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, 227 (6), 2542-55

Niflumic Acid Blocks Native and Recombinant T-type Channels

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Niflumic Acid Blocks Native and Recombinant T-type Channels

Enrique Balderas et al. J Cell Physiol.

Abstract

Voltage-dependent calcium channels are widely distributed in animal cells, including spermatozoa. Calcium is fundamental in many sperm functions such as: motility, capacitation, and the acrosome reaction (AR), all essential for fertilization. Pharmacological evidence has suggested T-type calcium channels participate in the AR. Niflumic acid (NA), a non-steroidal anti-inflammatory drug commonly used as chloride channel blocker, blocks T-currents in mouse spermatogenic cells and Cl(-) channels in testicular sperm. Here we examine the mechanism of NA blockade and explore if it can be used to separate the contribution of different Ca(V)3 members previously detected in these cells. Electrophysiological patch-clamp recordings were performed in isolated mouse spermatogenic cells and in HEK cells heterologously expressing Ca(V)3 channels. NA blocks mouse spermatogenic cell T-type currents with an IC(50) of 73.5 µM, without major voltage-dependent effects. The NA blockade is more potent in the open and in the inactivated state than in the closed state of the T-type channels. Interestingly, we found that heterologously expressed Ca(V)3.1 and Ca(V)3.3 channels were more sensitive to NA than Ca(V)3.2 channels, and this drug substantially slowed the recovery from inactivation of the three isoforms. Molecular docking modeling of drug-channel binding predicts that NA binds preferentially to the extracellular face of Ca(V)3.1 channels. The biophysical characteristics of mouse spermatogenic cell T-type currents more closely resemble those from heterologously expressed Ca(V)3.1 channels, including their sensitivity to NA. As Ca(V)3.1 null mice maintain their spermatogenic cell T-currents, it is likely that a novel Ca(V)3.2 isoform is responsible for them.

