this mutation had increased the affinity of the low-affinity site by bringing the backbone oxygens closer together. Methanethiosulphonate reagents were used to form positively (MTSET), negatively (MTSES), and neutrally (MTSM) charged side groups on the cysteine-substituted residues at the purported K(+) binding site(s) in the channel mouth and conductance and inactivation kinetic measurements made. The reduced affinity of the site produced by the mutation L369C was probably due to the increased hydrophobicity of cysteine, which changed the relative positions of carbonyl oxygens since MTSES modification did not form a high-field-strength site as might be expected if the cysteine residues project into the pore. Addition of the side chain -CH(2)-S-S-CH(3), which is similar to the side chain of methionine, a conserved residue in many potassium channels, resulted in an increased peak current and reduced inactivation rate, hence a higher affinity binding site. Modification of cysteine substituted mutants occurred more readily from the inactivated state confirming that side chains probably rotate into the pore from a buried position when no K ions are in the pore. In conclusion we were able to show that, as for certain potassium channels in higher taxonomic groups, the site(s) responsible for modulation by [K(+)](out) is situated just outside the selectivity filter and is represented by the residues L(369) and F(370) in the jellyfish Shaker channel, jShak2.

The effects of level of expression of a jellyfish Shaker potassium channel: a positive potassium feedback mechanism.

1. When jellyfish Shaker potassium channels (jShak2) are heterologously expressed in Xenopus oocytes at different levels they demonstrate density-dependent changes in electrical and kinetic properties of macroscopic currents. 2. The activation and inactivation properties of jShak2 channels depend on the extracellular potassium concentration. In this study we present experimental data which show that expression-dependent changes in kinetic and electrical properties of jShak2 macroscopic currents can be explained by the positive feedback effect of dynamic accumulation of K+ in the perimembranal space.

Genomic organization of a voltage-gated Na+ channel in a hydrozoan jellyfish: insights into the evolution of voltage-gated Na+ channel genes.

Voltage-gated Na+ channels are responsible for fast propagating action potentials. The structurally simplest animals known to contain rapid, transient, voltage-gated currents carried exclusively by Na+ ions are the Cnidaria. The Cnidaria are thought to be close to the origin of the metazoan radiation and thus are pivotal organisms for studying the evolution of the Na+ channel gene. Here we describe the genomic organization of the Na+ channel alpha subunit, PpSCN1, from the hydrozoan jellyfish, Polyorchis penicillatus. We show that most of the 20 intron sites in this diploblast are conserved in mammalian Na+ channel genes, with some even shared by Ca2+ channels. One of these conserved introns is spliced by a rare U 12-type spliceosome. Such conservation places the origin of the primary exon arrangement of Na+ channels and different intron splicing mechanisms to at least the common ancestors of diploblasts and triploblasts, approximately 600 million-1 billion years ago.

A putative voltage-gated sodium channel alpha subunit (PpSCN1) from the hydrozoan jellyfish, Polyorchis penicillatus: structural comparisons and evolutionary considerations.

Extant cnidarians are probably the simplest metazoans with discrete nervous systems and rapid, transient voltage-gated currents carried exclusively by Na+ ions. Thus cnidarians are pivotal organisms for studying the evolution of voltage-gated Na+ channels. We have isolated a full-length Na+ channel alpha subunit cDNA (PpSCN1) from the hydrozoan jellyfish, Polyorchis penicillatus, that has one of the smallest known coding regions of a four domain Na+ channel (1695 amino acids). Homologous residues that have a critical bearing on the selectivity filter, voltage-sensor and binding sites for tetrodotoxin and lidocaine in vertebrates and most invertebrates differ in cnidarians. PpSCN1 is not alternatively-spliced and may be the only pore-forming alpha subunit available to account for at least three electrophysiologically distinct Na+ currents that have been studied in P. penicillatus.

Voltage sensing in jellyfish Shaker K+ channels.

The S4 segment of the jellyfish (Polyorchis penicillatus) Shaker channel jShak1 contains only six positively charged motifs. All other Shaker channels, including the jellyfish Shaker channel jShak2, have seven charges in this segment. Despite their charge differences, both these jellyfish channels produce currents with activation and inactivation curves shifted by approximately +40 mV relative to other Shaker currents. Adding charge without changing segment length by mutating the N-terminal side of jShak1 S4 does not have a pronounced effect on channel activation properties. Adding the positively charged motif RIF on the N-terminal side of K294 (the homologue of K374 in Drosophila Shaker, which is a structurally critical residue) produced a large positive shift in both activation and inactivation without altering the slope of the activation curve of the channel. When IFR was added to the other side of K294, there was a small negative shift in activation and fast inactivation of the channel was prevented. Our results demonstrate that K294 divides the S4 segment into functionally different regions and that the voltage threshold for activation and inactivation of the channel is not determined by the total charge on S4.

A cardiac-like sodium current in motor neurons of a jellyfish.

1. Whole cell voltage-clamp recordings from isolated swimming motor neurons (SMNs) reveal a rapidly activating and inactivating sodium current. 2. Permeability ratios of PLi/PNa = 0.941 and P(guanidinium)/PNa = 0.124 were measured for the mediating channel, which was impermeable to rubidium. 3. The conductance/voltage and steady state inactivation curves are shifted in a depolarizing direction by approximately 45 mV relative to most neuronal sodium currents in higher animals. 4. Activation could be fitted with two exponents and maximal current peaked at 0.74 +/- 0.06 ms (mean +/- SD). 5. Inactivation could be fitted with fast (Tau 1 = 1.91 +/- 0.07 ms at +10 mV) and slow (Tau 2 = 11.65 +/- 0.55 ms at +10 mV) exponents. 6. Half-recovery from inactivation occurred slowly (52.6 +/- 2.9 ms). 7. A second class of identifiable neurons, "B" neurons, possesses a distinctly different population of sodium channels. they showed different inactivation kinetics and far more rapid recovery from inactivation (half-recovery < 5 ms). 8. We conclude that there was physiological diversification of sodium channels early in metazoan evolution and that there has been considerable cell-specific selection of channel properties.