Effect of mibefradil on sodium and calcium currents
Peter R. Strege,
Cheryl E. Bernard,
Yijun Ou,
Simon J. Gibbons, and
Gianrico Farrugia
Enteric NeuroScience Program, Mayo Clinic College of Medicine, Rochester, Minnesota
Submitted 19 January 2005
; accepted in final form 21 March 2005
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ABSTRACT
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Interstitial cells of Cajal (ICC) generate the electrical slow wave. The ionic conductances that contribute to the slow wave appear to vary among species. In humans, a tetrodotoxin-resistant Na+ current (NaV1.5) encoded by SCN5A contributes to the rising phase of the slow wave, whereas T-type Ca2+ currents have been reported from cultured mouse intestine ICC and also from canine colonic ICC. Mibefradil has a higher affinity for T-type over L-type Ca2+ channels, and the drug has been used in the gastrointestinal tract to identify T-type currents. However, the selectivity of mibefradil for T-type Ca2+ channels over ICC and smooth muscle Na+ channels has not been clearly demonstrated. The aim of this study was to determine the effect of mibefradil on T-type and L-type Ca2+ and Na+ currents. Whole cell currents were recorded from HEK-293 cells coexpressing green fluorescent protein with either the rat brain T-type Ca2+ channel
13.3b +
2, the human intestinal L-type Ca2+ channel subunits
1C +
2, or NaV1.5. Mibefradil significantly reduced expressed T-type Ca2+ current at concentrations
0.1 µM (IC50 = 0.29 µM), L-type Ca2+ current at > 1 µM (IC50 = 2.7 µM), and Na+ current at
0.3 µM (IC50 = 0.98 µM). In conclusion, mibefradil inhibits the human intestinal tetrodotoxin-resistant Na+ channel at submicromolar concentrations. Caution must be used in the interpretation of the effects of mibefradil when several ion channel classes are coexpressed.
ion channel; gastrointestinal tract; patch clamp
A SPECIALIZED SUBSET of interstitial cells of Cajal (ICC) generate the electrical slow wave, an important determinant of gastrointestinal motility. The inward ionic conductances involved in the generation of the slow wave appear to vary among species. In humans, a tetrodotoxin-resistant Na+ current, carried by the SCN5A-encoded NaV1.5 channel, is expressed in intestinal ICC and contributes to the rising phase of the slow wave (20). SCN5A has also been identified in human intestinal circular smooth muscle cells (18) but not in mice and pigs. In mice, T-type-like Ca2+ currents have been reported from mouse colonic myocytes and mouse intestine ICC (11, 12) and are considered to contribute to properties of the slow wave. T-type Ca2+ channels have also been described in canine colonic ICC (13).
Mibefradil is a tetralol Ca2+ channel antagonist with higher affinity for T-type (Kd = 0.14.7 µM) than L-type (Kd = 1.428 µM) Ca2+ channels (summarized in Ref. 15). Because the tetrodotoxin-resistant Na+ current and T-type Ca2+ currents have fairly similar electrophysiological properties, mibefradil would be a useful tool to dissect out inward tetrodotoxin-resistant Na+ currents and T-type Ca2+ currents. Indeed, mibefradil has already been used in the gastrointestinal tract to identify a T-type-like current (11). However, the selectivity of mibefradil for Ca2+ currents compared with Na+ channels expressed in human gastrointestinal smooth muscle cells and ICC has not been clearly demonstrated. Therefore, we sought to determine the effect of mibefradil on Ca2+ and Na+ channels expressed in HEK-293 cells.
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MATERIALS AND METHODS
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Expression vector construction and HEK-293 transfection.
The expression vectors containing the human jejunum L-type Ca2+ channel
1C (
11.2)- and
2-subunits, the rat brain T-type Ca2+ channel
13.3b-subunit, or the human jejunum Na+ channel SCN5A
-subunit were described previously (14, 17, 18). The
13.3b-subunit clone was a gift from Dr E. Perez-Reyes. Using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA), the green fluorescent protein (GFP) pEGFP-C1 (Clontech, Palo Alto, CA), and either the sodium or L- or T-type calcium channel, expression vectors were transiently cotransfected into HEK-293 cells (American Type Culture Collection, Manassas, VA). HEK cells expressing L- or T-type Ca2+ channel subunits were cultured for 2 days, whereas cells expressing SCN5A were cultured for 1 day posttransfection. Transfected cells were identified by fluorescent microscopy and patch clamped to record whole cell currents.
Patch clamp recordings.
