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A Fluorescence-Based High-Throughput Screening Assay for the Identification of T-Type Calcium Channel Blockers

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A Fluorescence-Based High-Throughput Screening Assay for the Identification of T-Type Calcium Channel Blockers
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  266    ASSAY  and Drug Development Technologies    JUNE 2009 DOI: 10.1089/adt.2009.191 ORIGINAL  ARTICLE Francesco Belardetti, 1, * Elizabeth Tringham, 1  Cyrus Eduljee, 1   Xinpo Jiang, 1  Haiheng Dong, 1, ** Adam Hendricson, 1,†   Yoko Shimizu, 1,‡   Diana L. Janke, 1,§   David Parker, 1  Janette Mezeyova, 1  Afsheen Khawaja, 1  Hassan Pajouhesh, 1  Robert A. Fraser, 1,‡   Stephen P. Arneric, 1,#   and Terrance P. Snutch 2 1 Neuromed Pharmaceuticals Ltd., Vancouver, British Columbia, Canada. 2  Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.*Present address: Panora Pharmaceuticals Inc., Vancouver, British Columbia, Canada.** Present address: WuXi Pharmatech, Shanghai, China. †  Present address: Bristol-Myers Squibb, Wallingford, Connecticut. ‡  Present address: Centre for Drug Research and Development, University of British Columbia, Vancouver, British Columbia, Canada. §  Present address: WorkSafeBC, Burnaby, British Columbia, Canada. #  Present address: Eli Lilly & Company, Indianapolis, Indiana.  ABSTRACT T-type voltage-gated Ca 2+  channels have been implicated in contributing to a broad variety of human disorders, including pain, epilepsy, sleep disturbances, cardiac arrhythmias, and certain types of cancer. However,  potent and selective T-type Ca 2+  channel modulators are not yet available  for clinical use. This may in part be due to their unique biophysical  properties that have delayed the development of high-throughput screening (HTS) assays for identifying blockers. One notable challenge is that at the normal resting membrane potential (  V  m  ) of cell lines commonly utilized for drug screening purposes, T-type Ca 2+ channels are largely inactivated and thus cannot be supported by typical formats of functional HTS assays to both evoke and quantify the Ca 2+  channel signal. Here we describe a simple method that can successfully support a fluorescence-based functional assay for compounds that modulate T-type Ca 2+ channels. The assay functions by exploiting the pore- forming properties of gramicidin to control the cellular  V  m  in advance of T-type Ca 2+  channel activation. Using selected ionic conditions in the presence of gramicidin, T-type Ca 2+  channels are converted from the unavailable, inactivated state to the available, resting state, where they can be subsequently activated by application of extracellular K  + . The fidelity of the assay has been pharmacologically characterized with sample T-type Ca 2+ channel blockers whose potency has been determined by conventional manual patch-clamp techniques. This method has the  potential for applications in high-throughput fluorometric imaging plate reader (FLIPR®, Molecular Devices, Sunnyvale, CA)    formats with cell lines expressing either recombinant or endogenous T-type Ca 2+ channels. INTRODUCTION oltage-gated Ca 2+  channels are integral membrane proteins that play unique roles in regulating intracellular Ca 2+  levels and are a key factor in the regulation of muscle contraction, neuronal excitability, neurotransmitter and hormone secretion, and gene expression. 1,2  Ca 2+  channels have been generally classified by their electrophysiological and pharmacological properties as either high-voltage activated (HVA) ABBREVIATIONS:   DMSO, dimethyl sulfoxide; E  K , potassium equilibrium potential; FLIPR® (Molecular Devices, Sunnyvale, CA), fluorometric imaging plate reader; Fluo-4-AM, Fluo-4 acetoxymethyl ester; hCa  V  3.1, hCa  V  3.2, and hCa  V  3.3, human Ca  V  3.1, Ca  V  3.2, and Ca  V  3.3, respectively; HEK, human embryonic kidney; HVA, high voltage-activated Ca 2+  channel; IC 50 , concentration producing half-maximal inhibition; [K + ] i , intracellular potassium concentration; [K + ] o , extracellular potassium concentration; LVA, low voltage-activated Ca 2+  channel; NMDG, N  -methyl- D -glucamine; RLU, relative light units; V  1/2 , membrane potential at which half the channels are activated or inactivated; V  con , amplitude of conditioning prepulse potential;  V  h , holding potential; V  m , membrane potential; V  test , amplitude of test pulse potential.  