Structural and biophysical determinants of single CaV3.1 and CaV3.2 T-type calcium channel inhibition by N2O

Cell Calcium 46 (2009) 293–302

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Structural and biophysical determinants of single CaV 3.1 and CaV 3.2 T-type calcium channel inhibition by N2 O Peter Bartels a,1 , Kerstin Behnke a,1 , Guido Michels c , Ferdi Groner a , Toni Schneider d , Margit Henry d , Paula Q. Barrett e , Ho-Won Kang f , Jung-Ha Lee f , Martin H.J. Wiesen a , Jan Matthes a , Stefan Herzig a,b,∗ a

Department of Pharmacology, University of Cologne, Germany Center for Molecular Medicine, University of Cologne, Germany Department of Internal Medicine III, University of Cologne, Germany d Institute of Neurophysiology, University of Cologne, Germany e Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA f Department of Life Science, Sogang University, Seoul 121-742, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 13 January 2009 Received in revised form 2 September 2009 Accepted 4 September 2009 Available online 26 September 2009 Keywords: Single-channel analysis CaV 3.1 CaV 3.2 T-type calcium channel Nitrous oxide

a b s t r a c t We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels CaV 3.1 and CaV 3.2 by nitrous oxide (N2 O). To identify functionally important channel structures, chimeras with reciprocal exchange of the N-terminal domains I and II and C-terminal domains III and IV were examined. In whole-cell recordings N2 O significantly inhibited CaV 3.2, and – less pronounced – CaV 3.1. A CaV 3.2prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cellattached patches containing ≤3 channels N2 O reduced average peak current of CaV 3.2 by decreasing open probability and open time duration. Effects on CaV 3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single CaV 3.1 channels were significantly less active than CaV 3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within CaV 3.2 (H191), are responsible for the subtype-prevalent N2 O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N2 O on CaV 3.2. Such a fingerprint of single channels can help identifying the molecular nature of native channels. This is exemplified by a characterization of single channels expressed in human hMTC cells as functional homologues of recombinant CaV 3.1. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction T-type calcium channels are encoded by three genes, CaV 3.1, CaV 3.2 and CaV 3.3 [1]. T-type calcium channels are involved in diverse physiological functions, including cardiac and neuronal pacemaking [2,3]. Stress conditions like hypoxia were shown to regulate expression of T-type calcium channels [4,5] and the relevance of these channels for certain disease states of the central and peripheral nervous system is increasingly appreciated [6,7]. More specifically, upregulation of CaV 3.2 channels in dorsal root ganglion neurons emerges as a common pathophysiological mechanism of neuropathic pain of different origin [8–10].

∗ Corresponding author at: Department of Pharmacology and Center of Molecular Medicine, University of Cologne, Gleueler Strasse 24, 50931 Koeln, Germany. Tel.: +49 221 478 6064; fax: +49 221 478 89049. E-mail address: [email protected] (S. Herzig). 1 These authors contributed equally to this work. 0143-4160/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2009.09.002

CaV 3.2 therefore could be a promising analgesic drug target [11]. Of note, several compounds have been detected to inhibit CaV 3.2 in a subtype-selective manner, including Ni2+ [12], Zn2+ [13], ascorbate [14], and nitrous oxide [15]. This subtype-selective action is dependent upon a histidine residue (H191) contained within domain I on an extracellular loop of the channel which is not conserved in CaV 3.1 and CaV 3.3 [14,16–18]. In a previous study on the single-channel mechanism of Ttype calcium channel block, we identified a dual inhibitory action of mibefradil, a non-selective T-type inhibitor [19]. In native and recombinant CaV 3.2 channels, this drug reduced the fraction of depolarizing pulses causing channel openings, indicative of longterm (s) effects on channel availability. In addition, it shortened the duration of individual openings, consistent with rapid (ms) “open-channel” block. The aim of this study was to identify the biophysical mechanism(s) of subtype-selective block of CaV 3.2. A more detailed understanding of channel function in the presence of a drug should aid in the design of new therapeutically useful subtypeselective compounds. In addition, unlike standard whole-cell

