Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Cell Calcium 54 (2013) 226–235

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Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav 3.2 T-type channel Hyun-Jee Park a,b,1 , So-Jung Park a,b,1 , Eun-Joo Ahn a,b,1 , So-Young Lee a,b,1 , Haengsoo Seo a,b , Jung-Ha Lee a,b,c,∗ a

Department of Life Science, Sogang University, Seoul 121-742, Republic of Korea Basic Science Institute for Cell Damage Control, Sogang University, Seoul 121-742, Republic of Korea c Integrative Biotechnology Program, Sogang University, Seoul 121-742, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 2 January 2013 Received in revised form 16 May 2013 Accepted 17 June 2013 Available online 9 July 2013 Keywords: Cav 3.2 T-type channel Permeation and selectivity Cd2+ blocking sensitivity Proton block Voltage clamping

a b s t r a c t Voltage-activated Ca2+ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca2+ selective filters of all high-voltage-activated Ca2+ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca2+ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Cav 3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Cav 3.2, including relative permeability between Ba2+ and Ca2+ , anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd2+ blocking sensitivity, and pH effects, in distinctive ways. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Voltage-activated Ca2+ channels (VACCs) play pivotal roles in a variety of physiological functions including cardiac and neuronal cell excitability, muscle contraction, synaptic transmission, and hormone secretion [1]. Numerous experimental approaches including biochemical purification, molecular cloning, and reconstitution of VACCs have demonstrated that a VACC is composed of an ␣1 subunit and auxiliary subunits [1]. An ␣1 subunit is a main structural component connected by four repeated domains, each of which contains six membrane-spanning segments (S1–S6) and a pore loop between S5 and S6. In general, S1 to S4 segments act as voltage sensors, and pore loops with the remaining S5 and S6 segments form a calcium permeation route, controlling Ca2+ -selective permeation. High-voltage-activated Ca2+ channel ␣1 subunits commonly have loci for a selectivity filter composed of four glutamate residues, each of which presents in each pore loop of domains I–IV. Point mutation experiments have shown that the four glutamate

∗ Corresponding author at: Department of Life Science, Sogang University, ShinsuDong 1, Mapo-Gu, Seoul 121-742, Republic of Korea. Tel.: +82 2 705 8791; fax: +82 2 704 3601. E-mail address: [email protected] (J.-H. Lee). 1 These authors contributed equally to this work. 0143-4160/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ceca.2013.06.006

residues (EEEE) in an ion-conducting route are the main structural determinants controlling calcium selectivity, but their individual contributions to selectivity for Ca2+ , anomalous mole fraction effect (AMFE), and cadmium blocking sensitivity were shown to be differential [2–4]. Other studies have reported that point mutations of adjacent residues of the Glu at the pore loops could alter relative permeability of Ba2+ versus Ca2+ for the Cav 1.2 channel [5], and point mutation of an EF hand-like locus in the domain III S5-H5 region could change permeability properties of Cav 2.2 [6]. In comparison, all low-voltage-activated Ca2+ channel isoforms (Cav 3.1, Cav 3.2, and Cav 3.3) commonly have EEDD residues at the corresponding pore loci [7]. The Nilius group recently reported that point mutations of the Asp residue(s) to Glu significantly altered Ba2+ /Ca2+ ion permeability, Cd2+ blocking sensitivities, AMFE, pH effects on channel activity, and gating of Cav 3.1 [8,9]. They suggest that the two Asp residues distinctively participated in those biophysical properties, but the Asp in the domain III pore loop affected those properties of Cav 3.1 more strongly than the Asp in the domain IV pore loop. Independently, Shuba and his colleagues broadly characterized the selectivity and permeation properties of the three recombinant T-type channel isoforms in Ca2+ , Ba2+ , and Sr2+ [10]. We here investigate how the pore Asp residues of domains III and IV contribute to the ion permeation properties of Cav 3.2 Ttype channels which have been reported to produce greater Ba2+ currents in equimolar Ba2+ and Ca2+ solutions [10,11]. Comparison

H.-J. Park et al. / Cell Calcium 54 (2013) 226–235

studies of the wild-type Cav 3.2 (Cav 3.2/WT) and the constructed pore mutants (Cav 3.2/EEED, Cav 3.2/EEDE, and Cav 3.2/EEEE) indicated that mutation(s) of the pore Asp(s) of Cav 3.2 significantly altered relative permeability between Ba2+ and Ca2+ . As shown in Cav 3.2/WT, EEDE and EEEE mutants showed AMFE, but EEED mutant did not show it. Replacement of the pore Asp residue(s) with Glu increased cadmium blocking sensitivity. Interestingly, the proton inhibition profile of Cav 3.2 currents suggests that Cav 3.2 has double proton binding sites with high and low affinity. 2. Materials and methods 2.1. Chemicals and preparation of solutions Most of the chemicals we used were purchased from Sigma–Aldrich (St. Louis, MO, USA). The Ca2+ (or Ba2+ ) recording solution contained (in mM) 10 CaCl2 (or BaCl2 ), 90 NMDG, 1 KCl, and 5 HEPES (pH 7.4). For mole fraction experiments, the divalent Ba2+ plus Ca2+ concentration in the mixtures was maintained at 10 mM with Ba2+ /Ca2+ ratios of 10/0, 9/1, 7.5/2.5, 5/5, 2.5/7.5, and 0/10. Serial cadmium solutions were prepared by diluting the 100 mM cadmium stock solution with 10 mM Ba2+ recording solution before every experiment. For the proton-blocking experiments, different pH solutions were prepared using MES, HEPES, and TAPS (5 mM each) to broaden the buffering range from pH 5.5–9.1. 2.2. Generation of the pore mutants of Cav 3.2 The pore Asp residues in domains III and IV of the human Cav 3.2 (␣1H ; GenBank accession number AF051946) channel were individually mutated into Glu by overlap extension PCR (polymerase chain reaction). All PCRs were performed using Pfu DNA polymerase (Vivagen, Seoul, Korea). Amplified PCR products were sequenced for confirmation. To facilitate construction of pore mutant channels, we first constructed Cav 3.2/G5184T in which a silent Hind III restriction enzyme site was introduced to Cav 3.2 by mutating G at 5184 (nucleotide no.) to T. Silent restriction enzyme site(s) are marked by asterisks (*). Restriction enzyme sites used are marked by numbers in parentheses. 2.2.1. Cav 3.2/D1504E (EEED) The forward and reverse primers to amplify the upper fragments were 5 -ACAGAACCGGTTCCGCGT-3 and 5 -ACCCATCCTTCCTTGGATGACAGCACG-3 , respectively. The forward and reverse primers to amplify the lower fragments were 5 -TCCAAGGAAGGATGGGTGAACATCATG-3 and 5  ATCTTCAGAAGCTTCAGCACACGGGCA-3 , respectively. The upper and lower fragments were overlapped and extended by additional PCR. Sequencing of amplified DNA fragments confirmed that the pore Asp of domain III was mutated into Glu. The Cav 3.2/D1504E in pGEM-HEA was subsequently constructed by ligating the PCR products digested with AgeI (3843, Cav 3.2) and SalI (4556, Cav 3.2) to the Cav 3.2 pGEM-HEA vector opened with AgeI (3843, Cav 3.2) and SalI (4556, Cav 3.2). 2.2.2. Cav 3.2/D1808E (EEDE) The forward and reverse primers to amplify the upper fragments were 5 -TGAAGCTTCTGAAGATGGCTACGGGC-3 and 5 -TTCCAGTTCTCCCCCGTCCACACGCG-3 , respectively. The forward and reverse primers to amplify the lower fragments were 5 -ACGGGGGAGAACTGGAACGGGATCATG-3 and 5  AGGGTGTCCCTAGGGCT-3 , respectively. The upper and lower fragments were overlap-extended by additional PCR. Sequencing of amplified DNA fragments confirmed that the pore Asp of domain IV was mutated into Glu. The Cav 3.2/D1808E in pGEM-HEA was constructed by ligating the fragments digested with SalI (4556,

