Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in CaV3 T-type channels alter the passage and block of monovalent and divalent ions

Abstract

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. 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

A lysine residue from an extracellular turret switches the ion preference in a Cav3 T-Type channel

“…Traditional Ca 2+ -selective channels generate a “ U-shaped ” response curve in response to increasing doses of calcium ions in the presence of external Na + (Senatore et al, 2014, Guan et al, 2020). The U-shaped response to increasing Ca 2+ is considered a reflection that Na + ions are unable to compete for the limited cation binding sites as Ca 2+ effectively repels the funneling of Na + through the Ca 2+ -selective pore in the presence of low 10 μM external Ca 2+ concentrations, and where Ca 2+ influx dominates Na + influx at physiological (mM) levels of external Ca 2+ through calcium-selective channels (Senatore et al, 2014, Guan et al, 2020).…”

“…We also observe an additional, intra-turret cysteine bridge within P2-S6 extracellular loop of Domain IV within all protostome invertebrates, balancing the reinforcement of extra cysteine bridges within their S5-P1 extracellular turrets in across from the Ca V 3 T-type channel pore in Domain II. We have illustrated that the extra cysteine bridge in D4P2-S6 extracellular turret of Domain IV is necessary for the D2S5-P1 extracellular turret contained in exon 12a from mollusks to impart the characteristically high Na + preference over Ca 2+ onto human Ca V 3.2 channel pores (Guan et al, 2020). Increases in cysteine bridge number over the basic complement of other Ca V 1 and Ca V 2 channels within extracellular turrets, first appears in the basal group of extant animals containing “ true ” nervous systems, the cnidarians, where there are two different Ca V 3 T-type channel genes in anthozoans and scyphozoan cnidarians, one resembling the vertebrate condition with a Cav3 T-type gene coding for one cysteine bridge in D4P2-S6 extracellular loop and another Cav3 T-type gene resembling the protostome invertebrate condition with a gene coding for two cysteine bridges in D4P2-S6 extracellular loop (Guan et al, 2020).…”

“…Traditional Ca 2+ -selective channels generate a "U-shaped" response curve in response to increasing doses of calcium ions in the presence of external Na + (Senatore et al, 2014, Guan et al, 2020. The U-shaped response to increasing Ca 2+ is considered a reflection that Na + ions are unable to compete for the limited cation binding sites as Ca 2+ effectively repels the funneling of Na + through the Ca 2+ -selective pore in the presence of low 10 µM external Ca 2+ concentrations, and where Ca 2+ influx dominates Na + influx at physiological (mM) levels of external Ca 2+ through calcium-selective channels (Senatore et al, 2014, Guan et al, 2020. The property of a high Na + selectivity over Ca 2+ in the molluscan LCa V 3 channel pore containing exon 12a is reflected in the transformation of the "U-shaped" response curve in response to increasing external Ca 2+ into a "reverse S" response curve, where low 10 µM external Ca 2+ concentration is ineffective in the blocking of the Na + influx, and rises in external Ca 2+ to external physiological (mM) concentrations does not lead to greater Ca 2+ passage through the Na + selective T-type channel pore, but rather there is an observed increasing block of the T-type channel current because Ca 2+ is not capable of permeating through the sodium-selective, LCav3-12a T-type channel pore at physiological (mM) concentrations of Ca 2+ (Senatore et al, 2014, Guan et al, 2020.…”

“…Exon 12b, which doesn’t express in the snail heart, but predominates in skeletal muscle tissue, confers the typical phenotype of mostly Ca 2+ passing currents onto the LCav3 T-type channel (Senatore et al, 2014). We demonstrate that the human calcium-selective Cav3.2 channel can be converted into T-type calcium channel with a preference for passage of Na + over Ca 2+ , in chimeras which includes the swapping of exon 12a from the snail LCa V 3 T-type channel (Guan et al, 2020). Exons 12a and 12b code for the L5 extracellular turret in Domain II (IIS5-P1) of the four domain Cav3 T-type channel, terminating just upstream of the hallmark pore selectivity filter residues known for conferring the characteristic ion selectivity of Ca V and Na V channels (Senatore et al, 2014, Guan et al, 2020).…”

“…We demonstrate that the human calcium-selective Cav3.2 channel can be converted into T-type calcium channel with a preference for passage of Na + over Ca 2+ , in chimeras which includes the swapping of exon 12a from the snail LCa V 3 T-type channel (Guan et al, 2020). Exons 12a and 12b code for the L5 extracellular turret in Domain II (IIS5-P1) of the four domain Cav3 T-type channel, terminating just upstream of the hallmark pore selectivity filter residues known for conferring the characteristic ion selectivity of Ca V and Na V channels (Senatore et al, 2014, Guan et al, 2020). What has eluded discovery is an atomic model to explain how a typically fast, Ca 2+ -selective T-type channel converts into a kinetically slow ion channel with a preference for passage of Na + over Ca 2+ without alterations to the hallmark pore selectivity filter or amino acids in the voltage sensor domain.…”