A Fluorescence-Based High-Throughput Screening  Assay for the Identification of T-Type Calcium Channel Blockers   V The first two authors contributed equally to this work.  © MARY ANN LIEBERT, INC. • VOL. 7 NO. 3 • JUNE 2009  ASSAY  and Drug Development Technologies 267  AUTOMATED SCREENING FOR T-TYPE CALCIUM CHANNEL BLOCKERS (L-, N-, P/Q-, and R- types) or low-voltage activated (LVA) (T-types) Ca 2+  channels. The various physiological classes of Ca 2+  channels are generated by a family of 10 different α 1  subunit proteins that largely determine channel properties (Ca  V  1.1–1.4, Ca  V  2.1–2.3, Ca  V  3.1–3.3). The HVA Ca 2+  channel α 1  subunits are associated with various α 2 δ , β , and γ   auxiliary subunits that modulate channel biophysical properties and trafficking, whereas T-type Ca 2+  channel properties can be fully reconstituted with any of the three distinct α 1  subunits alone: Ca  V  3.1, Ca  V  3.2, and Ca  V  3.3. 1,3–6  T-type Ca 2+  channels display several unique biophysical properties that readily distinguish them from HVA Ca 2+  channels and underlie their distinct physiological functions. These distinguishing T-type Ca 2+ channel properties include: (a) the midpoints of both the voltage dependence of channel activation and inactivation curves are substantially more negative than those for HVA Ca 2+  channels, such that a large proportion of channels are inactivated at the resting membrane potential ( V  m ) of excitable cells; (b) the voltage dependences of channel activation and inactivation profiles are overlapping, resulting in the generation of T-type Ca 2+  channel “window currents” at the voltages where the two relationships intersect; (c) fast and complete inactivation at positive potentials; (d) rapid recovery from inactivation; and (e) slow deactivation of open channels, generating characteristically long “tail currents” during repolarizations. Overall, these “window” and “tail” currents are responsible for steady-state and transient intracellular Ca 2+  increases, which in turn affect both cellular excitability and voltage-dependent Ca 2+ -second messenger processes. For example, angiotensin II-mediated aldosterone secretion from the surrenal cortex appears to be mediated via the window currents of Ca  V  3.2 channels. 7,8 Transient membrane hyperpolarizations generated during the normal activity of excitable cells, such as inhibitory postsynaptic potentials and after hyperpolarizations, are sufficient to first remove T-type Ca 2+ channel inactivation and then trigger rebound, T-type Ca 2+  channel-dependent low threshold spikes, which in turn may engage fast Na +  channels to generate full-size action potentials. 6  Depending upon the specific kinetic properties of the T-type Ca 2+  channels involved, together with other co-expressed  voltage-gated channels, low threshold spikes result in either burst firing or steady pacemaker activity. Examples include low threshold spike-mediated burst firing in thalamic relay neurons and their regulation of sleep patterns, 9,10  T-type Ca 2+  channel-dependent pacemaker activity supporting basal dopamine secretion in a subset of nigral dopaminergic cells, 11  and a contribution of T-type channels to the normal cardiac rhythm. 12 This unique role of T-type Ca 2+  channels in regulating Ca 2+  influx and cellular excitability, coupled to their wide distribution, implicates them in a variety of pathophysiological states. For example, knock-out of the Ca  V  3.1 gene leads to both enhanced and reduced pain sensitivity, depending on the modality examined, 13,14  as well as suppression of absence epilepsy. 15  Regional knock-down of Ca  V  3.2 expression in rats reduces chronic neuropathic and acute pain, 16  a discovery later expanded through other approaches, including conventional knock-out in mice. 17–20  Developmental Ca  V  3.2 gene knock-out also leads to endothelium-dependent coronary vasodilation and accompanying cardiovascular malformations. 