227

Cav 3.2) and HindIII* (5182, Cav 3.2/G5184T) and the fragments digested with HindIII* (5182, Cav 3.2/G5184T) and AvrII (6092, Cav 3.2) into the Cav 3.2 pGEM-HEA vector opened with SalI (4556, Cav 3.2) and AvrII (6092, Cav 3.2). 2.2.3. Cav 3.2/D1504,1808E (EEEE) The fragments of Cav 3.2/D1504E digested with AgeI (3843, Cav 3.2) and HindIII* (5182, Cav 3.2/G5184T) and the fragments of Cav 3.2/D1808E digested with HindIII* (5182, Cav 3.2/G5184T) and AvrII (6092, Cav 3.2/G5184T) were ligated to Cav 3.2 pGEM-HEA vector opened with AgeI (3843, Cav 3.2) and AvrII (6092, Cav 3.2). 2.3. Expression of the Cav 3.2 T-type channel and its pore mutants in Xenopus oocytes Female Xenopus laevis frogs were purchased from Xenopus Express (France). Ovary lobes were surgically removed and torn into small clusters of 4–10 oocytes in a standard oocyte solution (in mM: 100 NaCl, 2 KCl, 1.8 CaCl2 , 1 MgCl2 , 5 HEPES, 2.5 pyruvic acid, and 50 ␮g/ml gentamicin; pH 7.6). Clusters of oocytes were digested with collagenase (10 mg/ml, Gibco-GRL, Gaithersburg, MD, USA) as well as trypsin inhibitor (Type III-O, Sigma–Aldrich, St. Louis, MO, USA) in Ca2+ -free OR2 solution (in mM: 82.5 NaCl, 2.5 KCl, 1 MgCl2 , 5 HEPES; pH 7.6) for 50 min to eliminate follicle membranes. Complementary DNAs encoding wild-type Cav 3.2 and its pore mutants were digested at their 3 ends by AflII and the linearized cDNAs were used as templates for in vitro transcription using T7 RNA polymerase (Ambion, Austin, TX) according to the manufacturer’s instructions. The Cav 3.2 cRNAs were injected into defolliculated oocytes at concentrations of 1 ng/50 nl for wildtype Cav 3.2 and 30–50 ng/50 nl for pore-mutant channels using a Drummond Nanoject pipette injector (Parkway, PA) attached to a micromanipulator. 2.4. Electrophysiology using two-electrode voltage clamping and data analysis Ba2+ or Ca2+ currents were measured using a two-electrode voltage clamp amplifier (OC-725C, Warner Instruments, Hamden, CT) between the third and fifth days after cRNA injection. Microelectrodes were pulled using a pipette puller and filled with 3 M KCl. The electrode resistance was 0.5–1.0 M. The bath solution contained (in mM) 10 BaCl2 (or 10 CaCl2 ), 90 NMDG, 1 KCl, and 5 HEPES (pH 7.4 with HCl). All the oocytes used to record Ca2+ currents were injected with 50 nl of 50 mM BAPTA (1,2-bis[o-aminophenoxy] ethane-N,N,N ,N -tetraacetic acid) 30–60 min before recordings, to eliminate contamination of Ca2+ -activated chloride currents. The currents were sampled at 5 kHz and low pass-filtered at 1 kHz using the pClamp system (Digidata 1322A and pClamp 8; Axon Instruments, Foster City, CA). Data analysis and graphs were obtained with Clampfit software and Prism software (GraphPad, San Diego, CA), respectively. Data are presented as means ± S.E.M. Differences are tested for significance using a Student’s unpaired t-test and one-way ANOVA combined with a Newman–Keuls post-test, with P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***), as levels of significance. The maximal slope conductance was obtained by fitting the current–voltage (I–V) data with a modified Boltzmann equation:

I=

Gmax (V − Vr ) (1 + exp(−(V − V50 )/Sact ))

(1)

where I is the recorded peak current, Gmax is the slope conductance, V is the test potential, Vr is the apparent reversal potential, V50 is the potential of half-maximal activation, and Sact is the slope parameter for activation.

