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. 2010 Mar;135(3):275-95.
doi: 10.1085/jgp.200910316.

Activation of Slo2.1 channels by niflumic acid

Li Dai  1 , Vivek Garg, Michael C Sanguinetti
Affiliations

Activation of Slo2.1 channels by niflumic acid

Li Dai et al. J Gen Physiol. 2010 Mar.

Abstract

Slo2.1 channels conduct an outwardly rectifying K(+) current when activated by high [Na(+)](i). Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na(+). In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC(50) = 2.1 mM) or flufenamic acid (EC(50) = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K(+)](e), the conductance-voltage (G-V) relationship had a V(1/2) of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V(1/2) to more negative potentials (EC(50) = 2.1 mM) and increased the minimum value of G/G(max) (EC(50) = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V(1/2) of the G-V relationship was shifted to more positive potentials when [K(+)](e) was elevated from 1 to 300 mM (EC(50) = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K(+)](e) dependency (EC(50) = 23.5 mM). Conductance was also [Na(+)](e) dependent. Outward currents were reduced when Na(+) was replaced with choline or mannitol, but unaffected by substitution with Rb(+) or Li(+). Neutralization of charged residues in the S1-S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1-S4 segments. In contrast, mutation of R190 located in the adjacent S4-S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K(+)](e). Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K(+)](e) and [Na(+)](e), and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na(+).

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Figures

Figure 1.
Figure 1.
Incubation of oocytes with Nai-loading solution activates ISlo2.1. (A) Currents recorded from an uninjected oocyte before (control) and after 10-min incubation in Nai-loading solution. Vt was varied from −140 to +80 mV and applied in 20-mV increments from a holding potential of −80 mV. (B) Currents recorded from an oocyte injected with 3.2 ng Slo2.1 cRNA before (control) and after incubation in Nai-loading solution for 10 min. Vt was varied from −140 to +80 mV and applied in 20-mV increments from a holding potential of −80 mV. (C) Average ISlo2.1–V relationship determined after incubation of oocytes in Nai-loading solution for 10–15 min (n = 8). (D) Kinetics of ISlo2.1 activated by incubation of oocytes with Nai-loading solution for 15 min. Left plot summarizes the fast and slow time constants for activation (τfast and τslow) of the time-dependent component of current (n = 15). On the right is a plot of the amplitude of the instantaneous current (Iinst) divided by the total outward current (Itotal) (n = 15). (E) Currents recorded from oocytes injected with 3.2 ng Slo2.1 cRNA and incubated in Barth’s solution (top) or Barth’s solution plus 10 µM ouabain for 2 d (bottom). Vt was varied from −160 to +80 mV, applied in 40-mV increments. (F) I-V relationships for uninjected oocytes incubated in Barth’s solution plus 10 µM ouabain for 2 d (open triangle; n = 9) and in oocytes injected with 3.2 ng Slo2.1 cRNA and incubated in Barth’s solution alone (open circle; n = 7) or in Barth’s solution plus 10 µM ouabain for 2 d (filled square; n = 13).
Figure 2.
Figure 2.
NFA activates Slo2.1 channels heterologously expressed in oocytes. (A) Currents recorded from an uninjected oocyte before and after treatment with 1 mM NFA. (B) Currents recorded from an oocyte expressing Slo2.1 before (control) and after treatment with 1 mM NFA for 8 min. Oocytes were injected 2 d earlier with 0.32 ng Slo2.1 cRNA. In A and B, Vt was varied from −160 to +80 mV, applied in 20-mV increments. (C) Average I-V relationships for currents recorded before (control) and after treatment with 1 mM NFA (n = 35). (D) [NFA]– and [FFA]–response relationships for increase of ISlo2.1 in response to continuous pulsing to 0 mV. Data were fitted with a Hill equation (smooth curves) to determine EC50 (2.08 ± 0.04 mM) and Hill coefficient, nH (1.94 ± 0.05), for NFA (n = 12). For FFA, the EC50 was 1.4 ± 0.02 mM and nH was 1.92 ± 0.03 (n = 5).
Figure 3.
