Functional and pharmacological characterization of a Shal-related K+ channel subunit in Zebrafish
© Nakamura and Coetzee; licensee BioMed Central Ltd. 2008
Received: 03 September 2007
Accepted: 08 February 2008
Published: 08 February 2008
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.
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.
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.
Danio rerio is one of the few model organisms used to examine developmental regulation of vertebrate biological processes. Much has been leaned regarding transcriptional control of organogenesis, including development and growth of the heart  and brain . Although the zebrafish is often used to study vertebrate development, random mutagenesis screens have identified several important genes involved in physiology and behavior. The importance of ion channels is exemplified by mutants such as the slow mo mutant, which disrupts the heart rate due to changes in the pacemaker current (Ih) . Another mutation in zebrafish, the lethal island beat (isl), disrupts the α1C L-type Ca2+ channel subunit, which regulate heart growth independently of effects on contractions . The role of ion transporters and Ca2+ homeostasis is further emphasized by the arrhythmias observed in tre embryos, which have a mutation of the Na+-Ca2+ exchanger . The zebrafish may be an appropriate model for studying human inherited arrhythmias, for example in a recent study defining mutations of a delayed rectifier K+ current in contributing to long QT syndrome . Although the description and function of ion channels in zebrafish systems biology is beginning to emerge [7–11], little is known about the expression and functional characteristics of most zebrafish ion channels.
In Drosophila, Shaker was the first type of K+ channel to be described . This was soon followed by homology cloning of Shab, Shaw and Shal . Many more voltage-activated K+ channels have since been described in mammals , but the corresponding preferential mammalian nomenclature for these first four are Kv1, Kv2, Kv3 and Kv4 , with several members in each of these subfamilies. The Kv4 subfamily contains three members; Kv4.1, Kv4.2 and Kv4.3 . The Kv4.x K+ channels are expressed in a variety of tissue, with particularly high levels in the brain and heart. The Kv4 subunits form functional channels, with very similar biophysical and pharmacological properties, that are responsible for transient, voltage-dependent K+ currents in the nervous system (A currents) in the neuronal system. These somatodendritic subthreshold A-type K+ current in nerve cells contribute significantly to determine somatodendritic signal integration. The corresponding K+ current in the heart is called the transient outward K+ current (Ito), which contributes to action potential repolarization .
Shal-type K+ channel subunits are highly conserved evolutionary and are present even in primitive metazoans such as the jellyfish . We show using sequence and phylogenetic analysis that orthologs of all three mammalian Kv4 subfamily members exist in Danio rerio. We collectively named these subunits zShals, in keeping with the nomenclature where "Kv" is reserved for mammalian subunits . We also describe the biophysical and pharmacological properties of zShal3 (the ortholog of the mammalian Kv4.3), as well as its regulation by the Kv4 accessory subunit, NCS-1. Characterization of channel function and distribution is an important first step in understanding that the physiological role of ion channels in this model animal system.
Electrophysiological measurements in oocytes
Stage V-VI oocytes were prepared from Xenopus laevis and 50 nl of in vitro transcribed cRNA (0.1–18 ng) were injected using a 10 μl micropipette (Drummond Scientific Co, Broomall, PA). Two to three days after the injection, oocytes were voltage-clamped using the standard two-electrode voltage-clamp technique as described previously . Recordings were obtained using a Geneclamp 500 amplifier (Axon Instruments, Inc.) with data sampled at 5 kHz and filtered at 1 kHz. Currents were elicited by depolarizing steps from -100 to +60 mV in 10 mV increments every 15 s from a holding potential of -120 mV (to allow full recovery from inactivation). The recording chamber was continually perfused (1 ml/min). To avoid contamination with Ca2+-activated Cl- currents, a low Cl- recording solution containing in (mM) Na-glutamate 96, K-glutamate 2, CaCl2 1.8, MgCl2 1, HEPES 5 (pH 7.5 adjusted with NaOH) was used. All experiments were performed at room temperature (20 ± 2°C).
Data were analyzed using the pClamp suite of software (Axon Instruments) and Origin for Windows (Microcal Software). Leak subtraction was not performed.
the time constants of inactivation were obtained by fitting the current traces to a sum of two exponential functions.