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analysis, a combination of single-channel analysis and subtypeselective pharmacology may aid in defining a fingerprint of CaV 3.1 and CaV 3.2, and hence help to clarify the molecular identity of endogenous channels. For practical reasons, we chose N2 O as the most promising agent to be examined. First because N2 O acts as a general anesthetic agent, we did not expect major problems for the drug to reach its site of action (presumably the extracellular face of the channel) when applied during cell-attached recording. Second, due to its volatile nature, “wash-in” and “wash-out” procedures seemed feasible under recording conditions that maintained the signalto-noise conditions required to resolve unitary T-type currents. Finally, because this compound is a clinically useful analgesic, we felt convinced that at feasible N2 O concentrations the extent of CaV 3.2 inhibition attained would be therapeutically relevant. Thus we first tested and validated that N2 O can be applied under experimental conditions amenable to low-noise single-channel recording. Next, we examined the biophysical single-channel mechanisms of inhibition and finally focused on subtypeselectivity using recombinant human CaV 3.2 and CaV 3.1 channels. To address the role of channel structure regarding N2 O inhibition, we also examined two chimeric constructs with reciprocal exchange of the N-terminal (domains I and II) and C-terminal (domains III and IV) portion between CaV 3.1 and CaV 3.2, and a point-mutant lacking the histidine residue mentioned above (H191Q). 2. Materials and methods 2.1. Cell culture and CaV isoforms Culturing of human embryonic kidney HEK-293 cells, and of hMTC, a human medullary thyroid carcinoma cell line was done as described [19]. Stably transfected HEK-293 cells were used with either human CaV 3.1 (accession number, AF190860 [20]), or human CaV 3.2 (accession number, AF051946 [21]) containing a pcDNA3 vector carrying a penicillin/aminoglycoside resistance. Native HEK293 cells were transiently transfected with the same plasmids and GFP, using Superfect® or Effectene® reagent (both from Quiagen, Hilden, Germany) according to manufacturer’s instructions. For another set of experiments native HEK-293 cells were transiently transfected with the CaV 3.2 mutant H191Q in pcDNA3 (kindly provided by Prof. E. Perez-Reyes) using Effectene® reagent [16]. Transfection preparation was incubated for 4–24 h at 37 ◦ C, by 6% CO2 gassing. HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium (Biochrom KG) supplemented with fetal bovine serum (Sigma) and G418 (0.1%; Invitrogen, Karlsruhe, Germany) in case of stably transfected HEK-cells. Cells were used 1–3 days after plating on polystyrene dishes. 2.2. Construction of CaV 3 chimeras For creation of the construct GGHH (composed of domains I and II of CaV 3.1 and domains III and IV of CaV 3.2), CaV 3.2 (AF051946 modified, 7768 bp [22]) was digested by BamHI, cutting within the polylinker region and at positions 729 and 6184. CaV 3.1 (AF190860, 7349 bp [20]) was cut by BamHI at positions 560, 3863 and 6858. In case of the second construct HHGG (composed of domains I and II of CaV 3.2 and domains III and IV of CaV 3.1), HindIII cut at a site within the linker region between domain II of CaV 3.2 (AF051946 [21]) and domain III of CaV 3.1 (AF027984 [23]) [16,24]. The full-length cDNA of HHGG was constructed by ligating ClaI (5 -polylinker)PvuI (3725, CaV 3.2), PvuI (3728, CaV 3.2)-HindIII+ (3963, CaV 3.2), HindIII+ (4246, CaV 3.1)-KpnI (6170, CaV 3.1) into pcDNA3-CaV 3.1, which was opened by ClaI (5 -polylinker) and KpnI (6170, CaV 3.1).