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The smooth curves for channel activation and steady-state inactivation were derived by fitting the average data with a Boltzmann equation: G=

1 1 + exp[(V50 − V )/Sact ]

(2)

where V50 is the potential for half-maximal activation and Sact is the slope conductance. Dose–response curves for Cd2+ blocking of T-type channel currents were derived by fitting the data using a Hill equation: B=

1 1 + IC50 /[Cd2+ ]

(3)

n

where B is the normalized block, IC50 is the concentration of Cd2+ giving half maximal blockade, and n is the Hill coefficient. The smooth lines for proton blocking of Cav 3.2 and mutant currents were fitted with a Hill equation for independent doublebinding sites as shown below: % of block =

fract1 1 + 10(pH−pK1 )

+

1 − fract1 1 + 10(pH−pK2 )

(4)

where K1 and K2 are dissociation constants for two binding sites, and fract1 is the fraction of a site with dissociation constant K1 . 2.5. Ensemble fluctuation analysis Cav 3.2/WT and the pore mutants (EEED, EEDE, and EEEE) in pcDNA3 were individually co-transfected with EGFP-pcDNA3 into HEK293 cells using Lipofectamine 2000 reagent. Whole-cell patch clamp recordings were made using an Axopatch 200B amplifier (Molecular Devices, Foster City, CA), connected to a computer via a Digidata 1322A converter (Molecular Devices, Foster City, CA), and controlled using pCLAMP 9.2 software. Tail currents were repetitively evoked by test pulses of 10 mV from a holding potential of −90 mV in 10 mM Ba2+ solution (140 TEACl, 2.5 CsCl, 10 BaCl2 , 1 MgCl2 , 10 HEPES, 5 glucose, pH = 7.4 with TEAOH). The pipette solution contained the following (in mM): 130 CsCl, 10 HEPES, 10 EGTA, 5 MgATP, 1 NaGTP, pH = 7.4 with CsOH. Currents were filtered at 5 kHz and sampled at 20 kHz. The average variance ( 2 ) of the tail currents at −70 mV following +10 mV test pulse was subtracted with the average basal noise at −90 mV for correction. The average variances ( 2 ) were plotted against the mean current amplitude, and then the plotted data were fitted with the equation ( 2 = iI(t) − I(t)2 /N) using Prism software, where i is single-channel current amplitude and N is number of channels [12]. 3. Results 3.1. Comparison of Ba2+ and Ca2+ currents through Cav 3.2 and its pore mutants The positions of two Glu and two Asp residues (EEDD) comprising the pore filter were exhibited on the schematic four-domain structure of Cav 3.2 (Fig. 1A). The Asp residues at the pore loops of domains III and IV were individually mutated to Glu, to evaluate their contributions to ion permeation and selectivity of Cav 3.2. As a control, we first recorded currents from oocytes expressing wildtype Cav 3.2 (Cav 3.2/WT, EEDD) in 10 mM Ca2+ or 10 mM Ba2+ as a charge carrier. When step pulses to −20 mV from a holding potential of −90 mV were applied to the oocytes, the peak amplitude of Ca2+ currents through Cav 3.2/WT (EEDD) was smaller than that of Ba2+ currents by 32 ± 8% (ICa /IBa = 0.68 ± 0.02; n = 6; Fig. 1B). This property of Cav 3.2/WT is similar to that noted in a previous report [11]. The Ba2+ preferred permeation of Cav 3.2/WT is in contrast to that of Cav 3.1/WT, showing greater current in Ca2+ than Ba2+ [8].

In a similar way, we compared the permeation properties of pore mutant channels (EEED, EEDE, and EEEE) in 10 mM Ca2+ or 10 mM Ba2+ as a charge carrier. The relative permeation properties of all the pore mutants were significantly altered from those of Cav 3.2/WT. Cav 3.2/D1504E (EEED) generated by switching the Asp of the domain III with Glu, showed that ICa /IBa was 0.15 ± 0.03, indicating that Ca2+ through the EEED mutant is far less able to permeate than Ba2+ (n = 7; Fig. 1C). In contrast, Cav 3.2/D1808E (EEDE) generated by switching the pore Asp of domain IV with Glu, showed that ICa /IBa was 1.33 ± 0.08, indicating that Ca2+ is more permeable through EEDE than Ba2+ (n = 6; Fig. 1D). The Ca2+ preferred permeation was most strongly produced in Cav 3.2/D1504,1808E (EEEE, ICa /IBa = 4.52 ± 0.56, n = 6; Fig. 1E) among Cav 3.2/WT and its pore mutants. We further examined whether the above results obtained from the ICa /IBa ratios (Fig. 1F) are consistent with the maximal slope conductance ratios of Cav 3.2/WT and its pore mutants. Currents from oocytes expressing Cav 3.2/WT or a pore mutant were recorded in 10 mM Ca2+ or 10 mM Ba2+ solution using a voltage protocol consisting of serial step potentials to obtain their current–voltage (I–V) relationships. Their maximal conductance(s) were derived from fitting the I–V data with a modified Boltzmann equation (refer to Eq. (1) in Section 2; Fig. 2A–D). Maximal conductance ratios (GMaxCa /GMaxBa ) were calculated and are shown as bar graphs for comparison (Fig. 2E). The GMaxCa /GMaxBa ratios for Cav 3.2/WT and EEED are 0.79 ± 0.05 and 0.25 ± 0.05, respectively (n = 6–7). In contrast, the ratios for the domain IV pore mutant (EEDE) and the double mutant (EEEE) are 1.30 ± 0.08 and 2.41 ± 0.43, respectively (n = 6). Comparison of these GMaxCa /GMaxBa ratios to the ICa /IBa ratios (Fig. 1F) indicates that the maximal conductance ratios are consistent with the relative permeation ratios (ICa /IBa ). It is notable that expression of EEEE in oocytes was very weakly detected as a tiny inward current, especially in 10 Ba2+ solution as a charge carrier, and its reversal potential was negatively shifted compared to Cav 3.2/WT and the other mutants. These properties of EEEE were similar to the results previously addressed in the case of the double pore mutant of Cav 3.1 (Cav 3.1/EEEE) [8]. As suggested, the negatively shifted reversal potential as well as the strong outward currents in the range of high test potentials seems to interfere with the fitting of maximal conductance for the double mutant channel. 3.2. Voltage-dependent activation is affected by mutations of the pore Asp to Glu We next tested whether point mutation(s) of the pore Asp residues of domains III and IV can affect the voltage dependence of channel activation and steady-state inactivation. Activation curves of Cav 3.2/WT and the pore mutants were obtained by fitting the current–voltage data using a Boltzmann equation (Eq. (2)). Analyses of activation curves showed that the V50,act values for Cav 3.2/WT and the Asp mutants (EEED, EEDE, and EEEE) were −30.07, −21.34, −22.81, and −20.38 in 10 mM Ba2+ solution (Fig. 3A), while their V50,act values were −27.14, −19.25, −20.68, and −18.81 in 10 mM Ca2+ solution, respectively (Fig. 3B). These indicate that the pore Asp mutations caused shifts in activation curves to the positive direction by 7–10 mV (unpaired Student’s t-test, P < 0.05 or 0.01, n = 6–7). In addition, the slope factor(s) were significantly increased by the pore mutations (unpaired Student’s t-test, P < 0.01, n = 6–7). However, the pore mutations did not cause significant effects on voltage dependence of steady-state inactivation (Fig. 3E and F; n = 6–7). 3.3. Cd2+ blocking sensitivity is altered by pore Asp mutations Previous studies have shown that Cd2+ interacts with the EEEE motif in the selectivity filters of HVA Ca2+ channels [3], while nickel or zinc interacts with His191 in the IS3-IS4 loop