Recovery from inactivation
A double pulse protocol was used to access the time course of recovery from inactivation. Two depolarizing pulses to +20 mV were separated by intervals of increasing duration (from 20 to 460 ms in 40 ms increments) at -120 mV. The peak current amplitude during the test pulse (I) was normalized to that recorded during the first pulse (Io) and plotted as a function of the inter-pulse duration. Curve fitting of the data points was performed to , where Imax is the maximum current during the recovery time (not restrained to 1). A and τ are the relative amplitude and time constant respectively.
A pre-pulse voltage protocol was used. The membrane was held at voltages ranging between -150 mV and -10 mV for a period of 10 seconds (to obtain the complete steady-state inactivation) followed by a 1 s test pulse to +20 mV, during which peak current amplitude (I) was measured. Data were normalized to the peak current amplitude following a pre-pulse at -150 mV (I0), and plotted as a function of pre-pulse potential. The line through the data represents the best nonlinear least-squares fit to a Boltzmann function, , where Vh is the voltage where half-maximal inactivation was observed and k is the slope factor.
Normalized conductance-voltage curve
Conductance (G) was calculated with the following equation: G = Ipeak/(E-EK), where Ipeak is the peak current amplitude, E is the test potential and EK is the equilibrium potential for K+ (calculated to be -99 mV under our experimental conditions). The conductance (G) was normalized to the maximal conductance at +60 mV (Gmax) and plotted as a function of the test potential.
Comparisons between two groups were performed using the Student's t-test or the Rank Sum test. To test for overall significance when using more than two groups, we used the 1 way-ANOVA or, when tests for normality failed, the non-parametric Kruskall-Wallis test (Sigma Stat, Jandel Scientific). If the overall significance was obtained, multiple comparisons were performed to a control group respectively using the Dunnet's t-test or Dunn's test. Values of p < 0.05 were considered statistically significant.
Identification of zShal3
zShal3 corresponds to the short splice variant of mammalian Kv4.3. The long splice variant contains the sequence GLSYLVDDPLLSVRTSTIK within the intracellular C-terminus. We did not identify a genomic region on this zebrafish chromosome that translates to a similar sequence and it is unclear at this point whether zebrafish also generates two zShal3 splice variants.
Biophysical characterization of zShal3
Pharmacological blockade of zShal3 currents
Modification of zShal3 by accessory subunits
Comparison with mammalian Kv4 subunits
We identified a zebrafish homolog of the mammalian Kv4.3 subunit, which we named zShal3. It has a high degree of sequence similarity (65–77% identity) with members of the Kv4 subfamily. Within the transmembrane domains (S1 to S6) and pore region the sequence similarity is much higher (>90%), suggesting evolutionary conservation of these regions responsible for channel function. The three mammalian Kv4 subunits differ from each other in defined regions of the intracellular N- and C-termini. It is interesting that most of the sequence divergence between zShal3 and Kv4's also occur in these regions, suggestive of subunit-specific roles of these divergent regions.
Biophysical and pharmacological properties of zShal3
zShal3 behaved in most respects identically to mammalian Kv4.3 channels when expressed in Xenopus oocytes. The "threshold" voltage for activation is around -70 mV, which is similar to Kv4.3 when expressed in Xenopus oocytes . This is not entirely surprising given the sequence similarity on the core region (S1 to S6) of the polypeptides. Once activated, zShal3 inactivates at rates similar to Kv4.3. The molecular mechanisms involved in Kv4 inactivation are not well understood, but classic N-type ("ball and chain") or C-type (pore-collapse) inactivation may not occur. There may be a concerted interaction between the intracellular N- and C-termini in causing Kv4.1 inactivation  and it is likely that a similar mechanism may operate to cause zShal3 inactivation. The similarities in voltage-dependence of inactivation and rates of recovery from inactivation further support the notion of evolutionary conserved inactivation mechanisms. The zShal subunit may be useful in further elucidating Kv4 inactivation, which is thought to occur predominantly from a pre-open closed state . Pharmacologically, both zShal3 and Kv4.3 are blocked by 4-AP and flecainide. There is evidence that the 4-AP binding site is on the cytoplasmic surface of the Kv4.2 channel at, or adjacent to, the domains involved in channel inactivation [21, 29]. Flecainide sensitivity, in contrast, is mediated by a single residue in the S6 transmembrane domain of voltage-gated potassium channels . This residue is L392 in the case of Kv4.2, which is conserved in zShal3 (and also in the other two zShals; see later). Because of this sequence conservation, it is likely that these compounds may also block zShal3 currents by interaction with these homologous residues. Furthermore, as observed for Kv4.3 current , zShal3 current was also largely modulated by PKC activation, suggesting that a similar functional role for signal transduction-mediated modulation of A-type current may exist in zebrafish.