21  Moreover, certain point mutations in the human Ca  V  3.2 gene have been implicated in idiopathic generalized epilepsy. 22,23  Also, a  variety of experimental approaches suggest a role of T-type Ca 2+ channels in kidney microcirculation 24  and certain forms of cancer. 25,26  Interestingly, several established drugs have been found to block T-type Ca 2+  channels in a nonselective fashion, suggesting a possible role of T-type Ca 2+  channels in their therapeutic actions. Included among these are the antihypertensive mibefradil (now withdrawn from the market because of potential for off-target drug–drug interactions 27,28 ) and certain neuroleptics 29  and anticonvulsants. 30  Together, these data strongly support T-type Ca 2+  channels as attractive targets for therapeutic intervention. 25,28,31–35 To take advantage of the therapeutic potential of selective T-type Ca 2+  channel blockers, a functional HTS assay has been developed. The assay includes a step that generates a membrane hyperpolarization of sufficient amplitude and duration to remove T-type Ca 2+  channel inactivation and make the channels available for activation. To this purpose, the assay employs gramicidin, a linear pore-forming pentadecapeptide antibiotic selective for small monovalent cations with permeability sequence H +  > NH 4+  > Cs +  > Rb +  ≥ K  +  > Na +  > Li +  >> tetramethylammonium + . 36–39  Upon extracellular addition to human embryonic kidney (HEK) cells stably expressing cloned T-type Ca 2+  channels, gramicidin readily inserts into the plasma membrane and forms pores capable of conducting ions. Using the appropriate ionic conditions, gramicidin pores can be manipulated to solely conduct K  +  ions, thereby driving the cell V  m  close to the predicted potassium equilibrium potential ( E  K  ) and remove the inactivation of T-type Ca 2+ channels. Under these conditions, a subsequent increase in extracellular potassium concentration ([K  + ] o ) leads to T-type Ca 2+  channel activation and Ca 2+  influx. By preloading the HEK cells with a Ca 2+ -sensitive dye to measure Ca 2+  entry into the cell, under these conditions an effective fluorescence-based T-type Ca 2+ channel assay has been established and pharmacologically characterized. In comparison to alternative protocols, this approach offers a simple and inherently flexible assay that could be adapted to any cell line and even primary cells in culture.  268    ASSAY  and Drug Development Technologies    JUNE 2009 BELARDETTI et al. MATERIALS AND METHODS Construction of Stable Cell Lines Expressing Mammalian T–Type Channels Human Ca V  3.1 (hCa V  3.1).  A full-length hCa  V  3.1 cDNA construct was initially generated from five overlapping cDNA fragments isolated from a human thalamus cDNA library (Clontech, Mountain View, CA) and then subcloned into the mammalian expression vector pcDNA3.1/Zeo(+) (Invitrogen, Carlsbad, CA). DNA sequencing of the pcDNA3.1Zeo(+)/hCa  V  3.1 ae  construct confirmed the integrity of the full-length hCa  V  3.1 cDNA. The pcDNA3.1Zeo(+)/hCa  V  3.1 ae  was transfected into HEK-tsa201 cells using standard Ca 2+ -phosphate methodology, and the stable transfectants were selected and clonally expanded in complete growth medium consisting of 10% fetal bovine serum in Dulbecco’s Modified Eagle’s Medium (high glucose containing D -glucose, L -glutamine, and Na +  pyruvate [Invitrogen]), supplemented with 100 μ g/ml zeocin (Invitrogen) in a 5% CO 2 humidified incubator at 37 ° C over a 12–14-day period. Positive clones identified using Southern blotting were functionally characterized by manual whole-cell patch-clamp electrophysiology to confirm T-type Ca 2+ channel expression. The Ca  V  3.1 ae -expressing cell line was maintained at 80% confluence in a complete growth medium supplemented with 25 μ g/mL zeocin. Human Ca V  3.2 (hCa V  3.2).  