Chimeric constructs were fused into the pcDNA3 plasmid and used for transient transfection. The resulting plasmids were verified by sequencing. 2.3. Reverse-transcription PCR mRNA was isolated from hMTC cells and amplified after reversetranscription as described [25]. In brief, subtype-selective primers for CaV 3.1 (forward 5 -AGCCCCGGTTTCTTCTA-3 , reverse 5 TGAGCGGTCGAGCACAC-3 , expected size: 397 bp) and CaV 3.2 (forward 5 -CCCCGTCGCCCGTCTACTTCGTGA-3 , reverse 5 GGTGCCGGCCCCATAGGTCTCCAT-3 , expected size of product 380 bp) were used for 35 PCR cycles (94 ◦ C, 30 s; 60 ◦ C, 30 s; 72 ◦ C, 1 min), starting with an initial 15 min heating step at 95 ◦ C and with a final elongation period of 10 min at 72 ◦ C. The CaV 3.1 and CaV 3.2 amplification products were validated by direct sequencing. 2.4. Electrophysiological methods Whole-cell Ba2+ -currents were elicited by depolarizing test pulses delivered at 0.3 Hz, recorded at 5 kHz and filtered at 2 kHz (−3 dB, 4-pole Bessel) using an Axopatch 200A amplifier (Axon Instruments, Union City, CA, USA). Whole-cell bath solution contained [mM]: 130 NaCl, 10 BaCl2 , 1 MgCl2 , 10 glucose and 10 HEPES, pH 7.4 (NaOH). Pipette solution was composed of [mM]: 120 CsCl, 10 MgCl2 , 10 HEPES, 10 EGTA, 4 Mg-ATP, pH 7.2 (CsOH). Resistance of borosilicate pipettes was 5–7 M. Series resistance and capacitance values were taken directly from the amplifier. Series resistance was compensated to the maximum possible extent that still avoided current oscillation. Application of voltage commands and digitization of membrane currents were performed using Clampex 6.0 of the pClamp software (Axon Instruments, Union City, CA, USA) running on a personal computer. Cells were held at −110 mV and depolarized to respective maximum peak inward current (−30 to −10 mV) to check for current response over time. To analyze the current–voltage relationship, cells were typically held at a holding potential of −110 mV and pulsed to test potentials of −100 mV up to +50 mV, with increments of 10 mV applied every 3 s. Data were analyzed using Clampex 6.0 (Axon Instruments) and Origin 6.0 (Microcal Software, Northampton, MA, USA). Single calcium channels were recorded in the cell-attached configuration of the patch-clamp technique. Data were filtered at 2 kHz (−3 dB, 4-pole Bessel) and acquired at 10 kHz using an Axopatch 1D amplifier (Axon Instruments). Using holding potentials (HP) of −90 mV or −50 mV, patches were depolarized to a test potential of −20 mV for 150 ms at 0.5 Hz [19]. The calculated liquid junction potential was −18 mV in cell-attached experiments (data given in this work are not corrected for this value). Experiments were performed in an external solution containing [mM]: 120 K-glutamate, 25 KCl, 2 MgCl2 , 10 HEPES, 2 EGTA, 1 CaCl2 , 1 Na2 -ATP, 10 glucose, pH 7.4 (KOH). Pipettes (borosilicate glass, 6–7 M) were filled with 110 mM BaCl2 and 10 mM HEPES, pH 7.4 (TEA-OH). To reduce thermal noise, pipettes were coated using Sylgard® (Dow Corning GmbH, Wiesbaden, Germany). The pClamp software (Clampex 5.5, Fetchan 6.0, and pSTAT 6.0, Axon Instruments) and Origin 6.0 (Microcal Software) were used for data acquisition and analysis. All experiments were carried out at room temperature (21–23 ◦ C). 2.5. Single-channel analysis Linear leak and capacity currents were digitally subtracted. Openings and closures were identified by the half-height criterion. Unitary current amplitude i was estimated visually based on ≥10 fully resolved openings per experiment under each condition. In case of CaV 3.1 and CaV 3.2 analysis of all-point histograms based on raw data was performed. To elucidate the putative mechanisms

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underlying N2 O effects we analyzed several gating parameters indicative of distinct durations of the drug–channel interaction [19]. The availability (factive , fraction of sweeps containing at least one channel opening), the open probability (Popen , defined as the relative duration spent in the open state in active sweeps), and the peak ensemble average current (Ipeak , taken by eye from averaged current traces) were analyzed for one-channel and multi-channel patches (2 ≤ m ≤ 3). For such multi-channel patches examined at polarized holding potentials, values were corrected based on m, the number of channels observed in the patch (see below). m was defined as the maximum current amplitude observed divided by the unitary current. Peak current obtained from multi-channel patches was divided by the number of channels in the patch. To maximize the validity of estimating the channel number in multi-channel patches [26], only experiments with at least 180 consecutive sweeps under a given condition were analyzed. Furthermore, recordings with obviously more than three channels in a patch were excluded. Open time constants were obtained by maximum-likelihood estimation using pSTAT software on logtransformed open time histograms. Calculations were based on the following equations: Availability (fraction of active sweeps): f [%] =

Mactive × 100 M0

Open probability (Popen ):

 Popen [%] =

topen I...N

Mactive × t0

× 100

Mean open time (mot):

 mot [ms] =

topen I...N Nopen

Fig. 1. (A) Photograph of the experimental setup. Gassing was performed using an inverted glass funnel connected to gas lines (switched by three-way connectors). The funnel was put close (a few mm) to the recording chamber to allow for a constant equilibrium between gas atmosphere and liquid compartment (bath solution). This setup allowed for continuous gassing and immediate change from control gas to N2 O and vice versa. (B) N2 O concentration in cell-free bath solution was determined in 100 ␮L aliquots taken from the recording chambers at indicated time points. Concentration was determined by gas chromatography in n = 3 samples taken from separately superfused dishes. Gas application started at 0 min and was stopped after 16 min.

were performed on three separate days, with double determination of N2 O concentration each.

Availability in multi-channel patches (2 ≤ m ≤ 3):

2.7. Statistical analysis

f  = 1 − (1 − f )

Statistical difference was tested for using Student’s t-test. Comparison of gating parameters with more than three datasets was performed by using ANOVA with Bonferroni-corrected post-tests. A p-value below 0.05 was considered significant. All values are given as mean ± standard error of the mean, based on n as the number of independent experiments.

