Expanded Functional Diversity of Shaker K⁺ Channels in Cnidarians Is Driven by Gene Expansion

<div><p>The genome of the cnidarian <i>Nematostella vectensis</i> (starlet sea anemone) provides a molecular genetic view into the first nervous systems, which appeared in a late common ancestor of cnidarians and bilaterians. <i>Nematostella</i> has a surprisingly large and diverse set of neuronal signaling genes including paralogs of most neuronal signaling molecules found in higher metazoans. Several ion channel gene families are highly expanded in the sea anemone, including three subfamilies of the Shaker K⁺ channel gene family: Shaker (Kv1), Shaw (Kv3) and Shal (Kv4). In order to better understand the physiological significance of these voltage-gated K⁺ channel expansions, we analyzed the function of 18 members of the 20 gene Shaker subfamily in <i>Nematostella</i>. Six of the Nematostella Shaker genes express functional homotetrameric K⁺ channels <i>in vitro</i>. These include functional orthologs of bilaterian Shakers and channels with an unusually high threshold for voltage activation. We identified 11 Nematostella Shaker genes with a distinct “silent” or “regulatory” phenotype; these encode subunits that function only in heteromeric channels and serve to further diversify Nematostella Shaker channel gating properties. Subunits with the regulatory phenotype have not previously been found in the Shaker subfamily, but have evolved independently in the Shab (Kv2) family in vertebrates and the Shal family in a cnidarian. Phylogenetic analysis indicates that regulatory subunits were present in ancestral cnidarians, but have continued to diversity at a high rate after the split between anthozoans and hydrozoans. Comparison of Shaker family gene complements from diverse metazoan species reveals frequent, large scale duplication has produced highly unique sets of Shaker channels in the major metazoan lineages.</p></div

Comparison of inactivation in NvShak channels.

<p><b><i>(a)</i></b> Comparison of inactivation time course in NvShak1, NvShak4 and NvShak5 currents recorded from excised patches in response to a depolarization to +60 mV. Currents were recorded in 2 mM external K⁺ and were normalized by peak amplitude to facilitate comparison of inactivation. Scale bar indicates time. <b><i>(b)</i></b> Steady state inactivation curves are given for NvShak1, NvShak4, NvShak5 and NvShak6. Data points show mean peak current ± S.E.M. (n = 4–6) elicited during a +60 mV test pulse following a 5 s pre-pulse to the indicated voltage. Data from individual patches were normalized in amplitude prior to comparison. Curves represent single Boltzmann distribution fits; V₅₀ and slope values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051366#pone-0051366-t001" target="_blank">Table 1</a>. Currents elicited at +60 mV for wild type (WT, black) and N-terminal truncated (Trunc, red) versions of NvShak1, NvShak5 and NvShak4 are compared in <b><i>c-e</i></b>, respectively. Currents were normalized in amplitude for display. Truncations eliminated amino acid residues 2–30, 2–48 and 2–58 for NvShak1, NvShak4 and NvShak5, respectively. Truncation eliminated fast inactivation in NvShak1 and NvShak5, but only reduced the rate of inactivation in NvShak4. 140 mM K+ had little effect on the inactivation time course of WT NvShak4 (gray) but eliminated residual inactivation in the truncated form of NvShak4 (dark red), suggesting an unmasked C-type inactivation process in the truncated channel.</p

Expression patterns of Nematostella Shaker genes determined by in situ hybridization.