Modulation of zShal3 by accessory subunits
KChIPs and NCS-1 (frequenin) strongly modulate Kv4 currents by increasing their expression levels and slowing their inactivation [24–26]. Since NCS-1 may be the evolutionary ancestor of the Ca2+-binding protein family, we examined the effect of NCS-1 and found this Kv4 accessory protein to affect zShal3 in an identical manner. KChIPs (and likely also NCS-1) bind to Kv4 subunits by interacting with specific residues. A major interaction site for KChIPs binding within Kv4.2 lies within the N-terminus – particularly with the proximal N-terminal region [24, 32, 33]. The absolute conservation of residues and domains involved in interaction, despite divergence in other parts of the N-terminus, suggests an evolutionary conserved mechanism of modulation of Shal (Kv4) subunits by accessory subunits, which predates the vertebrate evolutionary branching point of mammals and fish. Indeed, inspection of zebrafish genome suggests orthologs of at least some of the KChIPs (zKChIPs, Genbank entries AAH96914, XP_687972 and XP_694748), as well as a NCS-1 ortholog (AAO34710). It remains to be seen whether these zKChIPs or zNCS-1 will have any influence on zShal currents.
Other Shal-related family members in zebrafish
There are three members in the mammalian Kv4 subfamily (Kv4.1, Kv4.2 and Kv4.3). Similarity searches of the zebrafish genome revealed homologs for each of these mammalian Kv4 subunits. The putative subunits zShal1, zShal2 and zShal3 are respectively located on zebrafish chromosomes 19 (Ensembl ID: ENSDART00000009506), 4 (ENSDART00000042673) and 24 (Zv5_scaffold1202.6) and correspond to Kv4.1, Kv4.2 and Kv4.3. The sequence similarity between zShals and Kv4's are greater than observed with other mammalian Kv subunits (Fig 1). Given this high degree of sequence similarity, as well as the similarities in the functional and pharmacological properties between zShal3 and mammalian Kv4's (this study), it is expected that these additional zShal members will have biophysical and pharmacological properties resembling their mammalian orthologs. However, this prediction (as well as their relative tissue-distributions) still needs to be examined.
Possible functional significance of zShal expression in Zebrafish
Without detailed electrophysiological recordings of native currents in Zebrafish or in the absence of knockout experiments, it is not possible to determine the functional significance of zShal expression in the fish at this point. Given the similarities in function and regulation of zShal to their mammalian orthologs (Kv4 subunits), one would expect these channels to have similar functions in the fish as they do in the mammal. Known functions of Kv4 subunits in mammals include a role in somatodendritic subthreshold A-type K+ currents, which operate in the subthreshold range of membrane potentials  and may be involved in determining the properties of dendritic, backpropagating action potentials and long-term potentiation . Preliminary whole-mount in-situ hybridization experiments suggest that zShal3 may be expressed in neural tissues of Zebrafish embryos (data not shown). Depending on the protein distribution and dentritic localization of zShals, they may have similar functions. If any of the zShals (see above) are expressed in the Zebrafish heart, then by analogy with the mammalian heart, one would expect these subunits to give rise to a 'transient outward K+ current', which helps to determine action potential shape and cardiac contractile properties . The regulation of zShals by accessory proteins such as NCS-1 (or possibly by KChIP orthologs) also suggests a similarity in how these channels are regulated in-vivo.
We identified a member of the Shal subfamily of K+ channel subunits in Zebrafish and named it zShal3, since it appears to be the ortholog of the mammalian Kv4.3 subunit. We report the biophysical and pharmacological characterization of zShal3. 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.
These studies were supported in part by the National Institutes of Health (R01- HL064838) and in part by the Seventh Masonic District Association, Inc. Further support was provided by a Grant-in-Aid for Scientific Research, 19590220 and Priority Areas 13142210 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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