A full-length sequence-verified hCa  V  3.2 cDNA construct was generated from seven overlapping cDNA fragments isolated from human fetal brain total RNA and a human thalamus cDNA library (Clontech) using reverse transcriptase-polymerase chain reaction and cDNA library screening and cloning techniques and then subcloned into pcDNA3.1/Zeo(+). DNA sequencing of the pcDNA3.1Zeo(+)/hCa  V  3.2 construct confirmed the integrity of the full-length hCa  V  3.2 cDNA. After transfection of pcDNA3.1Zeo(+)/hCa  V  3.2 into HEK-293 cells using Lipofectamine™ transfection reagent (Invitrogen), stable transfectants were selected and clonally expanded as described for the Ca  V  3.1 cell line. The positive clones identified by northern blot were functionally characterized by whole-cell patch-clamp electrophysiology, and the lines were maintained as described for the Ca  V  3.1 cell line. Human Ca V  3.3 (hCa V  3.3).   A full-length hCa  V  3.3 cDNA construct was generated from six overlapping cDNA fragments isolated from human hippocampus polyA+ RNA (Clontech) by reverse transcriptase-polymerase chain reaction and then subcloned into pcDNA5/FRT isogenic expression vector (Invitrogen), producing pcDNA5/FRT/hCa  V  3.3. DNA sequencing confirmed the integrity of the full-length hCa  V  3.3 cDNA construct. The pcDNA5/FRT/hCa  V  3.3 was transfected into Flp-In™-293 cells (Invitrogen) using FuGENE® 6 transfection reagent (Roche Applied Science, Laval, QC, Canada). Stable transfectants were selected and clonally expanded in complete growth medium supplemented with 100 µg/mL zeocin and 200 µg/mL hygromycin (Invitrogen) for 12–14 days. The positive clones identified by northern blot were functionally characterized by whole-cell patch-clamp electrophysiology, and the lines were maintained as described for the Ca  V  3.1 cell line with the addition of 200 µg/mL hygromycin in the medium. Preparation of Compounds The T-type Ca 2+  channel blocking test compounds as well as  valinomycin and gramicidin were dissolved in dimethyl sulfoxide (DMSO) at 5–30 m M   and stored in aliquots at -80 ° C. On the day of the experiments, aliquots were thawed and diluted to their final concentration in either fluorometric imaging plate reader (FLIPR®, Molecular Devices, Sunnyvale, CA) loading buffer for the fluorescence assay or extracellular patch-clamp buffer for the manual patch-clamp measurements ( Table 1 ). All samples were used on the same day as being prepared. The maximal final concentration of DMSO to which cells were exposed was 0.2% for both patch-clamp and FLIPR assays. Current-Clamp Voltage Recordings Prior to recording V  m , the culture medium was replaced with extracellular current-clamp buffer ( Table 1 ). Borosilicate glass patch pipettes, pulled on a P-97 micropipette puller (Sutter Instruments, Novato, CA), with typical resistances of 2–4 M   were backfilled with intracellular current-clamp buffer ( Table 1 ).  Voltages were recorded in the whole-cell configuration at room temperature (~21 ° C) using an Axopatch 200B (Molecular Devices) patch-clamp amplifier. Recordings were low-pass-filtered at 1 kHz (-3 dB four-pole Bessel filter), digitized at 2 kHz with a Digidata 1322A interface (Molecular Devices), and acquired using pClamp version 9.2 (Molecular Devices). Data were analyzed by comparing empirically derived V  m  values with a calculated E  K obtained using the Nernst equation (Eq. 1), where R  is the universal gas constant (1.987 cal/mol/K), T   is the absolute temperature, F   is the Faraday constant (9.6485 ×  10 4  cal/mol), and [K  + ] i  is the intracellular potassium concentration: (1)  E  K  =  RT F  ln[K + ] o [K + ] i Patch-Clamp Current Recordings Prior to recording T-type Ca 2+  currents, the culture medium was replaced with extracellular patch-clamp buffer ( Table 1 ). Recordings were made similarly to those in current-clamp mode except that patch pipettes were backfilled with an intracellular patch-clamp buffer ( Table  © MARY ANN LIEBERT, INC. • VOL. 7 NO. 3 • JUNE 2009  ASSAY  and Drug Development Technologies 269  AUTOMATED SCREENING FOR T-TYPE CALCIUM CHANNEL BLOCKERS 1 ) and leak and uncompensated capacitance currents were subtracted on-line with a P/4 protocol. Test compounds were applied through a gravity-driven multibarrelled array of capillaries (24 gauge), connected to reservoirs controlled by solenoid valves. Unless otherwise noted, T-type Ca 2+ channel currents were evoked by applying 50-ms square pulses to -40 mV from a holding potential ( V  h ) of -110 mV every 15 s. Data were analyzed and fitted using OriginPro version 7.5 (OriginLab, Northampton, MA) software. Experimental measurements of peak T-type Ca 2+ channel currents were fitted