Abstract
Invertebrate LCaV3 shares the quintessential features of vertebrate CaV3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca2+ channels, the CaV1 and CaV2 channels. Unlike the vertebrates though, CaV3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCaV3 channel into a higher Na+ and lower Ca2+ current passing channel, more resembling a classical NaV1 Na+ channel. Cnidarian CaV3 T-type channels can possess genes with alternative cysteine-rich, D4L6 extracellular loops in a manner reminiscent of the alternative cysteine-rich, D2L5 extracellular loops of non-cnidarian invertebrates. We illustrate here that the preferences for greater Na+ or Ca2+ ion current passing through CaV3 T-type channels are contributed by paired cysteines within D2L5 and D4L6 extracellular loops looming above the pore selectivity filter. Swapping of invertebrate tri- and tetra-cysteine containing extracellular loops, generates higher Na+ current passing channels in human CaV3.2 channels, while corresponding mono- and di-cysteine loop pairs in human CaV3.2 generates greater Ca2+ current passing, invertebrate LCaV3 channels. Alanine substitutions of unique D2L5 loop cysteines of LCaV3 channels increases relative monovalent ion current sizes and increases the potency of Zn2+ and Ni2+ block by ~ 50× and ~ 10× in loop cysteine mutated channels respectively, acquiring characteristics of the high affinity block of CaV3.2 channels, including the loss of the slowing of inactivation kinetics during Zn2+ block. Charge neutralization of a ubiquitous aspartate residue of calcium passing CaV1, CaV2 and CaV3 channels, in the outer pore of the selectivity filter residues in Domain II generates higher Na+ current passing channels in a manner that may resemble how the unique D2L5 extracellular loops of invertebrate CaV3 channels may confer a relatively higher peak current size for Na+ ions over Ca2+ The extracellular loops of CaV3 channels are not engaged with accessory subunit binding, as the other Na+ (NaV1) and Ca2+ (CaV1/CaV2) channels, enabling diversity and expansion of cysteine-bonded extracellular loops, which appears to serve, amongst other possibilities, to alter to the preferences for passage of Ca2+ or Na+ ions through invertebrate CaV3 channels.
Highlights
Abbreviations HSF site High field strength site D2L5 Extracellular loop spanning the end of transmembrane segment 5 to the start of the pore selectivity filter (S5-P) in Domain II D4L6 Extracellular loop spanning the end of the pore selectivity filter to the start of transmembrane segment 6 (P-S6) in Domain IV
Snail channels are notably different in possessing a 5–10 mV hyperpolarized operating range of voltage-sensitivity and possess faster activation and inactivation kinetics compared to the human T-type channels (Fig. 1, Supplementary Table S1)
Native splicing of a novel peptide fragment spanning the extracellular loop before the pore selectivity filter in Domain II of the four domain channel (Fig. 2a) generates large sized L CaV3 channel currents with exon 12a that is ~ 15 fold higher peak current size when [Na+]ex replaces weakly permeant monovalent ion [NMDG+] ex in the presence of [Ca2+]ex, compared to the same channel with exon 12b where there is approximately equal Na+ and C a2+ contributions to the total peak current size (Fig. 2b,c)
Summary
Abbreviations HSF site High field strength site D2L5 Extracellular loop spanning the end of transmembrane segment 5 to the start of the pore selectivity filter (S5-P) in Domain II D4L6 Extracellular loop spanning the end of the pore selectivity filter to the start of transmembrane segment 6 (P-S6) in Domain IV. LCaV3–12a and LCaV3–12b The alternative “a” and “b” splice isoforms spanning exon 12, which includes the the distal ends of Domain II, segment 5, the extracellular D2L5 loop, and the proximal, P1 helix before the selectivity filter in Domain II of Lymnaea CaV3 T-type channel. A critical difference from vertebrate C aV3 T-type channels was encountered after in vitro expression of an unusual, alternative splice isoform spanning exon 12, which generated high Na+ current passing T-type c hannels[9]. This splice isoform expresses as the only isoform in the invertebrate heart[9]. What started as an examination of curious T-type N a+ currents within invertebrates has led to the discovery of the influences of cysteine-enriched extracellular loops contributing to a unique structure above the channel pore in the regulation of passage of altered N a+ or C a2+ permeation and blockade through C aV3 T-type channels
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