1/m

Open probability in multi-channel patches (2 ≤ m ≤ 3):  Popen =

topen t0 × m × f  × M0

3. Results with Mactive , number of active sweeps;  M0 , total number of sweeps; topen I. . .N , sum of all open times Nopen , number of all openings; (ms); t0 , pulse duration (ms); m, number of channels. 2.6. Gas application Petri dishes were superfused for up to 15 min with 80% N2 O/20% O2 (Linde Gase, Leuna, Germany) or with 100% N2 O (Linde) by using an inversed glass funnel (Fig. 1A). To check for putative mechanical effects of the gassing procedure, dishes were superfused with control gas containing 80% N2 /20% O2 (Linde Gase, Leuna, Germany) up to 6 min before N2 O gassing. After N2 O treatment, dishes were reexposed to control gas to test for reversibility of the drug effects and to control for a putative “run-down” of the currents. N2 O concentration in the bath solution was checked by gas chromatography (Sigma 3B, PerkinElmer) according to U.S. Pharmacopeia XXII. Gas was applied (16 min 100% N2 O, followed by 8 min of 80% N2 , 20% O2 , flow rate 1 l/min as in the electrophysiological experiments) with 100 ␮l samples drawn at indicated time points. The measurements

3.1. Gas application In order to apply N2 O in a reversible manner, yet maintaining low-noise recording conditions essential for resolution of unitary T-type currents, we developed a system of gas application avoiding physical contact with the recording chamber (Fig. 1A). The gas line was connected to a glass funnel fixed above the recording dish. Thus, N2 O, N2 and O2 could equilibrate with the bath solution by diffusion through the bath surface only. Application of 1 l/min of pure N2 O (Fig. 1B) led to an equilibration within about 10 min. Switching back to our control gas (80% N2 , 20% O2 ) caused reduction, but not complete abolition of bath N2 O levels within the timeframe achievable during electrophysiological recordings. In conclusion, we planned our experiments to achieve at least a 10 min-exposure time towards N2 O, and to collect data for steady-state effects after 8–14 min of N2 O exposure. Furthermore, we were prepared to see partial, but not necessarily complete recovery of channel activity after “wash-out”.

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Fig. 2. Exemplary whole-cell Ba2+ currents mediated by either CaV 3.1 or CaV 3.2. Time courses on the left show peak currents at the test potential that gave rise to maximum currents in initial IV-curves. Exemplary IV-traces before and during N2 O exposure are displayed on the right. Horizontal scale bars indicate 50 ms. (A) Whole-cell Ba2+ currents through CaV 3.2 channels were significantly decreased by N2 O (100%) gassing and recovered during wash-out (holding potential −110 mV, test potential −10 mV). (B) Currents through CaV 3.1 channels revealed a significant decrease of whole-cell Ba2+ currents after exposure to 100% N2 O (holding potential −110 mV, test potential −30 mV). (C) Inhibition by 80% N2 O (plus 20% O2 ) of currents carried by CaV 3.1 channels (holding potential −90 mV, test potential −20 mV).

3.2. N2 O effects on whole-cell currents In order to validate the technique of gas application, we studied whole-cell currents through CaV 3.1 and CaV 3.2 channels transiently expressed in HEK-293 cells. As expected, CaV 3.2 was inhibited (by 41.1 ± 9.9%, n = 6, p < 0.05) in a partially reversible manner, with a time course (Fig. 2A) matching the concentration profile of N2 O in the bath (Fig. 1B). In contrast to our expectation [15], we also observed a smaller (28.1 ± 5.3%, n = 5, p < 0.05), yet significant, reversible inhibition of CaV 3.1 currents under similar conditions (Fig. 2B). In order to reproduce more closely the conditions used by Todorovic et al. [15], we modified our methods (−90 mV holding potential, mixture of 80% N2 O and 20% O2 ). Still here, N2 O caused a small (24.2 ± 1.4%, n = 3, p < 0.05 onesided), reversible current inhibition at all test potentials (Fig. 2C). In conclusion, our gas application system could be validated by con-

firming the known pharmacological response of CaV 3.2 against N2 O [15]. The data with CaV 3.1 suggested that the extent of subtypeprevalence of N2 O should be further explored at the single-channel level. 3.3. N2 O effect in the cell-attached configuration In order to examine whether N2 O can reach its site of action on channels in the cell-attached configuration, we applied N2 O to HEK-293 cells with stable overexpression of CaV 3.1 and CaV 3.2 channels. In cell-attached patches, current responses to fully activating voltage steps (holding potential −90 mV, test potential −20 mV) did not allow resolution of unitary events. Instead, the current waveform, arising from certainly more than 10 channels per patch, closely resembled the typical whole-cell current kinetics (Fig. 3, and see above). Under these conditions, N2 O (100%)