using the applicable equations: logistic fit to obtain values for concentration producing half-maximal inhibition (IC 50 ) (Eq. 2) or Boltzmann function for determining the  voltage dependence of channel activation and inactivation (Eq. 3): (2)   y =  max − min1 +  [ drug ]  IC  50       n H     + min  (3)    y =  max − min1 + e ( V  m − V  h  )/  k      Ca 2+ –Dependent Fluorescence Measurements Two days prior to conducting the assay, HEK cells stably expressing Ca  V  3.1, Ca  V  3.2, and Ca  V  3.3 were seeded into poly- D -lysine-coated 384-well clear-bottom plates (catalog number 356663, BD Biosciences, San Jose, CA) at ~14,000–20,000 cells per well (100 µl per well) using a 384 Multidrop dispenser (Thermo Scientific, Waltham, MA). The cell plates were incubated in a 5% CO 2 humidified incubator at 37°C and 1 day before conducting the assay were shifted to an incubator at 29°C in 5% CO 2 .On the day of the assay, the plates were washed with FLIPR loading buffer ( Table 1 ) using a plate washer (Bio-Tek Instruments, Winooski,  VT). A stock solution of 1 m M   Fluo-4 acetoxymethyl ester (Fluo-4- AM) in DMSO (catalog number F14202, Invitrogen) was diluted to 0.5 m M   with 20% (wt/vol) Pluronic-F127 in DMSO (catalog number Table 1. Composition of Solutions Extracellular patch-clamp bufferIntracellular patch-clamp bufferExtracellular current-clamp bufferNa + -free, extracellular current-clamp bufferIntracellular current-clamp bufferFLIPR loading bufferFLIPR data acquisition buffer NaCl132.5118KCl221304.72CsCl142Cs-methanesulfonate126.5MgSO 4 0.5KH 2 PO 4 1.2Glucose105511.75CaCl 2 26621MgCl 2 121111HEPES101016161018.416EGTA115Choline chloride132.5 a 140Na-ATP  a 2K-ATP  a 5Osmolarity (mOsm)  b 300295300300295300300pH 7.4  c 7.3  c 7.4  d 7.4  f  7.3  e 7.2  d 7.4  d Composition is given in m M unless otherwise noted. The high K +  solution used to elicit the FLIPR fluorescence signal (activating solution) was obtained by isotonically replacing a portion of choline chloride with KCl. a From 100 mM stock solution in the relevant intracellular solution, adjusted to pH 7.3 with CsOH or KOH, respectively, and stored at -80ºC, b adjusted with sucrose; c with CsOH; d with NaOH; e with KOH; f  in some experiments 132.5 mM NMDG replaced NaCl and pH was adjusted with HCl.          270    ASSAY  and Drug Development Technologies    JUNE 2009 BELARDETTI et al. P6867, Invitrogen). Using the FLIPR loading buffer, this Fluo-4- AM solution was then diluted to a final concentration of 5.5 µ M   in the cell-containing plate. Cells were incubated in the Fluo-4-AM solution for 45 min at 29°C in 5% CO 2 . The procedure for applying the test compounds and the specific requirements for the assay are described in Results. Ca 2+ -dependent fluorescence measurements were performed on the cell plate using the FLIPR TETRA  ® (Molecular Devices) or in initial experiments a Fluoroskan Ascent (Thermo Scientific). The illumination wavelength of the FLIPR light-emitting diode module was 470–495 nm, and the emissions were recorded at 515–575 nm. For the Fluoroskan platform, the excitation was performed at 485 nm, and the emissions read-out was at 518 nm.  Assay quality assessment was achieved by the implementation of the Z  -factor (Eq. 4) 40 : (4)   Z   = 1 − 3 SD sample  +  3 SD control mean sample  − mean control  Data are expressed as mean and SD values. RESULTS Biophysical and Pharmacological Properties of T–Type Ca 2+  Channels Stably Expressed in HEK Cells The cell lines expressing the three T-type Ca 2+  channel isoforms were characterized based on their efficiency of channel expression and fidelity of the channel biophysical and pharmacological properties as compared to the profiles previously reported for Ca  V  3.1, Ca  V  3.2, or Ca  V  3.3. 