H.-J. Park et al. / Cell Calcium 54 (2013) 226–235

229

Fig. 1. Alteration of ICa /IBa ratios by mutations of Asp residues at the third and fourth pore loops. (A) Schematic diagrams exhibit negatively charged amino acids at the pore loops of Cav 3.1 and its pore mutants. (B–E) Representative current traces through Cav 3.2/WT (B), Cav 3.2/EEED (C), Cav 3.2/EEDE (D), and Cav 3.2/EEEE (E) recorded in 10 mM Ca2+ or 10 mM Ba2+ solution were evoked by −10 mV voltage steps from a holding potential of −90 mV. Ca2+ current traces are presented in gray, while Ba2+ current traces are in black. (F) Ratios (ICa /IBa ) of Ca2+ and Ba2+ currents through Cav 3.2 and its mutants are exhibited as bar graphs for comparison (n = 6–7).

of Cav 3.2 [13,14]. Thus, we used Cd2+ rather than nickel or zinc to evaluate how the pore Asp-to-Glu mutations have an influence on blocking sensitivity of Cav 3.2. When serial Cd2+ solutions were applied to oocytes expressing Cav 3.2/WT or a pore mutant, currents through Cav 3.2/WT or each mutant were inhibited by Cd2+ in a concentration-dependent manner (Fig. 4). Analysis of the Cd2+ blocking data using a Hill equation (Eq. (3)) indicates that Cav 3.2/WT displayed the lowest Cd2+ blocking sensitivity (IC50 = 58.53 ± 4.23 ␮M, n = 1.02 ± 0.12). The single pore mutants (EEED and EEDE) displayed higher Cd2+ blocking sensitivities, with less steep slope factors in concentration-dependent blockade (IC50 and n for EEED = 25.24 ± 4.78 ␮M, 0.88 ± 0.12; IC50 and n for EEDE = 41.98 ± 3.76 ␮M, 0.84 ± 0.13), based on a one-way ANOVA (P < 0.01 and 0.05, n = 6–8). The IC50 value and slope factor for the double mutant (EEEE) are 4.65 ± 3.39 ␮M and 0.89 ± 0.10 (n = 7; Fig. 4E), respectively, suggesting that the double pore mutations further increased the Cd2+ blocking sensitivity, compared to the single pore mutations (P < 0.001, n = 6–8). 3.4. Anomalous mole fraction effect is modulated by pore Asp mutations Anomalous mole fraction effect (AMFE) is the phenomenon that smaller currents are recorded in mixtures including Ba2+ and Ca2+ than currents in either a Ba2+ or Ca2+ solution. AMFE caused by multiple interaction mechanisms between channels and two

permeable divalent charge carriers has been established as an important parameter distinguishing the selectivity and permeation properties of channels. Thus, we compared AMFE of currents through Cav 3.2/WT and the pore mutants at different Ca2+ –Ba2+ mole fractions. In contrast to Cav 3.1/WT, which was reported to lack AMFE [8], Cav 3.2/WT showed AMFE, evidenced by the data that currents at 10 mM mixtures including Ba2+ and Ca2+ were smaller than currents in either a 10 mM Ba2+ or 10 mM Ca2+ solution (Fig. 5A and E). Interestingly, AMFE was absent in EEED and EEEE mutants, suggesting that the switch of the domain III Asp with Glu abolished AMFE (Fig. 5B–E). In comparison, the EEDE mutant displayed AMFE in some mixtures, but not in all (Fig. 5C and E). These findings suggest that both Asp residues are involved in AMFE, but the domain III Asp plays a more pivotal role than does the domain IV Asp. 3.5. Extracellular proton effects on the channel activation and current amplitude of Cav 3.2 and the Asp-to-Glu mutants We examined how the pore Asp-to-Glu mutations modulate the extracellular pH effects on the activation properties of Cav 3.2/WT. In 10 mM Ca2+ solutions, strong currents were evoked from oocytes expressing Cav 3.2/WT or EEDE in response to a voltage protocol for I–V. In contrast, only small Ca2+ currents (less than 100 nA in peak current amplitude) were measured from oocytes injected with maximal amounts (50–100 ng) of EEED or EEEE cRNA (n = 50, 35), limiting collection of proper I–V data for pH effects. Thus we could

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in 10 mM BaCl2

Activation (%Gm ax)

A

100

50

50

0

in 10 mM CaCl2

B

100

0 -60

-40

-20

0

20

-60

Test potential (mV)

-40

-20

0

20

Test potential (mV)

C

D 10

**

**

Sact

**

V50

-10

-20 **

**

** **

** *

**

** **

5

0

-30

in 10 mM BaCl2

Percent Inhibition

E

100

75

75

50

50

25

25

0 -100

-80

-60

-40

Prepu lse po tential (mV)

Fig. 2. Comparison of current–voltage (I–V) relationships and relative slope conductances of Cav 3.2 and its pore mutants. (A–D) The I–V data of Cav 3.2/WT (A), EEED (B), EEDE (C), and EEEE (D) were obtained in 10 mM Ba2+ (filled symbols) and 10 mM Ca2+ (open symbols) as a charge carrier and their normalized I–V relationships were overlapped for comparison. The smooth lines are from fitting normalized I–V data in voltage ranges from −70 mV to +30 or +40 mV for all channels with Eq. (1) (refer to Section 2) except for EEEE. Ba2+ currents through the EEEE mutant were fitted from −70 up to 0 mV, because large outward currents at higher test potentials could not be compatible with Eq. (1). (F) The relative slope conductances (GMaxCa /GMaxBa ) in Ca2+ and Ba2+ are displayed as bar graphs. Significant differences in mutant channels compared to Cav 3.2/WT were at the P < 0.001 level (***; unpaired Student’s t-test, n = 6–7).

