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Channels (Austin)
2024 Dec 27;181:2420651. doi: 10.1080/19336950.2024.2420651.
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The endocannabinoid ARA-S facilitates the activation of cardiac Kv7.1/KCNE1 channels from different species.
Hiniesto-Iñigo I
,
Linhart VA
,
Kusay AS
,
Liin SI
.
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The endogenous endocannabinoid-like compound N-arachidonoyl-L-serine (ARA-S) facilitates activation of the human Kv7.1/KCNE1 channel and shortens a prolonged action potential duration and QT interval in guinea pig hearts. Hence, ARA-S is interesting to study further in cardiac models to explore the functional impact of such Kv7.1/KCNE1-mediated effects. To guide which animal models would be suitable for assessing ARA-S effects, and to aid interpretation of findings in different experimental models, it is useful to know whether Kv7.1/KCNE1 channels from relevant species respond similarly to ARA-S. To this end, we used the two-electrode voltage clamp technique to compare the effects of ARA-S on Kv7.1/KCNE1 channels from guinea pig, rabbit, and human Kv7.1/KCNE1, when expressed in Xenopus laevis oocytes. We found that the activation of Kv7.1/KCNE1 channels from all tested species was facilitated by ARA-S, seen as a concentration-dependent shift in the voltage-dependence of channel opening and increase in current amplitude and conductance over a broad voltage range. The rabbit channel displayed quantitatively similar effects as the human channel, whereas the guinea pig channel responded with more prominent increase in current amplitude and maximal conductance. This study suggests that rabbit and guinea pig models are both suitable for studying ARA-S effects mediated via Kv7.1/KCNE1.
Figure 1. Guinea pig and rabbit Kv7.1/KCNE1 channels showed overall similar biophysical behavior to the human Kv7.1/KCNE1 channel, when expressed in Xenopus laevis oocytes. A) Representative currents generated by Kv7.1/KCNE1 channels from indicated species in response to the voltage protocol shown as inset. The colored traces indicate the current sweep corresponding to a 0 mV test voltage. B) average conductance versus voltage (G(V)) curves for indicated constructs, determined by plotting the instantaneous tail current (Itail) as a function of the preceding test voltage (see methods for details). Data shown as mean ± sem. n = 18 for guinea pig and 31 for rabbit. The curves represent Boltzmann fits. Best fit for guinea pig Kv7.1/KCNE1: V50 = +33.5 mV, slope = 17.8 mV. Best fit for rabbit Kv7.1/KCNE1: V50 = +21.4 mV, slope = 19.4 mV. Data for human Kv7.1/KCNE1 is included for comparison and duplicated (from [8,18,19], with V50 = +30.0 mV, slope = 13.4 mV. C) comparison of V50 and slope for indicated constructs. Data shown as mean ± sem. n = 152 and 30 (for V50 and slope, respectively) for human Kv7.1/KCNE1 (from [8,18,19] and similar to panel B for guinea pig and rabbit channels. D) time-dependent effects on V50 and GMAX for indicated constructs (with EtOH vehicle only), during the same time-range as used to later assess ARA-S effects. Data shown as mean ± sem. n = 6 for guinea pig and 5 for rabbit. Statistics in panel C denote one-way ANOVA followed by Dunnett’s multiple comparisons test to compare to human. Statistics in panel D denote one-sample t test to compare to a hypothetical value of 0. * = 0.05, ** = 0.01, *** = 0.001, **** = <0.0001, ns = >0.05.
