Andrew N. Spencer

Background: Voltage-gated ion channels are membrane proteins containing a selective pore that allows permeable ions to transit the membrane in response to a change in the transmembrane voltage. The typical selectivity filter in potassium channels is formed by a tetrameric arrangement of the carbonyl groups of the conserved amino-acid sequence Gly-Tyr-Gly. This canonical pore is opened or closed by conformational changes that originate in the voltage sensor (S4), a transmembrane helix with a series of positively charged amino acids. This sensor moves through a gating pore formed by elements of the S1, S2 and S3 helices, across the plane of the membrane, without allowing ions to pass through the membrane at that site. Recently, synthetic mutagenesis studies in the Drosophila melanogaster Shaker channel and analysis of human disease-causing mutations in sodium channels have identified amino acid residues that are integral parts of the gating-pore; when these residues are mutated the proteins allow a non-specific cation current, known as the omega current, to pass through the gating-pore with relatively low selectivity. Results: The N.at-K _v 3.2 potassium channel has an unusual weak inward rectifier phenotype. Several mutations of two amino acids in the voltage sensing (S4) transmembrane helix change the phenotype to a typical delayed rectifier. The inward rectifier channels (wild-type and mutant) are sensitive to 4-aminopyridine (4-AP) but not tetra-ethyl ammonium (TEA), whereas the delayed rectifier mutants are sensitive to TEA but not 4-AP. The inward rectifier channels also manifest low cation selectivity. The relative selectivity for different cations is sensitive to specific mutations in the S4 helix, Conclusion: N.at-K _v 3.2, a naturally occurring potassium channel of the Kv3 sequence family, mediates ion permeation through a modified gating pore, not the canonical, highly selective pore typical of potassium channels. This channel has evolved to yield qualitatively different ion permeability when compared to all other members of this gene family.

Voltage-gated ion channels of the Kv4 subfamily produce A-type currents whose properties are tuned by accessory subunits termed KChIPs, which are a family of Ca²⁺ sensor proteins. By modifying expression levels and the intrinsic biophysical properties of Kv4 channels, KChIPs modulate the excitability properties of neurons and myocytes. We studied how a Kv4 channel from a tunicate, the first branching clade of the chordates, is modulated by endogenous KChIP subunits. BLAST searches in the genome of Ciona intestinalis identified a single Kv4 gene and a single KChIP gene, implying that the diversification of both genes occurred during early vertebrate evolution, since the corresponding mammalian gene families are formed by several paralogues. In this study we describe the cloning and characterization of a tunicate Kv4 channel, CionaKv4, and a tunicate KChIP subunit, CionaKChIP. We demonstrate that CionaKChIP strongly modulates CionaKv4 by producing larger currents that inactivate more slowly than in the absence of the KChIP subunit. Furthermore, CionaKChIP shifted the midpoints of activation and inactivation and slowed deactivation and recovery from inactivation of CionaKv4. Modulation by CionaKChIP requires the presence of the intact N terminus of CionaKv4 because, except for a minor effect on inactivation, CionaKChIP did not modulate CionaKv4 channels that lacked amino acids 2-32. In summary, our results suggest that modulation of Kv4 channels by KChIP subunits is an ancient mechanism for modulating electrical excitability.