analyze I–V data for only Cav 3.2/WT and EEDE in serial 10 mM Ca2+ solutions with different pH (9.1–5.5), exhibiting their representative I–V curves (Fig. 6A and B). Analysis of their I–V data in pH 9.1 Ca2+ solution (Fig. 6C) showed that the potential for half-maximal activation of EEDE was positively shifted by 9.8 mV, compared to that of Cav 3.2/WT (V50,act values for Cav 3.2/WT and EEDE = −33.23 ± 0.9 and −23.03 ± 0.5, n = 6). Further analyses of the I–V data of Cav 3.2/WT and EEDE recorded in different pH values (8.7, 8.4, 7.4, 6.6, and 5.5) displayed that the extracellular pH switch from 9.1 to 7.4 did not significantly alter the potential (V50,act ) for halfmaximal activation of Cav 3.2/WT or EEDE, but further acidification to pH 6.6 and 5.5 significantly shifted the V50,act for Cav 3.2/WT or EEDE to the positive direction (P < 0.05 or 0.01, unpaired Student’s t-test, n = 5–8). It is of interest that the V50,act values of EEDE are greater by roughly 10–12 mV than those of Cav 3.2/WT over the pH ranges examined (Fig. 6D), suggesting that the Asp-Glu mutation positively shifted the activation curve of Cav 3.2/WT to the positive direction regardless of extracellular pH. Similarly, gradual replacements of extracellular pH from 9.1 up to 7.4 did not significantly

F

100

0 -100

in 10 mM CaCl2

-80

-60

-40

Prepu lse po tential (mV)

Fig. 3. Activation curves and steady-state inactivation curves of Cav 3.2 and its pore mutants. (A and B) The voltage-dependent activation percentages (%Gmax ) of Cav 3.2 () and its pore mutants (EEED, ; EEDE, ; EEEE, ) in 10 mM Ba2+ (A) and 10 mM Ca2+ (B) solutions were from chord conductances obtained by dividing the current amplitude by the driving force, normalized and plotted against the test potential using the I–V data in Fig. 2. The smooth lines are from fitting the averaged activation data using a Boltzmann function (Eq. (2)). (C and D) V50 (C) and Sact (D) of Cav 3.2 and its mutants in 10 mM Ba2+ (gray) and 10 mM Ca2+ (white) are displayed in bar graphs. (E and F) The steady-state inactivation curves of Cav 3.2 and its mutants in 10 mM Ba2+ and 10 mM Ca2+ are shown. Steady-state inactivation percentages are plotted as a function of prepulse potentials. Smooth curves are from Boltzmann fits of the data.

change the slope factors for activation curves of Cav 3.2/WT or EEDE, but acidification to pH 6.6 and 5.5 significantly increased their slope factors (P < 0.05 or 0.01, unpaired Student’s t-test, n = 5–8; Fig. 6E). Over all the pH ranges tested, the slope factors of activation curves for EEDE were found to be significantly greater than those of Cav 3.2/WT (Fig. 6E). The I–V data for Cav 3.2/WT showed that an extracellular pH change from 9.1 to 8.7 inhibited ∼30% of Ca2+ current amplitude evoked by +10 mV test potential, but a pH change from 8.7 to 7.4 increased the blocking percentage much less (Fig. 6A and F). Further acidification by switching the extracellular solutions from pH 7.4 up to 5.5 blocked peak current amplitude in a pH dependent manner. Combined with a separate set of experiments to assess pH blocking effects on currents evoked by +10 mV test potential, the proton blocking percentages were plotted against pH values (Fig. 6F). Notably, the proton blocking percentages of Cav 3.2/WT plotted against pH values could not be fitted for a single binding site, but for double binding sites with a modified Hill equation (refer to Eq (4)). The pK1 and pK2 values for Cav 3.2/WT are 5.53 ± 0.19 and 8.96 ± 0.15, respectively. In comparison, the proton blocking data for the domain IV pore mutant (EEDE) showed that the Asp-to-Glu mutation significantly increased the proton blocking percentages

H.-J. Park et al. / Cell Calcium 54 (2013) 226–235

Cav3.2/WT

500 nA

10 µM 50 ms

50 ms

EEDE

C

EEEE

D

100 µM

250 nA

10 µM

10 µM

1 µM 50 ms

50 ms

100

% Inhibition

E

200 nA

100 µM

100 µM

10 µM

EEED

B

150 nA

A

231

50

0

1

10

100

1000

[Cd 2+ ], µ M Fig. 4. Cd2+ blocking sensitivity of Cav 3.2 and its pore mutants. (A–D) Representative currents of Cav 3.2 (A) and its mutants (B–D) before and after application of serial Cd2+ concentrations are shown as overlapped. Currents were evoked by voltage steps to −20 mV from a holding potential of −90 mV. (F) The dose–response curves of Cd2+ blocking on Cav 3.2 (䊉) and pore mutant channels (EEED, ; EEDE, ; EEEE, ). Currents were normalized to the peak current in the absence of Cd2+ , and the normalized blocking percentage was plotted against Cd2+ concentrations. The smooth curves represent the fits to the data using a Hill equation.

at pH 6.6 and lower pH solutions (Fig. 6F; unpaired Student’s ttest, P < 0.05, n = 7–9), with no significant changes in the proton blocking percentages at pH 7.4 and higher pH solutions. The pH blocking data for the EEDE pore mutant could also be fitted for double binding sites. The pK1 and pK2 values obtained from the fitting are 6.15 ± 0.18 and 9.05 ± 0.14. Statistical analyses showed that the pK1 value of EEDE is significantly bigger than that of Cav 3.2/WT (unpaired Student’s t-test, P < 0.05, n = 7–9), but their pK2 value is not significantly different. The significant increment of pK1 indicates that the pore Asp of domain IV is involved in a lowaffinity proton-binding site. In contrast, the similar pK2 values (∼9) between Cav 3.2/WT and EEDE indicate that Cav 3.2/WT and its pore mutants commonly contain a high-affinity proton-binding site of which binding affinity is not changed by the pore mutation(s). In 10 mM Ba2+ solution in which several fold greater currents through EEED was produced than in 10 mM Ca2+ solution, we exerted similar experiments to compare the extracellular pH effects on the channel activation and permeation of Cav 3.2/WT, EEED, and EEDE (Fig. 1). Cumulative I–V data for Cav 3.2/WT, EEED, and EEDE were obtained in serial 10 mM Ba2+ solutions with different pH values (pH 9.1–5.5, Fig. 7A–C). Analysis of I–V data recorded in pH 9.1 Ba2+ solution showed that the potentials for half-maximal activation of EEED and EEDE are positively shifted, compared to that of Cav 3.2/WT (V50,act values for Cav 3.2/WT, EEED, and