Figure 3.
Increased [Na+]i blocks outward ISlo2.1 at positive potentials. (A) Currents recorded from an oocyte expressing Slo2.1 under control conditions (a) after 8-min treatment with 1 mM NFA (b), after 25-min incubation in Nai-loading solution (c), and then after 8-min treatment with 0.1 mM NFA (d). Vt was varied from −160 to +80 mV and applied in 40-mV increments. (B) I-V relationships for the oocyte under the indicated conditions.
Figure 4.
Figure 4.
Activation of single Slo2.1 channels by extracellular NFA and intracellular Na+. (A) Slo2.1 channels recorded during voltage ramp from +60 to −120 mV. The top panel illustrates voltage clamp protocol. Currents were recorded from a single patch under three recording conditions: cell-attached (CA) patch mode (i) or inside-out (IO) mode (ii) with a bath solution that contained 140 mM KCl, and inside-out mode (iii) with a bath solution that contained 80 mM NaCl and 60 mM KCl. (B) Slo2.1 channel activity measured during gap-free recordings at –80 mV from the same patch as in A. The closed (C) and open (O1, O2, and O3) state levels are indicated to the right of the current traces. (C) Single-channel i-V relationships for the three recording conditions as indicated. Numbers in parenthesis indicate the number of patches summarized for each data point in the order indicated by the inset legend. The slope conductance for single channels (γ) determined for −40 to −120 mV was 144 ± 8 pS for CA patches, 144 ± 6 pS for IO patches and 140 KCl bath solution, and 130 ± 7 pS for IO patches and 80 NaCl/60 KCl bath solution.
Figure 5.
Figure 5.
Concentration-dependent effects of NFA on ISlo2.1 magnitude and kinetics. (A) I-V relationships determined before (control) and after treatment of oocytes with 1, 3, and 6 mM NFA as indicated. (B) G-V relationships based on I-V relationships plotted in A. Data were fitted with a Boltzmann function (smooth curves) to determine the half-point (V1/2) and effective valence, z, for ISlo2.1 activated by treatment of oocytes with NFA. The V1/2 was −1 ± 0.7 mV (n = 8), −43 ± 2.4 mV (n = 7), and −84 ± 6 mV (n = 5) for 1, 3, and 6 mM NFA, respectively. The z values were 0.71 ± 0.01, 0.57 ± 0.02, and 0.46 ± 0.02 for oocytes treated with 1, 3, and 6 mM NFA, respectively. (C) Kinetics of ISlo2.1 activated by NFA. A summary of the fast and slow time constants (τfast and τslow) for activation of the time-dependent component of current (n = 15 for 1 mM NFA and n = 14 for 3 mM NFA). (D) A plot of the amplitude of the instantaneous current (Iinst) divided by total outward current (Itotal) as a function of text potential, Vt (n = 14–15).
Figure 6.
Figure 6.
Voltage dependence of Slo2.1 channel activation is [K+]e dependent. (A) I-V relationships for ISlo2.1 activated with 1 mM NFA in oocytes bathed in an extracellular solution, where [K+]e was varied from 1 to 99 mM. [Na+]e was varied as indicated, so that the sum of [K+]e and [Na+]e was kept constant at 100 mM. Currents were measured from oocytes (n = 5) 3 d after they were injected with 0.84 ng cRNA. (B) Slo2.1 is K+ selective. Reversal potential of ISlo2.1 (Erev) was plotted as a function of log10[K+]e, and the data were fitted by linear regression. The slope of the line is 50.5 mV/decade increment of [K+]e (R2 = 0.99). (C) G-V relationships for Slo2.1 as a function of [K+]e. (D) V1/2 of G-V relationship plotted as a function of log10 [K+]e. The data were fitted with a Hill equation (smooth curve) with an EC50 of 21.2 ± 6.0 mM when the Hill coefficient, nH, was fixed to a value of 1.0 (R2 = 0.99). The data for 300 mM [K+]e was measured in a separate batch of oocytes. (E) I-V relationships for ISlo2.1 activated with 3 mM NFA in oocytes bathed in an extracellular solution, where [K+]e was varied from 1 to 99 mM. [Na+]e was varied as indicated, so that the sum of [K+]e and [Na+]e was kept constant at 100 mM. Currents were measured from oocytes (n = 6) 2 d after they were injected with 0.84 ng cRNA. (F) The slope conductance was determined at the 0 current potential (gErev) of the I-V relationship and plotted as a function of log10[K+]e. The data were fitted with a Hill equation (smooth curve) with an EC50 of 23.5 ± 3.5 mM when the Hill coefficient, nH, was fixed to a value of 1.0 (R2 = 0.99).