6,29,41–44  Whole-cell patch-clamp recordings from all three cell lines were routinely obtained, confirming the presence of functional inward Ca 2+  currents for each isoform. On average, cells expressing Ca  V  3.1 and Ca  V  3.2 channels displayed relatively large inward currents of 2.0 ± 1.5 and 1.7 ± 1.5 nA, respectively, at the peak of the current–voltage relationship curve (data not shown). In contrast, Ca  V  3.3-expressing cells on average yielded a smaller peak current of 0.7 ± 0.6 nA. Representative traces for each channel are shown in Fig. 1A . Following a similar pattern, the density of channel expression in the Ca  V  3.3 cell line was lower than that of either Ca  V  3.1 or Ca  V  3.2: Ca  V  3.1 = 120.8 ± 87.3 pA/pF; Ca  V  3.2 = 72.1 ± 55.6 pA/pF; Ca  V  3.3 = 36.4 ± 30.2 pA/pF ( Fig. 1B , n  = 20 cells per cell line).The voltage dependence of channel activation was examined for each channel type by constructing conductance–voltage relationship curves from normalized, average peak current–voltage relationship curves after adjusting for the driving force at each voltage tested ( Fig. 1C  , open circles). The fit of the voltage dependence of channel activation data with a Boltzmann function yielded a membrane potential at which half the channels are activated or inactivated  (V  1/2 ) of -55.3 ± 0.3 mV with a slope of 3.8 mV for Ca  V  3.1, a V  1/2  of -45.6 ± 0.1 mV with a slope of 5.0 mV for Ca  V  3.2, and a V  1/2 of -48.7 ± 0.1 mV with a slope of 3.4 mV for Ca  V  3.3. The voltage dependence of channel steady-state inactivation was obtained for each channel isoform by varying the amplitude of the conditioning prepulse potential   ( V  con ) preceding a fixed amplitude of test pulse potential   ( V  test ) and plotting the values of the evoked peak inward currents, normalized to the peak control amplitude, as a function of V  con  ( Fig. 1C  , solid circles). A fit of the voltage dependence of inactivation data with a Boltzmann function yielded a V  1/2 of -69.3 ± 0.2 mV with slope -4.1 mV, V  1/2  of -60.7 ± 1.8 mV with slope -4.5 mV, and V  1/2 of -61.9 ± 0.2 mV with slope -5.5 mV for Ca  V  3.1, Ca  V  3.2, and Ca  V  3.3, respectively. These values are similar to those reported in the literature for human T-type Ca 2+  channels under comparable experimental conditions, indicating that the cell lines stably express T-type Ca 2+  channels with the expected biophysical properties. 6,42 Initially, in order to evaluate the pharmacological profile of the expressed channels, the effects of established T-type Ca 2+  channel blockers mibefradil, penfluridol, and flunarizine were assayed. 29,43,44   Whole-cell current recordings were obtained while cells were perfused with increasing concentrations of test compounds. For each T-type Ca 2+  channel isoform, the value of the peak current after reaching steady-state block was plotted as a function of the concentration of test compound present in the bath, after normalization to the baseline peak current before block. Penfluridol was the most potent blocker among those tested, followed by flunarizine for Ca  V  3.1 and Ca  V  3.3 and mibefradil for Ca  V  3.2 ( Table 2 ). The IC 50  values for penfluridol based on logistic fits of the data were 10 n M   for Ca  V  3.1, 42 n M   for Ca  V  3.2, and 57 n M   for Ca  V  3.3 ( Fig. 2 ). These values are in qualitative agreement with previously reported values, further demonstrating the suitability of the cell lines for use in screening assays ( Table 2 ). 29,41,43,44 Gramicidin-Induced Hyperpolarization and T-Type-Dependent Ca 2+  Channel-Induced Fluorescence Signals In standard fluorescence-based high-throughput assays the V  m  parameter is not under experimental control; thus the spontaneous V  m  determines the extent to which T-type Ca 2+ channels are available for activation. The whole-cell current-clamp technique was used to measure the V  m  of untransfected HEK cells. Under basal conditions HEK cells displayed a spontaneous V  m  between -40 and -20 mV ( Figs. 3A–C  ), consistent with previous findings. 45  Within this relatively positive V  m  range, the T-type Ca 2+  channel isoforms are mostly in the inactivated state, making them unavailable for activation (compare the spontaneous V  m  in Fig. 3A–C   with the values of steady-state inactivation between -40 and -20 mV in Fig. 1C  ). In these HEK cells, a low spontaneous V  m  in the presence of a steep K  +  gradient ([K  + ] i  = 130 m M  , [K  + ] o  = 2 m M  ; see Table 1 ) suggests a modest contribution of K  +  channels to this parameter, and therefore changes in [K  + ] o  have only a limited effect on V  m  (data not shown). Given these conditions, we
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