Functional and pharmacological characterization of a Shal-related K+ channel subunit in Zebrafish

Abstract

Background: K+ channels are diverse; both in terms of their function and their molecular composition. Shal subunits were first described in Drosophila. There are three mammalian orthologs, which are members of the Kv4 subfamily. They are involved in neuronal firing patterns as well as control of the cardiac action potential duration.

Results: Here, we report the biophysical and pharmacological characterization of zShal3, which is the ortholog of the mammalian Kv4.3 subunit, which in mammals is involved in action potential repolarization and gives rise to neuronal A-type K+ currents involved in somatodendretic signal integration.

Conclusion: We demonstrate that zShal has similar functional and pharmacological characteristics compared to Kv4.3 and it is similarly regulated by pharmacological agents and by the Kv4 accessory subunit, NCS-1.

Figure 1

Comparison of zShals and mammalian Kv4s. A: Alignment of Danio rerio zShal3 amino acid sequence (NM_199802.1) with those of human Kv4.1 (NP_004970), human Kv4.2 (NP_036413) and the short splice variant of human Kv4.3 (AAF01045). Putative transmembrane domains and the pore region are indicated by horizontal bars. Potential PKC phosphorylation sites are indicated by open symbols, a potential PKA phosphorylation site is indicted by a solid symbol and a putative tyrosine phosphorylation site by a diamond. The T1 (or K⁺ channel tetramerisation domain) is a N-terminal, cytoplasmic tetramerisation domain of voltage-gated K⁺ channels encodes molecular determinants for subfamily-specific assembly of alpha-subunits into functional tetrameric channels. This domain is present between amino acids 42 to 131 of zShal3 (IILNVS ... PEIISDC). zShal3 also contains the K⁺ channel signature sequence, which is a highly conserved stretch of amino acids (G- [YF]-G.-D) in the P-region. B: Phylogenetic tree comparing zShals with representative mammalian Kv channel subunits. The mammalian (all human) sequences used were Kv4.1 (NP_004970), Kv4.2 (NP_036413) and Kv4.3 (AAF01045): Multiple sequence alignment, tree bootstrapping and tree generation was performed using ClustalW.

Figure 2

Electrophysiological properties of zShal3 expressed in Xenopus oocytes. A and B: Representative traces of zShal3 (A) and Kv4.3 currents (B). Currents were elicited by 1000 ms voltage steps from a holding potential of -120 mV to test potentials between -100 and +50 mV in 10 mV increments at every 15 s.C: Voltage dependence of activation and inactivation of zShal3 currents. The conductance-voltage curves were constructed by dividing the current by the driving force and normalizing to the maximal conductance (G_max) at +120 mV for activation and at -150 mV for inactivation curve.D: Recovery from inactivation of zShal3 currents at -120 mV.

Figure 3

Pharmacological characterization of z-Shal3 currents. A and B: Effect of 4-aminopyridine (4-AP, 10 mM)(A) and flecanide (300 μM) on zShal3 (left) and Kv4.3 currents (right). C and D: Effects of PKC activation on zShal3 currents. zShal3 currents before and 30 min after exposure to 10 nM PMA (C). Currents were elicited by 900 ms voltage steps from a holding potential of -120 mV to the test potential at +50 mV. Time course of the effect of PMA (10 nM) on zShal currents (D). The peak current amplitude was normalized to the current amplitude before PMA application (time = 0). Results are expressed as mean ± SEM of 8 experiments in each group.

Figure 4

Effects of NCS-1 on zShal3 currents. A and B: Representative traces of zShal3 currents expressed with or without NCS-1.C: Averaged current-voltage (I-V) relationship of peak zShal3 current with (●) or without (○) NCS-1. The inset depicts the conductance-voltage curves that were constructed by dividing the current by the driving force and normalizing to the maximal conductance (G_max) at +100 mV.D: zShal3 and zShal3/NCS-1 currents, normalized to the maximal peak amplitudes at +50 mV, to illustrate the effects of NCS-1 on the inactivation time course.E: Voltage dependence of steady-state inactivation for zShal3 (○) and zShal3/NCS-1 currents (●)(n = 8 for each experiment).F: Time course of the recovery from inactivation for zShal3 (○) and zShal3/NCS-1 currents (●)(n = 8 for each experiment).