<p>Expression in early stage polyps is shown for NvShak1-6 in <b><i>a,</i></b> and 7 NvShakR genes in <b><i>b</i></b>. Sagittal views are shown for all probes; coronal views are also shown for NvShak1, NvShak2, NvShak3, NvShak6, NvShakR2 and NvShakR12. Numbered arrows point to select regions of expression. (<b>1</b>) Homotetramer-competent inactivating subunits, NvShak1, NvShak4 and NvShak5, are expressed in developing tentacle bulbs, along with regulatory subunits NvShakR7, NvShakR9 and NShakR13. The same three regulatory subunits show light expression along with NvShak2 and NvShak4 in the vicinity of the developing oral nerve ring (<b>2</b>). Expression around the pharynx and pharyngeal nerve ring occurs for NvShak1, NvShak3, NvShak4, NvShak6 and the regulatory subunits NvShakR11 and NvShakR12 (<b>3</b>). All homotetramer-competent subunits (except NvShak5) and the regulatory subunits NvShakR2, NvShakR4, NvShakR11 and NvShakR12 show expression in subsets of cells in the mesenteries (<b>4</b>). NvShak6 expression is concentrated in body cells near the apical pole in early stage polyps (<b>5</b>), but transitions to mesenteric and pharyngeal expression as polyp development continues. The specific identity of Shaker-positive cells and cell-level expression overlap of Shaker transcripts remains to be determined.</p

Summary of Novel Current Properties in NvShak3 + NvShakRx Heteromers.

<p>Summary of Novel Current Properties in NvShak3 + NvShakRx Heteromers.</p

NvShak1-6 express a functionally diverse set of Shaker currents in <i>Xenopus</i> oocytes.

<p>Families of outward K⁺ currents recorded from excised inside-out patches taken from oocytes expressing NvShak1-6 are shown in a–f. Currents were recorded in response to 100 ms depolarizations ranging from −60 to +60 mV in 10 mV increments from a holding potential of −100 mV. Scale bars are given for time and current amplitude and arrows mark the current elicited at 0 mV. Note this is the first step with significant current for NvShak2 and NvShak3. K⁺ concentration for these recordings was 2 mM in the pipette (extracellular) and 140 mM in the bath (intracellular).</p

Voltage dependent activation of NvShak currents. <i>(a)</i>

<p>Voltage activation curves for NvShak1-6 and the mouse Shaker channel Kv1.2. Currents were activated with depolarizing pulses of 100 ms to the indicated voltages from a holding potential of −100 mV, and conductance measurements are taken from isochronal tail currents recorded upon repolarization to −100 mV. Experiments were conducted in symmetrical 140 mM K⁺. Data points are normalized and show mean ± S.E.M. (n = 4−8); curves show single Boltzmann distribution fits; V₅₀ and slope are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051366#pone-0051366-t001" target="_blank">Table 1</a>. <b><i>(b)</i></b> Comparison of current activation time course recorded in response to a voltage step to +60 mV for Kv1.2 and NvShak1-5. Example currents were normalized in amplitude for comparison and truncated versions of NvShak1, NvShak4 and NvShak5 were used to eliminate fast inactivation. The horizontal line marks 0.5 amplitude (half activation), and dotted vertical drop lines are used to mark half-activation times for each current. <b><i>(c)</i></b> Depiction of strategy to quantify activation time course in two parameters: sigmoidal delay and activation time constant. An example current is shown with a red line, current baseline is indicated with a dashed line and the voltage step is marked below. The activation time constant was determined from single exponential fit of the last 50% of the activation time course (black curve) and the delay was defined as the time between the start of the voltage pulse and the point at which the exponential fit intercepted current baseline (blue line). Delay of current occurs because of rate-limiting closed-closed transitions in the activation pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051366#pone.0051366-Zagotta1" target="_blank">[52]</a>. Time constants and delay values of activation for steps to the indicated voltages are shown for NvShak1-5 and mouse Kv1.2 in <b><i>d</i></b> and <b><i>e</i></b>, respectively. Values show mean ± S.E.M. (n = 4−8); all experiments were carried out on excised patches.</p

Voltage-dependent properties of NvShak1-6 Currents.

<p>Voltage-dependent properties of NvShak1-6 Currents.</p

Shaker Family Genes in Metazoan Species.

<p>Shaker Family Genes in Metazoan Species.</p