Figure 2. ARA-S facilitated the activation of Kv7.1/KCNE1 channels from all tested species. A) molecular structure of ARA-S. B) Representative example of traces of human Kv7.1/KCNE1 currents under control conditions and in the presence of 10 µM ARA-S and corresponding G(V) curve and normalized Itail for better visualization of the V50 shift effect. The colored traces indicate the current sweep corresponding to a 0 mV test voltage. For this specific cell: V50;ctrl = +30.2 mV, Itailmax;ctrl = 6.2 µA, V50;ARA-S = −23.0 mV, Itailmax;ARA-S = 10.1 µA. Currents were generated in steps from − 80 to +70 mV in 10 mV steps, followed by a tail voltage of −20 mV. The holding voltage was −80 mV. C) same as in B but for guinea pig Kv7.1/KCNE1. For this specific cell: V50;ctrl = +44.1 mV, Itailmax;ctrl = 4.4 µA, V50;ARA-S = −17.5 mV, Itailmax;ARA-S = 11.2 µA. D) same as in B but for rabbit Kv7.1/KCNE1. For this specific cell: V50;ctrl = +23.9 mV, Itailmax;ctrl = 3.7 µA, V50;ARA-S = −33.5 mV, Itailmax;ARA-S = 5.7 µA. E) Representative example of the time course of the wash-in and wash-out of ARA-S effects, assessed by determining the ARA-S effect on current amplitude at 0 mV. The horizontal bar indicates when ARA-S was applied. These examples are for the application and wash-out of 10 µM ARA-S on the human (left), guinea pig (middle) and rabbit (right) Kv7.1/KCNE1 channels.
Figure 3. ARA-S acted on Kv7.1/KCNE1 channels from all tested species in a concentration-dependent manner. A) concentration-response relationship for the V50 effect of ARA-S on indicated constructs. Best fit for human: EC50 = 8.9 µM, ΔV50, max = −79.6 mV; guinea pig: EC50 = 6.2 µM, ΔV50, max = −70.4 mV; rabbit: EC50 = 3.1 µM, ΔV50, max = −60.5 mV. B) same as in panel a but for the maximal conductance (GMAX). Best fit for human: EC50 = 5.7 µM, ΔGMAX, max = +83%; guinea pig: EC50 = 1.6 µM, ΔGMAX, max = +188%; rabbit = ambiguous. C) same as in a but for current amplitude (Iamp) at indicated voltages. Best fit at 0 mV for human: EC50 = 2 µM, ΔIamp, 0 mV, max = +497%; guinea pig: EC50 = 4.7 µM, ΔIamp, 0 mV, max = +1499%; rabbit: EC50 = 2.2 µM, ΔIamp, 0 mV, max = +515%. At 20 mV, human: EC50 = 1.4 µM, ΔIamp, 20 mV, max = +197%; guinea pig: EC50 = 3.0 µM, ΔIamp, 20 mV, max = +733%; rabbit: EC50 = 1.2 µM, ΔIamp, 20 mV, max = +225%. At 40 mV, human: EC50 = 0.6 µM, ΔIamp, 40 mV, max = +58%; guinea pig: EC50 = 4.2 µM, ΔIamp, 40 mV, max = +631%; rabbit = ambiguous. Statistics in A-C denote one-way ANOVA with Dunnett´s multiple comparison test to compare the fitted curves between human and species. Only statistically significant differences are indicated, all other comparisons are non-significant (ns). * = 0.05, ** = 0.01. Data shown as mean ± sem. n = 5-8 for guinea pig and 6-7 for rabbit Kv7.1/KCNE1. Data for human Kv7.1/KCNE1 is included for comparison and duplicated (from [8], n = 8-12). D) molecular docking of ARA-S into two sites (left) in Kv7.1 (PDB: 6UZZ) [16] that have been previously determined to underlie endocannabinoid effects [8]. A predicted amino acid sequence comparison of human, guinea pig and rabbit Kv7.1 is provided for these sites. The S4 site (referred to as site I) is related to the V50 effect of ARA-S, with the key arginine R228 in human Kv7.1 marked in blue. The S6 site (referred to as site II) is related to the GMAX effect of ARA-S, with the key lysine K326 in human Kv7.1 marked in orange. A close up of the two sites is provided with protein residues colored in light blue, except for R228 and K326. H-bond and salt bridge interactions to these residues are depicted as dashed black lines.