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Fig. 3. Ba2+ currents obtained from macro-patches recorded in the cell-attached configuration. The high expression levels in HEK-293 cells stably transfected with either CaV 3.1 or CaV 3.2 inevitably gave rise to multi-channel patches. Left panels depict the time course of peak values of inward current obtained within the individual current traces (right panels, scale bars indicate 50 ms). Kinetics of current traces resembles whole-cell recordings. (A) Data from cells expressing CaV 3.2, before, during and after gassing with 100% N2 O. (B) Data from cells expressing CaV 3.1 before during, and after gassing with 100% N2 O.

reduced the peak current of CaV 3.2 by 48 ± 4% (from −13.9 ± 3.3 pA to −7.0 ± 1.5 pA, n = 5, p < 0.05). CaV 3.1 was inhibited by 35 ± 2% (from −3.46 ± 0.49 pA to −2.22 ± 0.26 pA, n = 5, p < 0.05). These data indicate that T-type channels are accessible to N2 O inhibition in the cell-attached configuration. Furthermore, they corroborate the limited selectivity of N2 O to inhibit CaV 3.2 compared with CaV 3.1. The findings pave the way for further delineating the mode of N2 O action at the level of individual channel molecules. 3.4. Single-channel mechanism of N2 O inhibition As a first approach, we minimized multi-channel activity in HEK-293 cells with stable CaV 3.2 expression by taking advantage of voltage-dependent inhibition. At an inactivating holding potential of −50 mV, channel activity is largely reduced compared with −90 mV, but stable over recording time [19], allowing assessment of drug effects at the level of individual channel openings. As exemplified by a series of individual traces and the ensemble average of a typical experiment (Fig. 4 A and B), N2 O (100%) reduced peak current by 53.8 ± 10.7% (from −69.4 ± 12.3 fA to −33.4 ± 11.8 fA, n = 5, p < 0.05). This effect was caused both by diminishing the fraction of sweeps with channel openings (−35.8 ± 12.0%, n = 6, p < 0.05) and the open probability within active sweeps (−57.7 ± 8.2%, n = 6, p < 0.05). Single-channel current amplitude i remained unaffected (−2.4 ± 3.1%, n = 6, n.s.). Thus, N2 O exerted a similar inhibitory pattern as previously reported for mibefradil under the same conditions [19]. As an alternative means to examine the effect of N2 O, we transiently transfected HEK-293 cells with CaV 3.2 plasmid. Using the same pulse protocol (holding potential −50 mV, thus inactivating the majority of channels in the patch), we obtained inhibitory effects within the same range as in stably transfected cells. Peak

ensemble current tended to decrease by 43.5 ± 12.2% (n = 5, n.s., Fig. 5A), again due to a significant reduction of the fraction of active sweeps (−43.5 ± 10.4%, n = 5, p < 0.05, Fig. 5B) and due to inhibition of activity within such sweeps (Popen : −43.3 ± 16.9%, n = 6, p = 0.059, Fig. 5C). This was accompanied by a trend towards shortening of open times ( open : −21.9 ± 7.9%, n = 4, p = 0.057, Fig. 5D). Unitary current amplitude i again remained unaffected (−3.2 ± 3.7%, n = 5, n.s.). The extent and mode of action of N2 O on CaV 3.2 was evidently not influenced by hypoxia, since application of a mixture of 80% N2 O and 20% O2 caused significant effects, almost identical to 100% N2 O in size: peak current was changed by −39.5 ± 8.9% (n = 7, p < 0.05), availability −36.4 ± 5.6% (n = 7, p < 0.05), Popen −55.8 ± 13.7% (n = 6, p = 0.075),  open −6.8 ± 9.2% (n = 7, n.s.), and i +0.5 ± 3.8% (n = 5, n.s.). CaV 3.1, examined under identical conditions, appeared to be inhibited by 80% N2 O and 20% O2 in a similar way: peak current was altered by −48.9 ± 11.8% (n = 4, n.s.), availability −47.0 ± 14.4% (n = 4, p = 0.052), Popen −45.2 ± 4.8% (n = 4, p < 0.05),  open −11.2 ± 12.7% (n = 3, n.s.), and i −4.9 ± 1.3% (n = 4, n.s.). 3.5. Subtype-selectivity of channel function and pharmacology To further facilitate the recording and analysis of singlechannel behavior, we reduced the dose of plasmid DNA (from 6 to 0.5 ␮g/dish), lowered the channel:GFP plasmid ratio (from 3:1 to 1:1), and shortened the plasmid incubation time as well as the culturing time from transfection to recording (within the ranges indicated in Section 2). Patches then typically contained no channel activity, or one to three active channels. Under these conditions, we were able to resolve discrete unitary events at a holding potential of −90 mV (cf. Fig. 6A), enabling us to correct

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Fig. 4. Effects of N2 O on single-channel behavior in cells with stable expression of CaV 3.2, held at −50 mV and depolarized to −20 mV. 10 consecutive traces and ensemble average currents derived from one typical experiment are depicted. Scale bars indicate 20 ms and 0.5 pA (single traces) or 20 fA (ensemble average). (A) Control data obtained before of N2 O application. (B) Data obtained during N2 O (100%) application.

