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. 2001 Jun 15;21(12):4143-53.
doi: 10.1523/JNEUROSCI.21-12-04143.2001.

A novel extracellular calcium sensing mechanism in voltage-gated potassium ion channels

J P Johnson Jr  1 , J R Balser, P B Bennett
Affiliations

A novel extracellular calcium sensing mechanism in voltage-gated potassium ion channels

J P Johnson Jr et al. J Neurosci. .

Abstract

Potassium (K(+)) channels influence neurotransmitter release, burst firing rate activity, pacing, and critical dampening of neuronal circuits. Internal and external factors that further modify K(+) channel function permit fine-tuning of neuronal circuits. Human ether-à-go-go-related gene (HERG) K(+) channels are unusually sensitive to external calcium concentration ([Ca(2+)](o)). Small changes in [Ca(2+)](o) shift the voltage dependence of channel activation to more positive membrane potentials, an effect that cannot be explained by nonspecific surface charge screening or channel pore block. The HERG-calcium concentration-response relationship spans the physiological range for [Ca(2+)](o). The modulatory actions of calcium are attributable to differences in the Ca(2+) affinity between rested and activated channels. Adjacent extracellular, negatively charged amino acids (E518 and E519) near the S4 voltage sensor influence both channel gating and Ca(2+) dependence. Neutralization of these charges had distinct effects on channel gating and calcium sensitivity. A change in the degree of energetic coupling between these amino acids on transition from closed to activated channel states reveals movement in this region during channel gating and defines a molecular mechanism for protein state-dependent ligand interactions. The results suggest a novel extracellular [Ca(2+)](o) sensing mechanism coupled to allosteric changes in channel gating and a mechanism for fine-tuning cell repolarization.

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Figures

Fig. 1.
Fig. 1.
Direct observation of HERG channel inactivation and the effects of Ca2+ to slow the rate of channel activation. A, Activation and inactivation of HERG K+ currents measured during voltage-clamp steps to membrane potentials between +180 and +30 mV in 10 mV increments for 50 msec followed by a step to −50 mV for 100 msec. These recordings show rapid activation of HERG K+ currents followed by subsequent inactivation resulting in transient outward currents. On stepping to −50 mV, a large slow tail current is observed. The rate of activation (initial rising phase) is highly voltage dependent in this potential range, whereas the inactivation (decay) is not. The membrane potential was held at −80 mV before and after the test steps.B, Increases in the extracellular Ca2+ concentration caused a slowing of channel activation with no effect on inactivation, resulting in a truncation of the peak transient outward currents. The rate of tail current decay at −50 mV was also increased. Seven K+ currenttraces from the same cell, each in a different extracellular [Ca2+] (3 μm, 10 μm, 30 μm, 300 μm, 1 mm, 3 mm, and 10 mm), are superimposed. The membrane potential was held at −80 mV before stepping to +70 mV for 2 sec, then repolarized to −50 mV to measure tail currents. Note the two time scale barscorresponding to before and after the break in the current record. After ∼250 msec at +70 mV, the steady state current level was the same in all [Ca2+]; even the current in 10 mm Ca2+ had fully activated.
Fig. 2.
Fig. 2.
Comparison of HERG K+ currents in WT and S3–S4 charge neutralization mutants. Superimposed families of K+ current traces were recorded using the voltage-clamp protocol shown for each channel construct. Cells were held at −80 mV before stepping to test potentials between +70 and −60 mV for 2 sec. The membrane potential was then stepped to −50 mV for 2 sec to record tail currents. All records were made in the presence of 3 mm extracellular Ca2+.
Fig. 3.
Fig. 3.
S3–S4 charge neutralization did not affect inactivation. The voltage-clamp protocol is shown at thetop. A representative family of currenttraces is shown in the middle panel. The step to +60 mV was 2 sec long; the scale bar to theright applies only to the current traceto the right of the break in the record. Time constants were obtained by fitting an exponential function to the decaying current during the third voltage step. Error bars indicate the SEM for 4–8 cells for each mutant and at each membrane potential.
Fig. 4.
Fig. 4.
Effect of extracellular Ca2+ on the voltage dependence of activation of WT and mutant HERG channels. Voltage-dependent activation curves were determined from peak tail current amplitudes measured at −50 mV with a 2 sec step to the test potential (as in Fig. 2). Error bars indicate SEM (n = 3–15 for each concentration). Smooth curves correspond to the predicted values based on the three-dimensional fit of the voltage-dependent Monod–Wyman–Changeux model (see Fig. 6, Eq. 3).
Fig. 5.
Fig. 5.
Ca2+ dependence of the half maximal voltage (V1/2) of activation of WT and mutant HERG channels obtained from the curves in Figure 4. Error bars indicate SEM. Smooth curves correspond to the predicted values based on the three-dimensional fit of the voltage-dependent Monod–Wyman–Changeux model (see Fig. 6).
Fig. FS1.
Fig. FS1.
Fig. 6.
Fig. 6.
Ca2+-membrane voltage–response surface. Three-dimensional regression fits of the voltage-dependent Monod–Wyman–Changeaux model to relative K+currents for WT and mutant HERG measured in different Ca2+ concentrations and at different membrane potentials. The mean voltage– activation data for all Ca2+ concentrations are plotted as a function of membrane potential in a three-dimensional format. The vertical error bars on the symbols indicate the distance of the mean data point from the fitted plane.
Fig. 7.
Fig. 7.
Fitted parameters of the voltage-dependent MWC model for WT and mutant HERGs. L0 is the equilibrium constant for the allosteric transition between the closed and activated states in the absence of Ca2+ at 0 mV.Q is the equivalent gating charge ine−. Kc is the Ca2+ dissociation constant for the closed channel.Ka is the Ca2+ dissociation constant for the activated channel. The parameter uncertainties (SDs) are shown as error bars and were determined from the covariance matrix.
Fig. 8.
Fig. 8.
Top, Amino acid alignment of S3–S4 segments of several voltage-gated potassium channels. Standard single letter amino acid abbreviations are used. Glutamate 518 (E518) in HERG is indicated by an arrow. Kv1.1,Kv1.5, Kv2.1, Kv3.1, andKv4.3 indicate the human channels corresponding to KCNA1, KCNA5, KCNB1, KCNC1, and KCND3, respectively.eag is Drosophilaether-à-go-go, and HERG corresponds to KCNH2.Bottom, Diagram of a plausible physical explanation for the interactions between negative charges in S3–S4, positive charges in S4, and extracellular Ca2+. Ca2+ interacts at the surface of the channel at a site near the S4 voltage sensor by attraction to the negative charge of glutamate 519. The data suggest that E519 may participate in the Ca2+ binding site. The negative charge of glutamate 518 (E518) attracts or stabilizes positive charges in S4 and promotes or facilitates channel opening. Thus, E518 affects the Ca2+-independent voltage-dependence of the channel by electrostatic interaction with the voltage sensor. Adapted fromPapazian and Bezanilla (1999) for HERG channels.
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