Fig. 5. Anomalous mole fraction effects between Ba2+ and Ca2+ for Cav 3.2 and its pore mutants. The current traces were evoked by step pulses to +10 mV from a holding potential of −90 mV in a total 10 mM mixture containing various Ba2+ /Ca2+ ratios. (A–D) The currents through Cav 3.2/WT (A), EEED (B), EEDE (C), and EEEE (D) are recorded in the different Ba2+ /Ca2+ ratios, which are labeled beside current traces. (A) Cav 3.2/WT current was smaller in the 5/5 mixture than in the 10 mM Ca2+ or Ba2+ solution, indicating that AMFE is present in Cav 3.2/WT. (B–D) EEED and EEEE did not show AMFE. (F) Current amplitudes (mean ± SEM, n = 6–10) in various ratio mixtures normalized to current amplitude values in pure 10 mM Ba2+ for each channel (Cav 3.2, 䊉; EEED, ; EEDE, ; EEEE, ). The normalized amplitudes are plotted against various ratios of Ba2+ /Ca2+ .

EEDE = −35.6 ± 1.6, −27.1 ± 1.5, and −28.6 ± 1.1 mV, respectively; P < 0.05, unpaired Student’s t-test, n = 6–8) (Fig. 7D and E). The slope factors (Sact ) of activation curves for EEED and EEDE were greater than that of Cav 3.2/WT (Fig. 7F). The positive shifts and greater slope factors of activation curves for EEED and EEDE were similarly detected in all pH solutions (pH 9.1–5.5). Similarly in 10 mM Ca2+ solutions (Fig. 6), a change of extracellular solution from pH 9.1 up to pH 7.4 did change neither the half-maximal activation potential nor the slope factor for Cav 3.2/WT or each pore mutant. In contrast, application of pH 6.6 solution increased the half-maximal activation potential (P < 0.05) and further acidification to pH 5.5 increased both the half-maximal activation potential and the slope factor (P < 0.01 and 0.05, unpaired Student’s t-test, n = 6–8; Fig. 7E and F). In 10 mM Ba2+ solutions, we also characterized the proton blocking profiles of Cav 3.2 and the pore Asp mutants. When serial 10 mM Ba2+ solutions with different pH were superfused on oocytes expressing Cav 3.2/WT, EEED, or EEDE, individual channel currents elicited by +10 mV were blocked by extracellular protons. Similarly in 10 mM Ca2+ solutions (Fig. 6), the proton blocking percentages of Cav 3.2/WT plotted against pH values could be fitted for

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Fig. 6. pH effects on the activation properties of Cav 3.2/WT and EEDE in 10 mM Ca2+ . In serial 10 mM Ca2+ solutions with different pH, the currents through Cav 3.2/WT or EEDE channels were elicited by serial step potentials of −70 mV to +40 mV from a holding potential of −90 mV. (A and B) Representative I–V data for Cav 3.2/WT (A) or EEDE (B) recorded in serial 10 mM Ca2+ solutions with pH 9.1 (), 8.7 (), 8.4 (), 7.4 (), 6.6 (), and 5.5 (♦) were overlapped for comparison. The currents through EEED or EEEE in 10 mM Ca2+ solution were too small to be analyzed (not shown). (C) Activation curves of Cav 3.2/WT () and EEDE (䊉) in pH 9.1 Ca2+ solution. Chord conductance values obtained by dividing current amplitude by driving force were normalized and averaged, then plotted against test potentials and fitted using a Boltzmann function. The V50,act values of Cav 3.2/WT and EEDE are −33.23 ± 0.9 and −23.03 ± 0.5 (n = 6). (D and E) pH effects on the potentials (V50,act ) for half-maximal activation and slope factors of Cav 3.2/WT () and EEDE (䊉). I–V data at other different pH solutions were analyzed as above and then their V50,act values (D) and slope factors (E) were plotted against pH. (F) Proton blocking profiles of Cav 3.2/WT and EEDE currents. Serial changes of different pH solutions (pH 9.1, 8.9, 8.7, 8.4, 8.2, 7.4, 6.6, 5.8, and 5.5) inhibited currents through Cav 3.2/WT or EEDE evoked by +10 mV step pulses from a holding potential of −90 mV. Currents in different pH solutions were normalized to the current amplitude in pH 9.1 solution. Blocking percentages of currents of each channel were averaged and then plotted against pH. The blocking percentages were fitted with a Hill equation (Eq. (4)) for double binding sites with high affinity and low affinity. The estimated pK1 and pK2 for Cav 3.2/WT are 5.53 ± 0.19 and 8.96 ± 0.15 and the pK1 and pK2 values for EEDE are 6.15 ± 0.18 and 9.05 ± 0.14 (n = 7–9).

double binding sites with Eq. (4), but not for a single binding site. The estimated pK1 and pK2 values for Cav 3.2/WT are 5.92 ± 0.12 and 8.96 ± 0.13, respectively. Comparatively, the blocking percentages for the two pore mutants (EEED and EEDE) by pH 6.6 and lower pH solutions were greater than those for Cav 3.2/WT (Fig. 7G; unpaired Student’s t-test, P < 0.05, n = 7–9), but their blocking percentages by pH 7.4 and higher pH solutions were not significantly different. The pH blocking data for EEED and EEDE could also be

fitted for double binding sites of which pK1 and pK2 values for EEED are 6.36 ± 0.11 and 9.05 ± 0.15, and the pK1 and pK2 values for EEDE are 6.37 ± 0.10 and 9.11 ± 0.15, respectively. Statistical analyses showed that the pK1 values of EEED and EEDE are significantly bigger than that of Cav 3.2/WT (unpaired Student’s t-test, P < 0.05, n = 7–9), but their pK2 values were not significantly different. The significant increments of pK1 by the Asp-Glu mutations suggest that both Asp residues are involved in a low-affinity proton-binding site.