Fig. 5. N2 O (100%) effects on single-channel parameters obtained from HEK-293 cells transiently transfected with CaV 3.2. Holding potential was −50 mV to minimize activity arising from multiple channels. Test potential was −20 mV. Each symbol depicts a single experiment. Data were not corrected for channel number. (A) Peak ensemble average current. (B) Availability, i.e. fraction of depolarizing pulses causing at least one channel opening. (C) Open probability, i.e. relative time spent in the open state during depolarization. (D) Open time constant obtained by dwell time histogram analysis.

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Fig. 6. Basal single-channel gating properties of recombinant T-type Ca2+ channels and chimeras, obtained at low expression levels after transient transfection. Holding potential was −90 mV, test potential was −20 mV. For each experiment, 10 consecutive traces (scale bars: 20 ms and 1 pA) and the ensemble average current (scale bars: 20 ms and 20 fA) are shown. (A) CaV 3.2 (HHHH); (B) CaV 3.1 (GGGG); (C) Chimera GGHH; (D) Chimera HHGG.

the data from multi-channel patches for the number of channels detected. In order to narrow down the structural site of N2 O action, we also transiently transfected cells with plasmids encoding the chimeric constructs, GGHH and HHGG (Fig. 6C and D). When analyzing these experiments, we noticed some marked differences between CaV 3.2, CaV 3.1 and the respective chimeric constructs regarding singlechannel behavior. Therefore, we decided to first cross-compare the

baseline gating properties of the four channel types. The results of this analysis are compiled in Table 1, together with the statistical evaluation. It becomes evident that single CaV 3.2 channels are about twice as active as their CaV 3.1 relatives. Functionally, this difference can be attributed to a larger fraction of active sweeps, a higher open probability and longer open times with CaV 3.2. Interestingly, all these features of CaV 3.2 are shared by the chimera GGHH, whereas HHGG closely resembles CaV 3.1 (Table 1). In sum-

Table 1 Comparison of baseline values of single-channel parameters obtained from cells expressing native T-type Ca2+ channels or chimeras containing domains of CaV 3.1 and CaV 3.2. Recording conditions were as described in Fig. 6 (holding potential −90 mV, test potential −20 mV). Mean values and standard errors of n experiments per group are indicated. Baseline values *

Peak current [fA] Availability [%] Popen * [%]  open * [ms] n * ¶ #

p < 0.05 among groups (ANOVA). p < 0.05 versus CaV 3.2 (Bonferroni). p < 0.05 versus CaV 3.1 (Bonferroni).

CaV 3.2

CaV 3.1

−31.0 ± 5.0 53.6 ± 5.5 5.70 ± 0.52# 0.400 ± 0.019# 20–21 #

¶

−17.3 ± 2.8 37.9 ± 4.3 3.29 ± 0.34¶ 0.332 ± 0.015¶ 20

GGHH

HHGG

−20.9 ± 3.8 44.9 ± 6.6 4.80 ± 0.39 0.373 ± 0.014 13–14

−13.3 ± 2.8¶ 34.3 ± 5.1 2.38 ± 0.20¶ 0.306 ± 0.010¶ 11–12

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Table 2 Comparison of N2 O effects on single-channel parameters (% change from respective pre-drug control values) obtained from cells expressing recombinant CaV 3.1 and CaV 3.2 T-type Ca2+ channels or chimeras containing domains of CaV 3.1 and CaV 3.2. Recording conditions were as described in Fig. 6 (holding potential −90 mV, test potential −20 mV). Mean values and standard errors of n experiments per group are indicated. N2 O effect [% change]

CaV 3.2

CaV 3.1

GGHH

HHGG

Peak current Availability Popen  open i n

−44.1 ± 11.2* −15.1 ± 19.3 −17.8 ± 10.5 −14.5 ± 3.8* +5.3 ± 6.3 6

−19.5 ± 10.0 −38.1 ± 8.3* −10.7 ± 5.7 −1.3 ± 7.7 −5.6 ± 2.3 5

−1.5 ± 28.5 −4.1 ± 22.2 −9.3 ± 12.8 0.4 ± 3.3 −4.1 ± 2.4 4–6

−24.7 ± 14.9* −8.4 ± 11.2 −19.7 ± 7.5* −9.2 ± 4.6 −0.2 ± 2.4 5–6

*

p < 0.05 versus pre-drug control values.