Figure 7.
Figure 7.
I-V and G-V relationships measured from oocytes compared with relationships predicted by GHK current equation. (A) I-V relationships (top) and G-V relationships determined for [K+]e = 2 mM. (B) Relationships determined for [K+]e = 10 mM. (C) Relationships determined for [K+]e = 99 mM.
Figure 8.
Figure 8.
Slo2.1 channel rectification is greater than that predicted by GHK current equation. (A) Single Slo2.1 channel activity measured in an excised inside-out patch during a voltage ramp as indicated. Extracellular (pipette) solution contained (in mM): 90 NaCl, 10 KCl, 0.1 CaCl2, 2 MgCl2, 10 HEPES, and 1 NFA, pH 7.2; intracellular (bath) solution contained (in mM): 90 KCl, 10 NaCl, 2 EGTA, 10 HEPES, and 2 MgCl2, pH 7.2. The holding potential was −80 mV, and the voltage was stepped to +80 mV, and then ramped down to −120 mV over 775 ms. The slope conductance for this single channel at positive potentials was 55 pS. (B) Ensemble average currents recorded during 37 voltage ramps. The red curve represents the i-V relationship predicted by the GHK current equation for a voltage-independent conductance.
Figure 9.
Figure 9.
NFA decreases rectification of ISlo2.1. (A) Average ISlo2.1-V relationships in oocytes treated sequentially with 1, 2, and 3 mM NFA (n = 8). (B) Average ISlo2.1-V relationships in another batch of oocytes treated sequentially with 4, 5, and 6 mM NFA (n = 6). Extracellular solution was K104. (C) G-V relationships for ISlo2.1 for different concentrations of NFA as indicated. (D) Shift in V1/2 of the G-V relationship (filled circles) and minimum value of G/Gmax (open squares) is [NFA] dependent. Data were fitted with a Hill equation (smooth curves). For shift in V1/2, EC50 = 2.1 ± 0.2 mM and nH = 2.9 ± 0.1 (R2 = 1.0). For minimum G/Gmax, EC50 = 2.4 ± 0.2 mM and nH = 1.9 ± 0.3 (R2 = 0.93).
Figure 10.
Figure 10.
The effect of 1 and 6 mM NFA on single Slo2.1 channel activity. (A) Activity of a single Slo2.1 channel in an inside-out patch activated with 1 mM NFA in the pipette solution and recorded during voltage ramps from +80 to −120 mV. For these experiments, the extracellular (pipette) solution contained (in mM): 99 K gluconate, 1 Na gluconate, 2 MgCl2, 0.1 CaCl2, 0.1 GdCl3, 10 HEPES, and either 1 or 6 NFA. The intracellular (cell chamber) solution was the same, except that CaCl2 was replaced with 2 mM EGTA, and NFA was not present. Three different current traces (i–iii) are shown together with the ensemble average of 15 traces (iv) plotted on a reversed voltage scale. Note outward rectification of currents. (B) A different inside-out patch showing activity of at least two channels that were activated by 6 mM NFA in the pipette solution. Three different current traces (i–iii) are shown together with the ensemble average of 30 traces (iv) plotted on a reversed voltage scale. Note that currents do not exhibit rectification. (C) i-V relationships for single Slo2.1 channels activated by 1 mM (n = 3) or 6 mM (n = 6) NFA. The slope conductance determined from channels activated between −60 and −140 mV was 97 ± 11 pS for 1 mM NFA (n = 3) and 80 ± 2 pS for 6 mM NFA (n = 5).
Figure 11.
