Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in Ca3 T-type channels alter the passage and block of monovalent and divalent ions.

Invertebrate LCa3 shares the quintessential features of vertebrate Ca3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca channels, the Ca1 and Ca2 channels. Unlike the vertebrates though, Ca3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCa3 channel into a higher Na and lower Ca current passing channel, more resembling a classical Na1 Na channel. Cnidarian Ca3 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 ion current passing through Ca3 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 Ca3.2 channels, while corresponding mono- and di-cysteine loop pairs in human Ca3.2 generates greater Ca current passing, invertebrate LCa3 channels. Alanine substitutions of unique D2L5 loop cysteines of LCa3 channels increases relative monovalent ion current sizes and increases the potency of Zn and Ni block by ~ 50× and ~ 10× in loop cysteine mutated channels respectively, acquiring characteristics of the high affinity block of Ca3.2 channels, including the loss of the slowing of inactivation kinetics during Zn block. Charge neutralization of a ubiquitous aspartate residue of calcium passing Ca1, Ca2 and Ca3 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 Ca3 channels may confer a relatively higher peak current size for Na ions over Ca The extracellular loops of Ca3 channels are not engaged with accessory subunit binding, as the other Na (Na1) and Ca (Ca1/Ca2) 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 or Na ions through invertebrate Ca3 channels.