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Fig. 7. pH effects on the activation properties of Cav 3.2/WT, EEDE, and EEED in 10 mM Ba2+ solution. The currents through Cav 3.2/WT, EEED, or EEDE were elicited by the voltage protocol same as above, with serial perfusion of 10 mM Ba2+ solutions with pH 9.1 up to pH 5.5. (A–C) Representative I–V data for Cav 3.2/WT (A), EEED (B), or EEDE (C) in pH 9.1 (), 8.7 (), 8.4 (), 7.4 (), 6.6 (), and 5.5 (♦) solutions were overlapped. The currents through EEEE were too small to be analyzed (not shown). (D) Activation curves of Cav 3.2/WT (), EEED (), and EEDE () in pH 9.1 Ba2+ solution. Normalized chord conductance values were plotted against test potentials and fitted using a Boltzmann function. The V50,act values of Cav 3.2/WT, EEED and EEDE are −35.60 ± 1.60, −27.10 ± 1.50, and −28.60 ± 1.10, respectively (n = 6–8). (E and F) pH effects on the V50,act and slope factors of Cav 3.2/WT and EEDE. V50,act values (E) and slope factors (F) of Cav 3.2/WT (), EEED (), and EEDE () were plotted against extracellular pH. (G) Proton blocking profiles of Cav 3.2/WT, EEED, and EEDE currents. Application of serial pH recording solutions (from pH 9.1 up to 5.5) inhibited individual channel currents evoked by +10 mV step pulse. Currents in different pH were normalized to the current amplitude in pH 9.1 solution. Average blocking percentages are plotted against pH values and fitted with a Hill equation (Eq. (4)) for double binding sites. The pK1 and pK2 values for Cav 3.2/WT are 5.92 ± 0.12 and 8.96 ± 0.13, the pK1 and pK2 values for EEED are 6.36 ± 0.11 and 9.05 ± 0.15, and the pK1 and pK2 values for EEDE are 6.37 ± 0.10 and 9.11 ± 0.15, respectively (n = 7–9).

In contrast, the similar pK2 values (∼9) between Cav 3.2/WT and the pore mutants imply that they commonly contain a high-affinity site of which binding affinity is not changed by the pore mutations. 4. Discussion Different from the selectivity filters (EEEE) of HVA Ca2+ channels, those of LVA Ca2+ channels are made up of two Glu and two Asp at the pore loops of domains I–IV. We compared the

following properties of Cav 3.2 and its Asp mutants: ICa /IBa , GMaxCa /GMaxBa , voltage dependency of channel activation and steady-state inactivation, AMFE, Cd2+ blocking sensitivity, and extracellular pH effects. Our results indicated that both Asp residues at the pore loops of domains III and IV are distinctively involved in those biophysical properties of Cav 3.2. The peak amplitude of Ba2+ currents through most HVA 2+ Ca channels containing the “EEEE” filter is greater than Ca2+ currents through them, whereas Cav 2.3 shows the opposite [15]. In

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T-type channel isoforms, Cav 3.1 showed higher Ca2+ permeability than Ba2+ , while Cav 3.2 had lower Ca2+ permeability than Ba2+ . These indicate that although the “EEEE” and “EEDD” filters of VACC channels critically contribute to relative permeability of Ca2+ and Ba2+ , there exist additional regions affecting relative permeability (ICa /IBa ) in VACCs. For example, the EF hand-like region preceding the domain III pore loop was reported to affect ICa /IBa of the Cav 2.2 channel and its zinc blocking sensitivity as well [6,16]. In addition, point mutation of Phe or Tyr at the domain III pore loop could decrease Ba2+ conductance of Cav 1.2 channel [5]. These support the idea that, in addition to the pore locus of EEEE or EEDD, some residues adjacent to the pore locus can influence relative permeation between Ca2+ and Ba2+ in VACCs. Furthermore, it was reported that external Mg2+ could affect ICa /IBa of Cav 3.1, since Mg2+ in the external solution preferentially blocked Ba2+ over Ca2+ [17]. Although Cav 3.1/WT and Cav 3.2/WT commonly have EEDD in their pore structures, Cav 3.2/WT showed AMFE (Fig. 5A and E), while Cav 3.1/WT was reported not to show AMFE [8]. Interestingly, AMFE was abolished in Cav 3.2/EEED and Cav 3.2/EEEE in which the domain III Asp was replaced with Glu (Fig. 5B–E). Comparatively, the EEDE mutant displayed AMFE in some mixtures. On the contrary, the pore mutants (EEED and EEEE) of Cav 3.1 in which the domain III Asp was replaced with Glu gained AMFE [8]. The AMFE results from Cav 3.2/WT and the Cav 3.2 pore mutants are contradictory to those from Cav 3.1/WT and the Cav 3.1 pore mutants, suggesting that gain or loss of AMFE of Cav 3.1 and Cav 3.2 could not be simply interpreted by the presence of domain III Asp (or Glu) residue. Our tentative explanation for Cav 3.1 or Cav 3.2 to gain or lose AMFE is that not only the presence of EEEE and/or EEDD in their pore loops, but also other residues adjacent to the pore signature residues are likely to affect AMFE as well as relative permeability between Ba2+ and Ca2+ , discussed as above. Notably, the expressed currents of the mutant channels (EEED, EEDE, and EEEE) had much smaller amplitude than Cav 3.2/WT current. When an equal amount (10 ng) of Cav 3.2/WT cRNA or individual mutant cRNA was injected into oocytes, currents through the single mutants in response to +0 mV test potential were about ∼10-fold smaller than Cav 3.2/WT, respectively. Moreover, EEEE currents were about 300-fold smaller than Cav 3.2/WT. To chase the underlying mechanism(s), we tagged EGFP to the amino termini of Cav 3.2/WT and the mutant channels to compare their membrane targeting. Under a confocal microscope, we measured fluorescence intensities of the oocytes where 10 ng of EGFP-Cav 3.2 cRNA or an EGFP-tagged mutant cRNA was injected. Comparison of fluorescence intensities at the membrane showed that the average for EGFP-tagged Cav 3.2 was ∼2-fold higher than that of EGFPCav 3.2/EEDE or EGFP-Cav 3.2/EEEE (data not shown). The results indicate that the lower fluorescence levels of mutant channels are not sufficient to account for their smaller currents than Cav 3.2/WT. Whole-cell current can be expressed as a product of the number of channels in the membrane, opening probability, and singlechannel conductance. We speculate that the low expression of the mutant channels in current amplitude might come additionally from decreases of opening probability and/or single-channel conductance compared to Cav 3.2/WT. Considering that the pore mutants were constructed by replacing the pore Asp with Glu, which contains one carbon more in the side chain, we speculate that the longer side chain of Glu might distort the optimized structure of Cav 3.2, reducing single current amplitude, number of channels, and/or opening probability. To test these relevant factors, we expressed Cav 3.2/WT and the pore mutants in HEK 293 cells for ensemble fluctuation analysis [12], because currents recorded from oocytes by the two-electrode voltage clamping were known to be improper for fluctuation analysis. Compared to Cav 3.2/WT,