Figure 11.
Effects of replacing extracellular Na+ with mannitol, choline, or monovalent cations on Slo2.1 channel currents. (A–E) ISlo2.1-V relationships for oocytes bathed in KCM411 extracellular solution and activated by 1 mM NFA before and after replacement of extracellular Na+ with either mannitol or indicated monovalent cation. (A and B) Currents recorded from oocytes 3 d after injection with 0.32 ng cRNA (n = 6). (C and D) Currents recorded from oocytes 3 d after injection with 0.84 ng cRNA (n = 5–6). (D, inset) Currents for Vt of −60 to −160 mV at an expanded scale. (F) ISlo2.1–V relationships for oocytes activated by 3 mM NFA and bathed in an extracellular solution containing the indicated level (in mM) of NaCl. [Na+]e was varied from 0 to 90 mM by substitution with mannitol to maintain constant osmolarity (n = 6). Currents were recorded 3 d after injection of oocytes with 0.32 ng cRNA. Inset shows plot of maximum slope conductance as a function of log10[Na+]e. Data were fitted to a Hill equation (smooth curve; EC50 = 10.5 ± 4.2 mM, nH = 0.72 ± 0.13, and R2 = 0.997).
Figure 12.
Figure 12.
Location of charged residues in the S1–S4 segments of Slo2.1 channel subunits. (A) Sequence alignment of S4 segments for Slo and Kv1.2 channels. Numbering on top refers to residues in hSlo2.1, and box indicates the boundaries of the S4 segment. Basic residues are colored red, and acidic residues are colored blue. (B) Diagram of a single Slo2.1 subunit showing location of charged amino acids in the S1–S4 segments.
Figure 13.
Figure 13.
Neutralization of charged residues in the S1–S4 segment does not eliminate voltage-dependent gating of Slo2.1 channels. (A and B) I-V relationships for currents recorded from oocytes expressing S1 mutant K70A Slo2.1 (n = 14) or R80A Slo2.1 (n = 7) channels before and after treatment with 1 mM NFA. Extracellular solution was K104. Oocytes were recorded 2 d after injection of 2.5 ng cRNA. (C) I-V relationships for currents recorded from oocytes expressing the S2 double mutant (E118A/E143A) Slo2.1 channels before and after treatment with 1 mM NFA (n = 7). Oocytes were recorded 2 d after injection of 2.5 ng cRNA. (D) G-V relationships for WT and S1–S3 mutant channels. Dotted curve shows relationship predicted by GHK current equation for voltage-independent channel. The data were fitted with a Boltzmann function (smooth curves) to obtain V1/2 and z values as follows. WT: V1/2 = +95 ± 0.6 mV and z = 0.48 ± 0.01; K70A: V1/2 = +159 ± 8 mV and z = 0.30 ± 0.04; R80A: V1/2 = +116 ± 3 mV and z = 0.48 ± 0.02; E118A/E143A: V1/2 = +114 ± 4 mV and z = 0.51 ± 0.04. (E) I-V relationships for currents recorded from oocytes expressing the S4 neutral mutant (K174A/E178A/D183A/R186A/H175A/H185A) Slo2.1 channels before and after treatment with 1 mM NFA (n = 9). Oocytes were recorded 3 d after injection of 9.2 ng cRNA. (F) G-V relationships for WT and S4 neutral mutant channels (+84 ± 1 mV; z = 0.69 ± 0.02). Dotted curve shows relationship predicted by GHK current equation for voltage-independent channel. (G) Normalized I-V relationships for WT and S1–S4 mutant channels. Currents for each channel type were normalized to their average values measured at +120 mV.
Figure 14.
Figure 14.
