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. 1998 Jun;111(6):781-94.
doi: 10.1085/jgp.111.6.781.

HERG-like K+ channels in microglia

W Zhou  1 , F S Cayabyab, P S Pennefather, L C Schlichter, T E DeCoursey
Affiliations

HERG-like K+ channels in microglia

W Zhou et al. J Gen Physiol. 1998 Jun.

Abstract

A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance-voltage relation was half maximal at -59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (-14 vs. -39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795-805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with Ki = approximately 40 microM, and the HERG-selective blocker, E-4031, blocked with Ki = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.

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Figures

Scheme I
Scheme I
Figure 1
Figure 1
Whole-cell currents in rat microglial cells in KCl saline (pipette and bath). (A) Currents were elicited by voltage steps from −120 to 60 mV in 20-mV increments, from Vhold = −80 mV. (B) Currents in the same cell elicited by identical voltage steps, but from Vhold = 0 mV. Voltage steps were given at 15-s intervals, from negative to positive potentials. Calibration bars apply to both parts.
Figure 2
Figure 2
Currents recorded in standard (low K+) saline (A) and in the same cell in KCl saline (B), with KCl in the pipette. Voltage steps in both were applied from 80 to −160 mV in −20-mV increments every 15 s from Vhold = 0 mV. Calibration bars apply to both parts.
Figure 3
Figure 3
(A) Average normalized peak current–voltage relationship in KMeSO3 salines (pipette and bath). Families of currents were obtained by applying voltage steps between −140 and 60 mV in 20-mV increments from Vhold = 0 mV, and then repeating the same pulses in reverse order. There was little consistent hysteresis, so the currents plotted are the average from both protocols in each of five cells. Data were normalized to the current at −140 mV and are plotted without leak correction as mean ± SEM. (B) The average chord conductance–voltage relationship in K+ saline. The curve shows the best-fitting Boltzmann relationship: formula image where g K is the peak K+ conductance, g K,max is the fitted maximal g K, V1/2 is the midpoint of the curve, and k is a slope factor. The values obtained from averaged g K data were −59 mV (corrected for junction potentials) for V1/2 and 18.6 mV for k. Leak subtraction was based on the current at large positive potentials.
Figure 5
Figure 5
Tail current and instantaneous current–voltage relationship measurements in KMeSO3 (pipette and bath). (A) Tail currents were recorded with voltage steps from −100 to 80 mV in 20-mV increments at 15-s intervals, after a 30-ms prepulse to −120 mV, from Vhold = 0 mV. (B) The average instantaneous current–voltage relation (±SEM), normalized to the initial current at −80 mV (n = 7). The currents during test pulses were fitted with a single exponential, and extrapolated to the beginning of the voltage step.
Figure 4
Figure 4
Hysteresis in the voltage dependence of quasi–steady state inactivation (inverse of availability) in KMeSO3 (pipette and bath). (A) Superimposed are currents recorded during test pulses to −120 mV, from various Vhold. Vhold was changed progressively from 40 to −100 mV in −20-mV increments and was maintained for ∼20 s at each potential before the test pulse was applied. (B) Test currents from analogous measurements in the same cell as in A in which Vhold was increased from −100 to 40 mV. (C) Average peak test current amplitudes (n = 4 cells) are plotted as a function of Vhold, normalized to the test current from Vhold = 40 mV. In a few cells in which Vhold was changed up to 80 mV, there was no further enhancement of the test current beyond that at Vhold = 40 mV. In each cell, measurements were made both by changing Vhold from −100 to 40 mV (•) and from 40 to −100 mV (□), not always in the same order. The “up” and “down” relationships (arrows) were fitted to a Boltzmann function: formula image where I max is the peak test current when Vhold = 40 mV. For measurements in which Vhold was made progressively more positive, starting at Vhold = −100 mV (B, •), the midpoint, V 1/2, was −14 mV and the slope factor, k, was 7.7 mV. When Vhold was initially 40 mV, and then progressively hyperpolarized (A, □), V 1/2 was −39 mV and k was 9.5 mV. (D) Window currents from the experiment illustrated in A and B. The current at Vhold was measured just before each test pulse, and is plotted vs. Vhold. Distinct window currents were seen consistently in other cells when the protocol in A was used, whereas the window currents measured using the protocol in B were very small.
Figure 8
Figure 8
Mean time constants of inactivation, τi (•), and recovery, τrecovery (○), measured in KMeSO3 (pipette and bath). Decaying currents during hyperpolarizing pulses from Vhold = 0 mV were fitted with a single exponential to obtain τi (n = 7), and each envelope of peak currents during paired-pulse recordings like those illustrated in Fig. 7 was fitted with a single exponential to obtain τrecovery (n = 6). Measurements of τi in standard saline are also plotted for 16 cells (▪). The values of τi in standard and K+ saline differ significantly at both voltages (P < 10−5). In ∼10–20% of the cells studied, inactivation appeared to be qualitatively slower than usual, although in other respects the conductance resembled HERG rather than IR. The reason for this behavior was not determined and those cells were excluded from analysis.
Figure 7
Figure 7
Recovery from the inactivation at 0 mV in KMeSO3 (pipette and bath). Superimposed are currents recorded during pairs of identical 300-ms pulses to −120 mV applied from Vhold = 0 mV, separated by an interval of variable duration. The whole pulse protocol was given once every 30 s. To explore recovery at other voltages, the potential in the interval between pulses was varied. The time constant of recovery, τrecovery, was obtained by fitting the envelope of peak test currents to a single exponential. In this experiment, τrecovery was 0.74 s.
Figure 6
Figure 6
Time constants of activation (τact, □) and deactivation (τtail, •) in KMeSO3 (pipette and bath). The rising phase of currents during hyperpolarizing pulses from Vhold = 0 mV was fitted with a single exponential to obtain τact. Tail currents like those in Fig. 5 A were fitted with a single exponential to obtain τtail. Plotted values are the mean ± SEM of n = 22 and 7 cells for τact and τtail, respectively.
Figure 9
Figure 9
Pharmacological sensitivity of HERG-like currents. (A) Block by Ba2+. Identical pulses to −80 mV from Vhold = 0 mV were applied to a cell exposed to KCl saline (pipette and bath) with the indicated concentration of BaCl2. (B) Block by La3+ in KCl saline with K aspartate in the pipette. From Vhold = −80 mV, a 300-ms prepulse to 80 mV to remove inactivation was followed by a test pulse to −120 mV. In this cell, 30 μM La3+ had a substantial effect and 100 μM virtually abolished the current, with partial recovery upon washout. (C) Block by 100 nM E-4031 in KCl saline with K aspartate in the pipette. Pulses to −120 mV were applied after a prepulse to 80 mV from Vhold = 0 mV. To explore a range of concentrations, control currents were recorded for at least 5 min, the lowest concentration of E-4031 was added to the bath and sufficient time was allowed for steady state blockade (usually 5 min) before the next concentration was added. The time-independent outward currents at 80 mV in B and C are most likely anion currents.
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