Patch clamp recordings were obtained at 22°C using standard whole cell techniques (5, 6). Microelectrodes were pulled from Kimble KG-12 glass on a P-97 puller (Sutter Instruments, Novato, CA). Electrodes were coated with R6101 (Dow Corning, Midland, MI) and fire polished to a final resistance of 3 to 5 M
. Currents were amplified, digitized, and processed using an Axopatch 200B amplifier, Digidata 1322A, and pCLAMP 8 software (Axon Instruments, Foster City, CA). Whole cell records were sampled at 10 kHz and filtered at 5 kHz with an eight-pole Bessel filter. In T-type Ca2+ current experiments, cells were held at 100 mV and pulsed from 90 to +35 mV in 5-mV intervals for 150 ms each. To record L-type Ca2+ current, cells were held at 100 mV and pulsed from 60 to +40 mV in 5-mV intervals for 50 ms each. For Na+ current experiments, we used a holding potential of between 100 to 75 mV and stepped from 80 to +35 mV in 5-mV intervals for 50 ms each. Most cells were held at 100 or 90 mV, but when the Na+ currents were too large to be recorded accurately, the holding voltage was changed to a more depolarized voltage. Three cells were held at 80 mV and two cells at 75 mV. A 7080% series resistance compensation (lag of 60 µs) was applied during each recording. Cell capacitance of HEK-293 cells tranfected with
13.3b +
2 was 23 ± 4 pF (n = 12),
1C +
2 was 19 ± 3 pF (n = 10), and SCN5A was 20 ± 2 pF (n = 18). The differences in cell capacitance between these groups were not significant (P > 0.05). Resistance after access was established was 510 M
.
Drug and solutions.
Intracellular solution contained (in mM) 130 Cs+, 125 methanesulfonate, 20 Cl, 5 Na+, 5 Mg+, 5 HEPES, 2 EGTA, 2.5 ATP, and 0.1 GTP. Extracellular solution was normal Ringer solution (in mM); 149.2 Na+, 159 Cl, 4.74 K+, 2.54 Ca2+, 5 HEPES, and 5 glucose, and replaced with increasing concentrations of mibefradil 0, 0.03, 0.1, 0.3, 1, 3, 10, and back to the control solution without the drug (Sigma-Aldrich, St. Louis, MO). Up to three concentrations were applied to any one cell. The bath chamber was rinsed with 1.5 ml NaCl Ringer + mibefradil at 10 ml/min for
9 s. Two recordings were taken at each concentration immediately after rinse and 2 min later. Intra- and extracellular solutions were equilibrated to pH 7.0, 300 mmol/kg and pH 7.35, 300 mmol/kg, respectively. We did not normalize for rundown in our experiments. We did not apply more than three concentrations to a particular cell to avoid long experiments. We have not observed rundown in cells transfected with SCN5A. Rundown for the L-type Ca2+ current was about 15% over 10 min after peak inward current.
Data analysis.
Data were analyzed using Clampfit (Axon Instruments, Union City, CA), Excel (Microsoft, Redmond, WA), and SigmaPlot (SPSS, Chicago, IL). For current-voltage graphs, the peak inward current of a single control trace was normalized to one using the equation Inorm = 1·(IV/Imax), where Inorm is normalized current, IV is measured current at a given voltage, and Imax is maximum peak inward current of the control trace. Thus peaks of all other traces per cell were expressed as a fraction of 1. Statistical comparisons were made using a paired, two-tailed Students t-test. Statistical significance was accepted when P values were < 0.05. Data are expressed as means ± SE. Sigmoidal dose-response curves were built in SigmaPlot by inserting normalized peak current data into the equation: y = min + (max min)/[1 + 10(logEC50 x)Hillslope].
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RESULTS
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Effect of mibefradil on heterologously expressed T-type Ca2+ channel subunit
13.3b.
To compare the selectivity of mibefradil against L-type Ca2+ or Na+ currents, we first determined its effects on the T-type current under the same recording conditions. After coexpressing GFP with rat brain T-type Ca2+ channel
-subunit
13.3b and a Ca2+ channel
2-subunit in HEK-293 cells, we recorded standard whole cell currents (Fig. 1). Adding extracellular solution containing 0.03 µM mibedfradil did not change expressed T-type Ca2+ current (804 ± 180 pA 0 µM control, to 752 ± 192 pA 0.03 µM, 8 ± 7% decrease, n = 6, P > 0.05). However, 0.1 µM mibefradil significantly reduced T-type Ca2+ current by 24 ± 6% compared with controls (485 ± 143 pA 0 µM control, to 398.5 ± 126 pA 0.1 µM, n = 6, P < 0.05). Subsequent additions of 0.3, 1, 3, and then 10 µM mibefradil further reduced the current by 52 ± 5, 75 ± 7, 94 ± 1, and 98 ± 0.4%, respectively, compared with predrug (644 ± 119% pA control, to 395 ± 111 pA 0.3 µM, to 131.5 ± 52 pA 1 µM, to 48 ± 9 pA 3 µM, and 8 ± 2 pA 10 µM, n = 6 for each concentration, P < 0.05 at 0.110 µM). Only 10 ± 3% of the control current returned 2 min after washing out mibefradil with multiple exchanges of fresh Ringer solution (78 ± 25 pA, n = 6, P < 0.05 compared with predrug).