mary, the subtype-specific gating properties can be structurally attributed in large part to the C-terminal half of the pore subunit. Single-channel amplitudes (unitary currents) obtained by all-point histograms and single-channel conductance were not different between CaV 3.1 and CaV 3.2 (Fig. S5). The effects of N2 O (100%) on CaV 3.1, CaV 3.2, and the chimeric constructs are compiled in Table 2. Of note, under this condition of a hyperpolarized holding potential, a subtype-prevalent inhibition of CaV 3.2 peak ensemble current was observed. The open state duration  open as an underlying component of inhibition was significantly reduced in CaV 3.2 but not in CaV 3.1 (exemplified

in Fig. S1). Interestingly, N2 O inhibition of CaV 3.2 was mimicked by HHGG, both in qualitative and in quantitative terms. In contrast, GGHH remained unaffected by N2 O under these conditions, resembling this feature of CaV 3.1 (Table 2). These findings indicate that the N2 O effects on fast gating of CaV 3.2 are subtype-selective at a hyperpolarized holding potential. They disappear when the N-terminal half of the channel molecule is derived from CaV 3.1. Single-channel recordings with a CaV 3.2 mutant where the histidine 191 is replaced by a glutamine (H191Q) revealed small inhibitory effects, similar to those obtained with CaV 3.1 lacking H191 (Table 3). This data confirms the idea of an important role

Fig. 7. (A) After reverse-transcription of RNA isolated from hMTC cells, PCR yielded specific amplification products of the predicted size for both CaV 3.1 (397 bp) and CaV 3.2 (380 bp). ␤-Actin (expected size 310 bp) and no RNA (not shown) were used as controls. (B–E) Single-channel recordings were obtained from hMTC cells. In six experiments, data before and after application of 100% N2 O were obtained and analyzed as described above (holding potential −90 mV, test potential −20 mV). Results were compared with CaV 3.1 and CaV 3.2, obtained under identical experimental conditions (see Fig. 6 and Table 2). We then plotted the basal gating property (abscissa) against the pharmacological N2 O effect (ordinate) of each single-channel gating parameter. Symbols (: hMTC, : CaV 3.1, : CaV 3.2) and lines indicate mean values and standard error (shaded area: 95% confidence region), regarding (B) peak ensemble average current (n = 5–6), (C) availability (n = 5–6), (D) open probability (n = 5–6) and (E) open time constant (n = 3–6).

P. Bartels et al. / Cell Calcium 46 (2009) 293–302 Table 3 Effect of N2 O on CaV 3.1, CaV 3.2, and a mutant CaV 3.2 where the histidine at position 191 is replaced by a glutamine (H191Q). Single-channel activity was obtained by applying depolarizing pulses to −20 mV starting from a holding potential of −50 mV. Gassing with 100% N2 O led to only small inhibition of single-channel currents carried by H191Q. N2 O effect [% change]

CaV 3.2

CaV 3.1

H191Q

Peak current Availability Popen  open n

−43.5 ± 12.2 −43.5 ± 10.4* −43.3 ± 15.0 −21.9 ± 7.9 4–5

−18.6 ± 20.0 −24.5 ± 19.7 −28.7 ± 13.0 −14.9 ± 5.2* 4–5

−28.1 ± 4.0* −18.3 ± 6.1 −17.9 ± 3.0* −26.1 ± 7.3 4

*

p < 0.05 versus pre-drug control values.

of H191 for N2 O effects [18], as it was also suggested for interaction with Ni2+ [16], Zn2+ [17], and ascorbate [14]. As with CaV 3.1 and CaV 3.2 (Fig. 7) plotting basal gating parameters (cf. Table 1) against amount of inhibition by N2 O (cf. Table 2) allows for discrimination of chimeric channels in terms of a functional fingerprint (Fig. S2). 3.6. Characterization of endogenous channels Given this pattern of biophysical and pharmacological properties of human recombinant CaV 3.1 and CaV 3.2, it is tempting to try to identify the molecular identity of native T-type channels using single-channel recording. As a first test of this approach, we investigated the human cell line hMTC, known to express T-type channels [19]. At the mRNA level, transcripts of both CaV 3.1 and CaV 3.2 were detected by PCR (Fig. 7A). Single channels recorded in the cellattached configuration (again, holding potential of −90 mV, test potential of −20 mV), however, appeared as a rather homogenous population of low-activity channels, with minor inhibition exerted by N2 O. In the plots of Fig. 7 B-E, we co-visualized pharmacological N2 O effects (cf. Table 2 for means and standard errors) and the respective biophysical baseline values for each single-channel parameter (see Fig. S4 for this analysis at the level of individual experiments). By this approach, we compared the results obtained with hMTC cells with those of recombinant CaV 3.1 and CaV 3.2. In all graphs, the behavior of channels from hMTC cells is localized within the confidence limits of CaV 3.1, but not of CaV 3.2. In confirmatory experiments with hMTC cells N2 O had no significant effect on T-type calcium currents at the whole-cell level at all (Fig. S3). In conclusion our data argue strongly that molecular channel identification can be achieved using single-channel recording. 4. Discussion The present study demonstrates that N2 O inhibits single T-type channels by reducing the fraction of active sweeps (“availability”) and by diminishing channel open probability. Unitary current amplitude remains unaffected under all conditions tested, excluding blocking events that are faster than the recording bandwidth would allow to resolve. The N2 O effect is very reminiscent of mibefradil inhibition [19] and could be interpreted in terms of a dual block, suggestive of two sites of action with different kinetics. The kinetics of “slow” block may be further analyzed in one-channel patches, but we have too few such data to pursue this approach at present. As an alternative explanation, both pharmacological effects may be exerted through a common binding site, functionally coupled to both the rapid and slow gating properties of the channel. Remarkably, CaV 3.2 is intrinsically more active in both aspects than CaV 3.1 under control conditions, suggesting some coupling between fast and slow gating across different channel structures.

