Inverse Modulation of Neuronal Kv12.1 and Kv11.1 Channels by 4-Aminopyridine and NS1643

Abstract

The three members of the ether-à-go-go-gene-like (Elk; Kv12.1-Kv12.3) family of voltage-gated K+ channels are predominantly expressed in neurons, but only little information is available on their physiological relevance. It was shown that Kv12.2 channels modulate excitability of hippocampal neurons, but no native current could be attributed to Kv12.1 and Kv12.3 subunits yet. This may appear somewhat surprising, given high expression of their mRNA transcripts in several brain areas. Native Kv12 currents may have been overlooked so far due to limited knowledge on their biophysical properties and lack of specific pharmacology. Except for Kv12.2, appropriate genetically modified mouse models have not been described; therefore, identification of Kv12-mediated currents in native cell types must rely on characterization of unique properties of the channels. We focused on recombinant human Kv12.1 to identify distinct properties of these channels. We found that Kv12.1 channels exhibited significant mode shift of activation, i.e., stabilization of the voltage sensor domain in a “relaxed” open state after prolonged channel activation. This mode shift manifested by a slowing of deactivation and, most prominently, a significant shift of voltage dependence to hyperpolarized potentials. In contrast to related Kv11.1, mode shift was not sensitive to extracellular Na+, which allowed for discrimination between these isoforms. Sensitivity of Kv12.1 and Kv11.1 to the broad-spectrum K+ antagonist 4-aminopyridine was similar. However, 4-AP strongly activated Kv12.1 channels, but it was an inhibitor of Kv11 channels. Interestingly, the agonist of Kv11 channels NS1643 also differentially modulated the activity of these channels, i.e., NS1643 activated Kv11.1, but strongly inhibited Kv12.1 channels. Thus, these closely related channels are distinguished by inverse pharmacological profiles. In summary, we identified unique biophysical and pharmacological properties of Kv12.1 channels and established straightforward experimental protocols to characterize Kv12.1-mediated currents. Identification of currents in native cell types with mode shift that are activated through 4-AP and inhibited by NS1643 can provide strong evidence for contribution of Kv12.1 to whole cell currents.

Histidine at position 462 determines the low quinine sensitivity of ether‐à‐go‐go channel superfamily member Kv12.1

“…By applying hyperpolarised (−60 mV) and depolarised (+40 mV) conditioning voltages before the activating pulse potentials, we then analysed effects of quinine on the voltage dependence of K v 11.1 channels that also exhibit mode shift of activation (Figure b,c). As previously reported (Dierich et al, ; Tan, Perry, Ng, Vandenberg, & Hill, ), depolarised conditioning potentials (+40 mV) significantly shifted the voltage dependence of K v 11.1 channels by about −50 mV compared with the hyperpolarised conditioning potential (Figure d–f). Following the hyperpolarised conditioning potential (−60 mV), quinine (50 μM) significantly shifted the voltage dependence of K v 11.1 channels by −13 mV to more negative potentials (Figure d–f).…”

“…Noteworthy, K v 12.1 channels exhibit a mode shift of activation (also termed pre‐pulse facilitation or voltage‐dependent potentiation; Dai & Zagotta, ; Dierich et al, ; Dierich & Leitner, ; Li et al, ), which designates stabilisation of the voltage‐sensing domain in a “relaxed” open state after prolonged depolarisation of the membrane potential (Bezanilla, Taylor, & Fernandez, ; Villalba‐Galea, Sandtner, Starace, & Bezanilla, ). Most prominently, mode shift manifests through a large shift of activation voltages to hyperpolarised potentials following membrane potential depolarisation (Dai & Zagotta, ; Dierich et al, ; Dierich & Leitner, ; Li et al, ). To induce mode shift, we applied conditioning potentials of −60 or 0 mV (200 ms) before a series of voltage steps to activate K v 12.1 (pulse potentials: −140 to +20 mV; 600 ms; Figure b,c; c.f.…”

“…To induce mode shift, we applied conditioning potentials of −60 or 0 mV (200 ms) before a series of voltage steps to activate K v 12.1 (pulse potentials: −140 to +20 mV; 600 ms; Figure b,c; c.f. Dierich & Leitner, ; Dierich et al, ). Following the hyperpolarised conditioning potential (−60 mV), half‐maximal voltage ( V h ) and slope factor of K v 12.1 channel activation were −30.0 ± 2.0 mV and 14.9 ± 1.1 mV, respectively ( n = 6; Figure d–f).…”

“…When we applied the conditioning potential of 0 mV, V h was −75.9 ± 1.7 mV and s was 10.6 ± 0.5 mV ( n = 6; Figure d–f). Depolarised conditioning potentials thus induced a large (and significant) shift of the voltages of activation of K v 12.1 channels to hyperpolarised potentials by about −45 mV, representing their mode shift of activation (Dai & Zagotta, ; Dierich et al, ; Dierich & Leitner, ; Li et al, ). Application of quinine (1 mM) shifted the voltage dependence of K v 12.1 to depolarised voltages for both conditioning potentials.…”

“…K v 12.2 regulates excitability of hippocampal neurons (Zhang et al, ), but no physiological role has been assigned to K v 12.1 and K v 12.3 channels yet. Recent studies provided significant insight into functional characteristics of K v 12.1 (Dai & Zagotta, ; Dierich, Evers, Wilke, & Leitner, ; Dierich & Leitner, ; Kazmierczak et al, ; Li et al, ), but it is unknown whether K v 12 channels are equipped with a high affinity drug‐binding pocket as K v 10.1 and K v 11.1.…”