Effect of mibefradil on heterologously expressed human jejunum L-type Ca2+ channel subunit.
Previous studies testing the effects of mibefradil on L-type Ca2+ channels used Ba2+ instead of Ca2+ as the charge carrier (summarized in Ref. 15). To make a direct comparison between the doses necessary to block Ca2+ current of T- and L-type channels, we used Ca2+ in the extracellular solution. HEK-293 cells were transfected with the human jejunum L-type subunits
1C and
2 (Fig. 2), and whole cell currents were measured. Concentrations of 0.1 or 0.3 µM mibefradil did not significantly change L-type Ca2+ current [157 ± 39 pA control; to 151 ± 36 pA at 0.1 µM (1 ± 6% decrease), to 147 ± 40 pA at 0.3 µM (9 ± 6% decrease), n = 5; P > 0.05]. In separate experiments adding 1 µM mibefradil to the extracellular solution resulted in a nonsignificant 23 ± 11% decrease in L-type Ca2+ current (55 ± 11 pA control, to 38 ± 4 pA 1 µM, n = 5, P > 0.05). However, increasing the dose to 3 or 10 µM significantly blocked L-type current by 52 ± 4 and 83 ± 2%, respectively (157 ± 39 pA control to 69 ± 12 pA 3 µM, and 55 ± 11 pA control to 9 ± 1 pA 10 µM, n = 5 for each concentration, P < 0.05). Similar to T-type current, washout of mibefradil did not return L-type Ca2+ current to normal (46 ± 10% of predrug current, n = 5, P < 0.05 compared with predrug). Therefore, an eightfold lower dose of mibefradil was needed to block T- than L-type current, supporting the literature that mibefradil is more specific for T-type Ca2+ channels than L-type Ca2+ channels.
Effect of mibefradil on heterologously expressed
-subunit NaV1.5 of the human jejunum Na+ channel encoded by SCN5A.
Because Na+ and T-type Ca2+ current both reach maximum peak inward current at potentials more negative than 20 mV, it would be useful to have a pharmacological tool to discern these currents. We determined the sensitivity to mibefradil of the human jejunum Na+ channel
-subunit expressed in HEK cells (Fig. 3). Switching the concentration of mibefradil in the extracellular solution from 0 to 0.1 µM resulted in a nonsignificant change in Na+ current (1096 ± 202 pA 0 µM control, to 1033 ± 174 pA 0.1 µM,
4 ± 3%, n = 6, P > 0.05). Increasing the concentration to 0.3 µM significantly decreased Na+ by 34 ± 4% (1096 ± 202 pA 0 µM, to 657 ± 139 pA 0.3 µM, n = 6, P < 0.05). A further increase in extracellular mibefradil concentration to 1, 3, and then 10 µM significantly blocked Na+ current by 47 ± 9, 72 ± 4, and 96 ± 2%, respectively (883 ± 102 pA 0 µM, to 495 ± 103 pA at 1 µM, to 346 ± 86 pA at 3 µM, to 33 ± 16 pA at 10 µM, n = 6, P < 0.05 comparing either concentration to control). Of the initial Na+ current, 42 ± 8% recovered after a 2-min washout (1052 ± 123 pA predrug to 395 ± 83 pA postdrug, n = 15, P < 0.05).

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Fig. 3. Block of Na+ channel -subunit SCN5A by mibefradil. A: representative whole cell Na+ channel currents recorded in NaCl Ringers solution with 0, 1, or 10 µM mibefradil. B: current-voltage relationship for peak inward Na+ current was normalized to the maximum peak inward current of the control record. C: summary of the mean normalized peak Na+ current in cells exposed to 010 µM mibefradil (n = 6 per concentration, *P < 0.05).
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Selectivity of mibefradil to T-type Ca2+, L-type Ca2+, and Na+ currents.
