Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in Ca(V)3 T-type channels alter the passage and block of monovalent and divalent ions

Invertebrate LCa(V)3 shares the quintessential features of vertebrate Ca(V)3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca(2+) channels, the Ca(V)1 and Ca(V)2 channels. Unlike the vertebrates though, Ca(V)3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCa(V)3 channel into a higher Na(+) and lower Ca(2+) current passing channel, more resembling a classical Na(V)1 Na(+) channel. Cnidarian Ca(V)3 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 Ca(2+) ion current passing through Ca(V)3 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 Ca(V)3.2 channels, while corresponding mono- and di-cysteine loop pairs in human Ca(V)3.2 generates greater Ca(2+) current passing, invertebrate LCa(V)3 channels. Alanine substitutions of unique D2L5 loop cysteines of LCa(V)3 channels increases relative monovalent ion current sizes and increases the potency of Zn(2+) and Ni(2+) block by ~ 50× and ~ 10× in loop cysteine mutated channels respectively, acquiring characteristics of the high affinity block of Ca(V)3.2 channels, including the loss of the slowing of inactivation kinetics during Zn(2+) block. Charge neutralization of a ubiquitous aspartate residue of calcium passing Ca(V)1, Ca(V)2 and Ca(V)3 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 Ca(V)3 channels may confer a relatively higher peak current size for Na(+) ions over Ca(2+) The extracellular loops of Ca(V)3 channels are not engaged with accessory subunit binding, as the other Na(+) (Na(V)1) and Ca(2+) (Ca(V)1/Ca(V)2) 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 Ca(2+) or Na(+) ions through invertebrate Ca(V)3 channels.