Transient and big are key features of an invertebrate T-type channel (LCav3) from the central nervous system of Lymnaea stagnalis

Abstract

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.

FIGURE 1.

Full-length snail LCa_v3 is the largest identified voltage-gated ion channel expressed to date. It is coded by a 9031-bp cDNA transcript that forms a 2886-amino acid protein with a molecular mass of 322 kDa. A, the N terminus closely matches with a putative start site derived from marine snail A. californica EST (EB302921) and slightly resembles the N and C termini of human Ca_v3.1–3.3. B, LCa_v3 is 1.25× larger than human Ca_v3 channels and 1.5× larger than nematode T-type, cca-1B, and all other four repeat ion channels. C, LCa_v3 is larger than human Ca_v3 channels in the N and C terminus and also the I-II and II-III cytoplasmic linkers.

FIGURE 2.

Singleton, snail T-type Ca²⁺ channel gene is distantly related to vertebrate homologs and is the most abundant Ca²⁺ channel transcript in the snail brain. A, shown is the most parsimonious gene tree generated using multiple aligned sequences, analyzed in PAUP4.0 (D. L. Swofford) and illustrated with TreeView (R. D. M. Page). Sequences include official human sequences (IUPHAR database); LCa_v3 (GenBank^TM accession no. AF484084) and yeast gene Cch1 from Schizosaccharomyces pombe (GenBank^TM accession no. CAB11726) and Saccharomyces cerevisiae (GenBank^TM accession no. CAA97244). Numbers at branch points represent bootstrap values based on 100 replicates in heuristic search. Phylogram branches are scaled by their length and rooted with Cch1 Ca²⁺ channel homologs from fungi species. C, percent amino acid similarity scores were generated from EMBOSS NEEDLE (EMBL). B, Southern blot indicates a single copy gene in the Lymnaea genome. A T-type probe hybridized to create a banding pattern (white arrows) on the blot was created from membrane transfer of genomic DNA digested with either EcoRV(a), HindIII (b), EcoRI (c), or XhoI (d). The probe contained an EcoRV restriction site, so the probe hybridized to two genomic DNA fragments digested with EcoRV. D, densitometric intensity of RT-PCR bands (illustrated in the inset) was generated from Lymnaea brain tissue.

FIGURE 3.

Running window of similarity (A) and alignments (B and C) between amino acid sequences of distant T-type channel homologs (snail LCa_v3 and human Ca_v3.3) reveal that the invariant structures for T-type channels are harbored in six membrane-spanning segments in all four domains (I, II, III, and IV), including an ion conducting pore (S5-P-loop-S6) and voltage sensor (S1-S4). Illustrated is the position in the I-II linker where LCa_v3 polyclonal antibody (Ab) was generated in rabbits against a 200-amino acid peptide. B, shown is amino acid sequence alignment of the re-entrant P-loop located between S5 and S6 of each of the four domains illustrating the signature sequence (EEDD locus) that influences Ca²⁺ ion permeation and selectivity. The conserved aspartate residue (1097 in LCa_v3) in a position downstream of the selectivity filter glutamate residue is positioned to attract incoming Ca²⁺ ions to the pore.³ LCa_v3 contains a neutral isoleucine in the outer pore at position 468 where mammalian T-type channels have a negatively charged residue (Glu or Asp) that influence pore blocking drugs. C, alignment of the cytoplasmic gating brake in proximal I-II linker is shown. The gating brake is thought to prevent T-type channel gating at more hyperpolarized potentials.

FIGURE 4.

Transient transfection of HEK-293T cells harboring the pIRES2-EGFP plasmid containing invertebrate T-type channel cDNA reveal highly abundant channels and characteristic T-type channel properties. A, membrane delimited staining of LCa_v3 (inset) is evident in EGFP-positive cells but only with LCa_v3-specific antibody and not with preimmune serum or with LCa_v1-transfected cells. B, the box chart indicates the current density (pA/pF (picofarads)) of LCa_v3 expression on 3 or 6 days after transfection. The box chart also illustrates mean, median ± 1 S.D., min/max current densities. C, sample LCa_v3 currents are shown in response to 5-ms voltage steps from a −110-mV holding potential. Illustrated is an ensemble of rapidly activating and inactivating Ca²⁺ currents where each trace “crosses over” the previous one from rest to peak, and the resulting normalized peak currents are plotted as a function of voltage step, indicating low threshold of activation (−65 mV) and maximal currents generated at a step to −35 mV (D). Current-voltage relationships were curve-fitted with an Ohmic-Boltzmann function. E, the increase in inactivation kinetics (τ_inact) closely follows the increasing speed at which the current approaches peak (t_peak), also reflected in the faster rate of activation, curve-fitted and represented by τ_act.

FIGURE 5.

Invertebrate LCa_v3 has a large, persistent window current up to 1.8% of the total current near the resting membrane potential. A, sample current traces of maximal Ca_v3 currents (step to −35 mV) in response to a 1-s inactivating prepulse. B, a Boltzmann-fitted inactivation curve was generated by plotting the fraction of maximal current as a function of prepulse voltage. The fraction of maximal conductance at each voltage was plotted as an activation curve, curve-fitted with a Boltzmann function. The activation curve was derived from the current-voltage relationship minus the ohm-changes due to the driving force (illustrated in Fig 4D). C, calculation of the window currents were based on the product of the fraction of the whole cell conductance and fraction of available, non-inactivated channels at each voltage. Inset, a window current was measured at the end of a long, 1-s voltage-step. At 1 s, the majority of open channels will have been inactivated, leaving only open channels that persist under steady-state conditions, with a maximum at the resting membrane potential (−65 mV).

FIGURE 6.

Invertebrate LCa_v3 slowly deactivates similar to mammalian T-type channels. Sample tail currents and curve fitting of decay rate of tail currents (τ, ms) were generated by hyperpolarizing steps between −110 and −60 mV for 450 ms from a 7-ms depolarizing step to −35 mV.

FIGURE 7.

LCa_v3 currents are larger and faster when Ba²⁺ is the charge carrier. Sample traces (A) and current-voltage relationships (B) of LCa_v3 currents were generated from depolarizing voltage steps from a holding potential of −110 mV while microperfusing extracellular solution containing either 5 mm Ba²⁺ or 5 mm Ca²⁺. Whole cell Ba²⁺ conductance was estimated to be ∼50% greater than Ca²⁺ conductance at all voltages (C). Kinetics of activation (time to peak current, ms) (D) and inactivation decay (tau curve fit, ms) (E) are faster when barium instead of calcium is the charge carrier.

FIGURE 8.

Invertebrate T-type channels have similar Ni²⁺ and mibefradil sensitivity as mammalian T-types. A, shown is the time course of Ni²⁺ inhibition of normalized LCa_v3 peak currents (inset, representative traces). B, cumulative dose-response is illustrated, with an IC₅₀ (300 ± 29.2 μm) value that overlaps with IC₅₀ of Ca_v3.1 (304.8 ± 6.2 μm; Kang et al. (16)). Inset, a better fit illustrated with a biphasic dose-response curve is shown. C, T-type channel alignments in the region of the S3b-S4 paddle of Domain I illustrate the His-191 required for high Ni²⁺ sensitivity of Ca_v3.2 channels. LCa_v3 has an eight-amino acid insert with additional charged residues in the relative position of the His-191 residue in Ca_v3.2. D, shown is a cumulative dose-response curve of mibefradil block of LCa_v3 (inset, representative traces), indicating an IC₅₀ (300 ± 29.2 μm) value that is reminiscent of the IC₅₀ for mammalian Ca_v3 channels.