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The higher activity of CaV 3.2 can by itself render this channel isoform more susceptible to pharmacological block, especially with respect to an open-channel block mechanism. We are able to reject this idea, however, because the GGHH chimera largely retains the high-activity gating profile of CaV 3.2, yet it has lower sensitivity towards N2 O, thus in turn resembling a CaV 3.1 feature. To our surprise, CaV 3.1 was not found to be entirely resistant to N2 O inhibition. A somewhat weaker but significant inhibition was observed throughout all conditions tested. This discrepancy with earlier work [15,27] may be due to the fact that we used a human isoform of the CaV 3.1 channel, while previous studies were done with a rat clone. Given the conserved absence of the important H191 in CaV 3.1 of both species, demonstrated also in this study, we consider this explanation less likely. Alternatively, one should take into account that the bath solution in our experiments fully equilibrates with the gas atmosphere above the dish (Fig. 1). In contrast, conventional application of pre-equilibrated solution to the recording chamber may lead to lower concentration due to evaporation through the bath surface, but this speculation should be tested by concentration measurements. In conclusion, pharmacological testing with N2 O as a means to identify unknown native channel subtypes should be done with caution. Measurement of a full concentration–response curve seems advisable for whole-cell studies, and single-channel studies require extensive sets of data, given the subtle subtype difference we could detect. We could not fully resolve the biophysical mechanism of subtype-prevalence of N2 O block. The data obtained at a hyperpolarized holding potential suggest that the effects on fast gating are more prominent with CaV 3.2 and HHGG, supporting the important role of the H191 residue for N2 O inhibition [18]. However, the small size of N2 O effects (which cannot be overcome by an increase in concentration at normobaric conditions), and the notoriously large variance among different single-channel experiments should lead to a cautious interpretation of these data. Subtypespecific single-channel block may be followed up using other agents (e.g. ascorbate, Ni2+ ), but these would likely require excised-patch, outside-out recording. Perhaps more interestingly, an unexpected structure–function relation was disclosed regarding the basal gating properties of CaV 3.1 and CaV 3.2. The twofold higher activity of CaV 3.2 is mainly conferred by the C-terminal portion of the channel, in line with functional effects of natural splice variation in the III–IV linker region [28]. The marked influence of the I–II linker on CaV 3.2 gating [29] is shared by CaV 3.1 [30], supported by our single-channel data indicating similarity of gating with conserved N-termini. Our results provide a first hint that careful head-to-head comparison of channel isoforms may unravel functional distinctions that would otherwise elude discovery even by careful wholecell studies. In particular, the two-dimensional, combined analysis (Fig. 7B–E, Fig. S2) of baseline single-channel biophysics and pharmacological assessment emerges as a valuable tool to characterize and compare unknown native channels with their putative recombinant counterparts. We cannot fully explain why N2 O showed an only transient and insignificant inhibition of whole-cell currents in hMTC cells. Yet this finding is consistent with our general interpretation that T-type calcium channels in hMTC cells are phenotypically CaV 3.1 (see also [15]). One may speculate that the predominant – if not exclusive – membrane expression of CaV 3.1like channels in hMTC cells (Fig. 7) reflects the more efficient membrane targeting of CaV 3.1 channel [30], as compared with CaV 3.2 [29]. It seems very worthwhile to cross-validate our functional approach of channel identification with direct assays based on specific antibodies. In conclusion, single-channel analysis of Ttype calcium channel biophysics and N2 O pharmacology reveals important structural and functional insights, and may aid to identify native channels of hitherto unknown molecular composition.

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