To determine the relative selectivity of the T- and L-type Ca2+ vs. Na+ current, we plotted the log concentration/response relationship for mibefradil against normalized peak currents (Fig. 4). Mibefradil was most selective for the T-type Ca2+ channel
13.3b +
2 (IC50 = 0.29 µM), followed by the Na+ channel SCN5A (IC50 = 0.98 µM), and the L-type Ca2+ channel
1C +
2 (IC50 = 2.7 µM).

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Fig. 4. Dose-response curves representing mibefradil inhibition of Na+ and L- and T-type Ca2+ channels. Summary of mibefradil concentration vs. normalized peak currents as shown in Figs. 1C, 2C, and 3C. IC50 values for T-type Ca2+, Na+, and L-type Ca2+ channels were (in µM) 0.29 ( 13.3b + 2, n = 6), 0.98 (SCN5A, n = 6), and 2.7 ( 1C + 2, n = 5), respectively.
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DISCUSSION
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Coordinated gastrointestinal motility requires a complex interaction between several cell types, including nerves, smooth muscle cells, and ICC. Different classes of ICC are present in the gastrointestinal tract and appear to subserve different functions. At least three different functions have been ascribed to ICC (1, 9, 20, 2325). ICC act to modulate neuronal signals to smooth muscle cells (1, 23, 25), may serve as mechanotransducers (20), and generate and amplify the pacemaker signal and the slow wave (9, 24). The slow wave recorded from smooth muscle cells originates from ICC and is an important determinant of smooth muscle function, with the frequency of the slow wave setting the frequency of smooth muscle contraction, and the amplitude of the slow wave contributing to the control of the strength of smooth muscle contractions. The rhythmic depolarization of smooth muscle membrane potential opens L-type Ca2+ channels expressed in smooth muscle allowing Ca2+ entry and initiation of contractions (4).
Ionic conductances that underlie the pacemaker signal and upstroke of the slow wave are not well understood and appear to vary between species. In cultured mouse small intestinal ICC, both chloride channels (10, 26) and Ca2+-permeable nonselective cation channels of the TRP family have been proposed as the pacemaker channel (22). In dogs (13) and mice (11), a T-type channel has been reported to be expressed in ICC and in humans, a Na+ channel (20). The Na+ channel is not the pacemaker channel but contributes to the upstroke of the slow wave and its regulation by stretch (19, 20). With several different candidates with overlapping electrophysiological properties, it is difficult to interpret intact muscle strip experiments, yet these experiments are necessary to integrate information obtained at a channel level often under culture conditions that may alter the expression of ion channels. Pharmacological and electrophysiological experiments at the muscle strip level are therefore often needed, and drugs that discriminate between the candidate ion channels are required. QX314 is a quaternary membrane-impermeant derivative of lidocaine that is used as a Na+ channel blocker (3, 7, 21) and when applied externally has different effects on neuronal and cardiac Na+ channels, including Nav1.5 (2, 8, 20). QX314 is therefore useful to discriminate between nonselective cation channels, Ca2+-selective channels, and Na+ channels; however, QX314 at 500 µM also inhibits about 30% of T-type Ca2+ current (unpublished data) and its mechanism of action requires a prolonged application time (8, 20). Mibefradil has been used as a selective T-type Ca2+ channel blocker (11, 12), and the data provided in this report and the literature (summarized in Ref. 15) suggests that at concentrations below 1 µM it indeed can discriminate between
1C-L-type and the
13.3b-T-type Ca2+ channels.
Published data for the IC50s (in 2 mM Ca2+) for
13.1 (
1G, 0.27 µM) and
13.2 (
1H, 0.14 µM) also suggest that mibefradil can discriminate between T-type Ca2+ channels containing other
-subunits and
1C-L-type Ca2+ channels (15). However, the data from this report and a recently published report (16) also suggest that, with an IC50 of 0.29 µM for the
13.3b-T-type Ca2+ channel and an IC50 of 0.98 µM for Nav1.5, mibefradil cannot be used to discriminate between the two channel types, and caution must be used in the interpretation of the effects of mibefradil as a blocker of T-type Ca2+ channels when, as is nearly always the case, more than one channel type is expressed in a given cell type.
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GRANTS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52766 and DK-57061.
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ACKNOWLEDGMENTS
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We thank Dr. Perez-Reyes for providing us with the
13.3b-subunit clone and Kristy Zodrow and Beverly Colbenson for secretarial assistance.
Present address of Y. Ou: Department of Immunology, Mayo Clinic, Rochester, MN 55905.
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FOOTNOTES
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Address for reprint requests and other correspondence: G. Farrugia, 8 Guggenheim Bldg., Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905 (e-mail: farrugia.gianrico{at}mayo.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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