Figures

Fig. 1
Fig. 1. Blockade of T-type currents by NA in mouse spermatogenic cells
A) A family of currents recorded in whole cell configuration in control conditions in response to depolarized pulses from a −80 to +70 mV in +10 mV increment from a HP of −90 mV (traces from −80 to +30 mV are show for clarity). B) Dose-response curve. Data points are (mean ± SEM) the fraction of blocked current at −20 mV for each NA concentration tested. Fit of experimental points with a Hill equation (solid line) produces an estimated IC50 for NA of 73.5 µM. C) Currents recorded as in (A) but in the presence of 100 µM NA. D) Current-voltage (I–V) curve was constructed with the peak current normalized and averaged from 12 individual cells under control (filled circles) and in presence of 100 µM NA (open circles). In both conditions, threshold of currents was observed around −60 mV.
Fig. 2
Fig. 2. Steady-state inactivation of spermatogenic cell T-type currents is not affected by NA
A) Family of currents recorded with the voltage protocol depicted at the top. Channels were inactivated with 350-ms prepulses to potentials between −110 and −40 mV in 5 mV steps, from a HP = −90 mV. Then, channel availability was tested at −20 mV. B) Family of currents recorded as in A, but in presence of 100 µM NA. C) Steady-state inactivation curves in the absence and the presence of NA. Data points show the normalized peak amplitude of currents elicited under control (filled circles) and NA (open circles) by the testing pulse at −20 mV, and plotted against the prepulse potentials. Continuous lines are the best fits with a Boltzmann function. Values of V50 and k obtained from this curves are summarized in Table 1.
Fig. 3
Fig. 3. Effect of NA on the activation and inactivation kinetics of T-type currents
A) Calcium currents recorded under control conditions with the voltage protocol displayed at the top. B) Same as in A, but in presence of 100 µM NA. Tau of activation (τact) and tau of inactivation (τact) were obtained by fitting single exponentials to the traces as shown at the inset. C) Plot of mean values for τact against command voltage (n = 6). Control condition is represented by filled symbols and NA by open circles. D) Plot of mean values for τinact in control (filled circles) and NA (open circles) against test potential (n = 6). Note that NA induced a slow down effect on the current inactivation kinetics at all potentials more positive than −50 mV. Bars represent SEM.
Fig. 4
Fig. 4. NA Blockade of T-type channels is state-dependent
A) Left panel, traces of current recorded with the protocol depicted above to maintain the T-type channels in the closed state. A test pulse of −20 mV was applied with at = 120 s from a HP = −90 mV. Right panel, normalized current was plotted against time; white and grey bars represent time to exposure to control solution and NA, respectively. Note that ~ 25% of inward current amplitude was reduced by NA. B) Effect of NA on inactivated state of T-type channels. Left panel, current recorded in response to a depolarization to −20 mV from a 400 ms hyperpolarized pulse to −90 mV. Cells were held at −20 mV to maintain the channels inactivated. Right panel normalized current versus time of exposure to NA. Note a reduction of more than 50% in presence of the drug by using the inactivated state protocol. C) Left panel, blockade of T-type currents by NA in open state. Cells were clamped at −90 mV during the whole experiment with a depolarization to −20 mV with a Δt = 5 s. Inset, continuously stimulated cells with the protocol previously described during 500 s without drug did not modify the currents during the whole experiment. Right panel, normalized current shows a decrement in the amplitude of the current of more than 50% during the time of exposure. Error bars represent SEM, (n = 6). Note that in the three conditions 85% of the original current was recovered by washing the drug.
Fig. 5
Fig. 5. NA delays recovery from inactivation of T-type channels
Current traces recorded with two pulses voltage protocol, depolarizing pulses to −20 mV from a HP = −100 mV were applied at different inter-stimulus interval (Δt), 10 ms increments for control condition (A), and 100 ms for NA (B). Time courses of recovery from inactivation (right panels) were fitted by single exponential functions with time constant (τh) of 67.6 ± 3.05 ms for control (A) and 318.01 ± 20.5 ms in the presence of 100 µM of NA (B). Error bars represent SEM (n = 9).
Fig. 6
Fig. 6. Blockade of T-type channels by NA is not use dependent
Cells were stepped to −20 mV from a HP of −90 mV with a stimulation frequency of 3 (A), 2 (B), and 1 (C) Hz. Under control (left panels) and 100 µM NA condition (middle panels). Only pulses 1, 2 and 30 are shown for illustrative purposes. Right panels, plots of normalized current against time for control (filled circles) and NA (open circles) in the three frequencies tested. Note that reduction of ~35 % in the current was due to the accumulation of inactivation at least in 3 and 2 Hz. Error bars represent SEM (n = 6).
Fig. 7
Fig. 7. Recombinant CaV3 channels are sensitive to NA
A) Examples of calcium currents recorded at −40 mV before (Control), during (NA, thick trace), and after (Wash) exposure to 100 µM NA from HEK-293 cells stably expressing CaV3.1, CaV3.2 and CaV3.3 channels. Depolarization steps lasted 300 ms, although for clarity, only the first 150 ms are shown. B) Blockade percentage of peak calcium currents at −40 mV induced by 100 µM NA in CaV3 channels. The NA blockade percentage was calculated for each cell, averaged and plotted as a mean ± SEM for each type of channel. The respective values were 56 ± 3, 34 ± 5 and 42 ± 3% for CaV3.1, CaV3.2 and CaV3.3, respectively. The number of assayed cells is indicated in parentheses.
Fig. 8
Fig. 8. NA blockade of recombinant CaV3 channels is voltage-independent
A) Representative family of traces obtained in Control and 100 µM NA experimental conditions. Currents were recorded from a HEK-293 cell expressing CaV3.2 channels in response to the I–V protocol illustrated at the bottom. B–D) Current-voltage relationships for CaV3.1, CaV3.2, and CaV3.3 channels, respectively, in the absence (Control) and the presence of 100 µM NA. Data points representing inward currents were fitted with a modified Boltzmann function (continues line) and the obtained parameters are summarized in Table 1. Note that the amplitude of outward currents was practically unaffected by the presence of NA. E) Blockade percentage of CaV3 channels by NA and Vm relationships. The blockade percentage of peak current by NA was calculated for each potential. Data was obtained from 7 (CaV3.1), 6 (CaV3.2) and 5 (CaV3.3) HEK-293 cells.
Fig. 9
Fig. 9. Current activation kinetics of CaV3 channels is slowed down by NA
Current recordings as shown in Fig. 8A were fitted by the sum of two exponentials, one for activation and the other for current decay. The resulting time constants (taus) were plotted as function of the test potential for each channel as indicated. The larger modifications were observed in the current activation of CaV3.2 and CaV3.3 channels. Insets: Tau of inactivation versus test potential for the same channels.
Fig. 10
Fig. 10. Recovery from inactivation of recombinant CaV3 channels is drastically modified by NA
A) Recovery from inactivation at −100 mV of CaV3.2 channels in control conditions. The two-pulse protocol used is shown at the top. Ca2+ currents were inactivated by a 300 ms pulse to −40 mV, after which the membrane potential was stepped to −100 mV for periods ranging from 1 to 4200 ms, at that time a 20 ms activating voltage step to −40 mV was applied. Only current traces at −40 mV obtained after 5, 40, 80, 200, 800, 1600, 2400, 3200 and 4200 ms at −100 mV are shown, and only the tail current (generated by repolarizing at −100 mV) of the latter is partially plotted. B–D) Time course of recovery from inactivation at −100 mV for the indicated CaV3 channels in Control and 100 µM NA conditions. The values are the peak current during the 20 ms pulse, normalized to the peak current in the 300 ms pulse. Data were pooled from 6 (CaV3.1), 6 (CaV3.2) and 5 (CaV3.3) HEK-293 cells. Smooth curves are fits to the data using a one phase exponential association equation. Dashed line in (C) corresponds to the same kind of fit to data from 3 cells where current recovery from NA blockade was above 90%. Tau values are given in Table 1.
Fig. 11
Fig. 11. Niflumic acid docking to CaV3 channels
A) Average binding energies (kcal/mol) and distance (in Å, Angstroms), for niflumic acid (NA) as ligand docked to CaV3 pore domain models. Distance data was obtained from RMSD l.b. values, which is the relative value to the best mode using only movable heavy atoms or lower bound. Columns are averages of the minimum or best binding energy for the docking results. The 1K4C coordinates were used as control for the interaction with TEA. NA docked with 1K4C showed a low interaction value (~ −0.4), serving as low affinity control. Error bars represent SEM from n = 36 interaction models of each CaV3 channel. B) Docking analysis was clustered as shown, in order to search the whole grid spacing of the protein models and control structures. In and Out denote the intra and extracellular side of the cell. C) The ratio between kcal/mol and RMSD l.b. (in Å) for each cluster are illustrates as follows: cluster 1, clear blue, involves the upper region of the proteins; cluster 2, green, the pore; cluster 3, red, the middle transmembrane region of the proteins; and cluster 4, dark blue, the intracellular region of the protein.

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