Arrhythmogenic calmodulin mutations impede activation of small-conductance calcium-activated potassium current

Abstract

Background: Apamin-sensitive small-conductance calcium-activated potassium (SK) channels are gated by intracellular Ca(2+) through a constitutive interaction with calmodulin.

Objective: We hypothesize that arrhythmogenic human calmodulin mutations impede activation of SK channels.

Methods: We studied 5 previously published calmodulin mutations (N54I, N98S, D96V, D130G, and F90L). Plasmids encoding either wild-type or mutant calmodulin were transiently transfected into human embryonic kidney 293 cells that stably express subtype 2 of SK protein channels (SK2 cells). Whole-cell voltage-clamp recording was used to determine apamin-sensitive current densities. We also performed optical mapping studies in normal murine hearts to determine the effects of apamin in hearts with (n=7) or without (n=3) pretreatment with sea anemone toxin.

Results: SK2 cells transfected with wild-type calmodulin exhibited an apamin-sensitive current density of 33.6 pA/pF (31.4-36.5 pA/pF) (median and confidence interval 25th-75th percentile), which was significantly higher than that observed for cells transfected with N54I (17.0 pA/pF [14.0-27.7 pA/pF]; P = .016), F90L (22.6 pA/pF [20.3-24.3 pA/pF]; P = .011), D96V (13.0 pA/pF [10.9-15.8 pA/pF]; P = .003), N98S (13.7 pA/pF [8.8-20.4 pA/pF]; P = .005), and D130G (17.6 pA/pF [13.8-24.6 pA/pF]; P = .003). The decrease in SK2 current densities was not associated with a decrease in membrane protein expression or intracellular distribution of the channel protein. Apamin increased the ventricular action potential duration at 80% repolarization (from 79.6 ms [63.4-93.3 ms] to 121.8 ms [97.9-127.2 ms]; P = .010) in hearts pretreated with anemone toxin but not in control hearts.

Conclusion: Human arrhythmogenic calmodulin mutations impede the activation of SK2 channels in human embryonic kidney 293 cells.

Keywords: Arrhythmias; Catecholaminergic polymorphic ventricular tachycardia; Ion channels; Long QT syndrome; Patch clamp.

Figure 1

HEK 293 cells that stably express SK2 current. (A) Representative tracings of SK2 currents obtained with ramp protocol shown in the inset at time points indicated by arrow a and b in (B). (B) The time course of SK2 current measured at 0 mV. (C) Immunofluorescence staining for SK2 protein (red) in SK2 cells (left panel) and HEK 293 cells without SK2 (right panel). All cells express SK2 proteins. Blue shows DAPI stain that identifies the nuclei. Scale bar, 25 µm.

Figure 2

SK2 current densities in HEK 293 cells expressing calmodulin alleles. As compared with SK2 cells, transfection of WT calmodulin significantly increased the current in SK2 cells, while transfection with green fluorescent protein (GFP) or mutant calmodulins failed to increase the current. Numbers in parentheses indicate the number of cells studied. * p<0.05.

Figure 3

SK2 current densities as a function of WT to D96V ratio. Numbers in parentheses indicate the number of cells studied. SK2 current density was dependent on the amount of WT calmodulin transfected. * p<0.05.

Figure 4

Steady-state calcium dependency of SK2 current transfected with WT- and D96V calmodulins in SK2 cells. (A) SK2 current densities with different intracellular calcium concentrations. (B) SK2 current densities were normalized to the maximal SK2 current with a free calcium of 10 µM and plotted as a function of calcium concentration. The data were fitted with Hill equation: y=1/(1+[K_d/x]ⁿ), where y indicates the normalized SK2 current and x is the intrapipette free calcium concentration; K_d is the concentration of intrapipette free calcium at half-maximal activation of SK2 current; n is the Hill coefficient. Error bars represent standard error. Numbers in parentheses indicate the number of cells patched. * p<0.05.

Figure 5

Immunofluorescence of SK2 protein. Cells with and without green fluorescence represent SK2 cells with and without transfected calmodulin, respectively. Red fluorescence shows the staining of SK2 protein. Note that not all cells with SK2 protein express transfected calmodulin. Asterisks were used to label cells without calmodulin transfection. In cells expressing calmodulin, the protein was evenly distributed throughout the cell. Scale bar, 10 µm.

Figure 6

SK2 protein expression in SK2 cells transfected with different types of calmodulin plasmids. (A) A representative immunoblot showing the expression of SK2 proteins in SK2 cells transfected with either WT or mutant calmodulin plasmids (Top). (B) A plot showing normalized SK2 protein expression in different types of cells (based on at least 3 experiments).

Figure 7

Apamin prolongs APD₈₀ in hearts pretreated with ATXII but not in untreated control (ctrl) hearts. (A) Typical examples of optical action potential at baseline (a), after ATX-II (b) and after apamin (c). (B) A summary of APD changes in 7 ATX-II pretreated and 3 control hearts. Bars indicates ranges from 25% percentile to 75% percentile. * p<0.05 comparing the APD between control and ATXII groups. †p<0.005 comparing the APD between baseline and after ATXII. ‡p<0.005 comparing the APDs between after ATXII and apamin.