Nonconserved mutations of R190 in Slo2.1 induce constitutive channel activity and alter the sensitivity to NFA. (A) R190A current traces recorded at +40 mV before (control) and after treatment of oocytes with 1 mM NFA. (B) I-V relationships for R190A Slo2.1 channels determined in the presence and absence of 1 mM NFA (n = 5). Oocytes were recorded 2 d after injection of oocytes with 0.32 ng cRNA. (C) R190Q current traces recorded at +40 mV before (control) and after treatment of oocytes with 1 mM NFA. (D) I-V relationships for R190Q Slo2.1 channels determined in the presence and absence of 1 mM NFA (n = 8). Oocytes were recorded 2 d after injection of oocytes with 0.42 ng cRNA. (E) R190K Slo2.1 current traces recorded at +40 mV before (control) and after treatment of oocytes with 1 mM NFA. (F) I-V relationships for R190K Slo2.1 channels determined in the presence and absence of 1 mM NFA (n = 8). Oocytes were recorded 2 d after injection of oocytes with 0.84 ng cRNA. (G) 1 mM NFA does not activate R190E Slo2.1 currents recorded at +40 mV. Note that unlike the other R190 mutant channels, R190E channel currents are time independent in the absence of NFA. (H) NFA does not alter the I-V relationships for R190E Slo2.1 channels (n = 9). Oocytes were recorded 1 d after injection of oocytes with 0.84 ng cRNA.
Figure 15.
Figure 15.
Activation of R190 mutant Slo2.1 channels by NFA or Nai loading in oocytes bathed in high K+ extracellular solution. (A) I-V relationships for R190E Slo2.1 channels recorded using the indicated extracellular solutions. Currents were recorded from the same oocyte as extracellular solution was changed in the order (from top to bottom) indicated in the symbol legend. Oocytes were recorded 1 d after injection with 0.84 ng cRNA (n = 8). After recording currents when oocytes were bathed in KCM211 and K104 solutions, oocytes were then incubated in Nai-loading solution for 15 min before again measuring I-V relationship using K104 solution (“K104, Nai-loaded”). Finally, the bathing solution was switched to KCM211, and currents were recorded once again (“KCM211, Nai-loaded”). (B) Example of currents recorded from an oocyte before and after treatment with 1 mM NFA. Arrows indicate 0 current level. (C) I-V relationships for R190E Slo2.1 channels recorded from oocytes bathed in K104 extracellular solutions before and after treatment with 1 mM NFA. Oocytes were recorded 1 d after injection with 0.84 ng cRNA (n = 6). (D and E) I-V relationships for R190A (D) and R190Q (E) Slo2.1 channels recorded from oocytes bathed in K104 extracellular solutions before and after treatment with 1 mM NFA. Oocytes were recorded 1 d after injection with 0.92 ng R190A (n = 8) or 0.32 ng R190Q (n = 10) Slo2.1 cRNA.
Figure 16.
Figure 16.
Concentration-dependent effects of NFA on R190A and R190E Slo2.1 channels. (A) I-V relationships for oocytes expressing R190A Slo2.1 channels and bathed in K104 solution. Oocytes were studied 2 d after injection of 0.84 ng cRNA (n = 9). (B) I-V relationships for oocytes expressing R190A Slo2.1 channels and bathed in K104 solution. Oocytes were studied 2 d after injection of 0.84 ng cRNA (n = 6). (C) Concentration–response relationships for oocytes expressing WT, R190A, or R190E Slo2.1 channels and bathed in K104 extracellular solution. Currents were measured at a Vt of +80 mV. The data were fitted with a modified Hill equation (smooth curves) to estimate EC50 and nH values as follows: WT: EC50 = 2.24 ± 0.02 mM and nH = 1.73 ± 0.02; R190E: EC50 = 1.28 ± 0.09 and nH = 0.83 ± 0.06; R190A: EC50 = 0.97 ± 0.02 and nH = 1.28 ± 0.04. (D) Concentration–response relationships for oocytes expressing WT, R190A, or R190E Slo2.1 channels and bathed in KCM211 extracellular solution. Currents were measured at a Vt of 0 mV. Hill equation parameters were as follows: WT: EC50 = 2.1 ± 0.87 mM and nH = 1.74 ± 0.50; R190E: EC50 = 1.98 ± 0.43 and nH = 2.15 ± 0.80; R190A: EC50 = 0.71 ± 0.13 and nH = 1.34 ± 0.24. For C and D, currents were normalized to the extrapolated maximum response for each cell (I/Imax).
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