functional expression of EEDE was detected as small currents (less than 100 pA) in 10 mM Ba2+ solution by whole cell patch clamp. In comparison, expression of EEED and EEEE was not detected as any inward currents (n = 22, 18). We analyzed noise of the tail currents of Cav 3.2/WT and EEDE (see Section 2.5). The mean variances ( 2 ) were plotted against their mean current amplitude, and then the plotted data were fitted with “ 2 = iI(t) − I(t)2 /N”. The best fitting result for Cav 3.2/WT data was i of 0.5996 pA and N of 4342 (Supplementary Fig. 1A). In comparison, the result of the best fit for EEDE data is i of 0.2599 pA and N of 2171, in which N was set on the basis of the above fluorescence data (Supplementary Fig. 1B). These data suggest that the smaller current amplitude of EEDE mutant than Cav 3.2/WT comes from decrements of both single channel current and number of channels. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2013.06.006. The proton inhibition of Cav 3.2 currents was suggested to be due to a positive shift in the activation properties as well as an increment of the monovalent ion/Ca2+ relative permeability by channel protonation [18]. The activation properties of Cav 3.1 were reported to be similarly modulated by proton and the proton modulation profiles on channel gating were to be positively shifted by the pore Asp-to-Glu mutations [8,9]. A following study showed that extracellular Ca2+ counteracts the proton modulation effects on gating properties via competition of Ca2+ with proton for binding to surface charges [19]. Different from the proton blocking profile of Cav 3.1 fitted with a single binding site [8], we here report that the proton inhibition profile of Cav 3.2 in 10 mM Ca2+ or Ba2+ solutions was fitted with double proton binding sites with low- and high-affinity. Extracellular pH changes from 9.1 up to 7.4 inhibited the currents through Cav 3.2/WT and the Asp-Glu mutants with similar potencies, without significant shifts in the potentials of half-maximal activation. On the while, lower pH solutions (pH 6.6 up to 5.5) inhibited the Asp-Glu mutants more sensitively than Cav 3.2/WT, with positive shifts in the activation curves of Cav 3.2/WT and the Asp-Glu mutants in similar ways (Figs. 6 and 7). The result that the Asp-to-Glu mutations increased the inhibition sensitivity for only low affinity, implies that the Asp residues are involved in composing of the low affinity site. However, it should be considered that the increased pH sensitivity for the low affinity site could be influenced by the positive shift in channel activation by the pore mutations [18]. By the way, the similar pH modulation effects on the activation properties of Cav 3.2/WT and the Asp-to-Glu mutants suggest that the differences in the activation properties between Cav 3.2/WT and the pore mutants come from gating alterations by the structural modifications by the pore mutations, but not from altered proton binding affinity, as previously reported [9]. With regarding to the high affinity site, a recent report addressed which His residues in the S3-S4 loop and pore loop of domain I play crucial roles in the external proton effects on current amplitude as well as the activation properties of Cav 2.3 [20]. Based on the conservation of His residues at the two locations of Cav 2.3 and Cav 3.2, we hypothesize that the high-affinity proton binding site(s) may be related to His residues in the IS3-IS4 loop and I pore loop of Cav 3.2, of which the former was identified to be a critical site for nickel and zinc inhibition [13,14]. Our preliminary data showed that currents through Cav 3.2/His191Gln mutant were less potently inhibited by extracellular pH changes from pH 9.1 to 8.4 (data not shown), suggesting that His191 in the IS3-IS4 loop of Cav 3.2 is critical for the high affinity site. However, it remains to be further investigated how individual His residues in the IS3-IS4 loop and I pore loop contribute to the proton inhibition profile and the channel activation process of Cav 3.2 and whether extracellular protons compete with nickel (or zinc) ions on the His residues of Cav 3.2.

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We conclude that the Asp residues in the third and fourth pore loops of Cav 3.2 pore loops distinctively contributed to the biophysical properties of Cav 3.2, including divalent ion permeation, AMFE, Cd2+ blocking sensitivity, and extracellular proton effects. Additionally, we suggest that Cav 3.2 has two proton interaction sites, of which the pore Asp residues in the 3rd and 4th pore loops contribute to forming the low-affinity proton binding site. Acknowledgements This work was supported by Priority Research Centers Program (2012-0006690) through the National Research Foundation of Korea and a Sogang University Special Research Grant 2012 (201214003) to J.H. Lee. References [1] W.A. Catterall, Voltage-gated calcium channels, Cold Spring Harbor Perspectives in Biology 3 (2011) a003947. [2] D.D. Friel, R.W. Tsien, Voltage-gated calcium channels: direct observation of the anomalous mole fraction effect at the single channel level, Proceedings of the National Academy of Sciences of the United States of America 86 (1989) 5207–5211. [3] J. Yang, P.T. Ellinor, W.A. Sather, J.F. Zhang, R.W. Tsien, Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels, Nature 366 (1993) 158–161. [4] P.T. Ellinor, J. Yang, W.A. Sather, J.F. Zhang, R.W. Tsien, Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions, Neuron 15 (1995) 1121–1132. [5] X. Wang, T.A. Ponoran, R.L. Rasmusson, D.S. Ragsdale, B.Z. Peterson, Amino acid substitutions in the pore of the Ca(V)1.2 calcium channel reduce barium currents without affecting calcium currents, Biophysical Journal 89 (2005) 1731–1743. [6] Z.P. Feng, J. Hamid, C. Doering, S.E. Jarvis, G.M. Bosey, E. Bourinet, T.P. Snutch, G.W. Zamponi, Amino acid residues outside of the pore region contribute to Ntype calcium channel permeation, Journal of Biological Chemistry 276 (2001) 5726–5730.

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[7] E. Perez-Reyes, Molecular physiology of low-voltage-activated T-type calcium channels, Physiological Reviews 83 (2003) 117–161. [8] K. Talavera, M. Staes, A. Janssens, N. Klugbauer, G. Droogmans, F. Hofmann, B. Nilius, Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G), Journal of Biological Chemistry 276 (2001) 45628–45635. [9] K. Talavera, A. Janssens, N. Klugbauer, G. Droogmans, B. Nilius, Pore structure influences gating properties of the T-type Ca2+ channel alpha1G, Journal of General Physiology 121 (2003) 529–540. [10] A. Shcheglovitov, P. Kostyuk, Y. Shuba, Selectivity signatures of three isoforms of recombinant T-type Ca2+ channels, Biochimica et Biophysica Acta 1768 (2007) 1406–1419. [11] T. Kaku, T.S. Lee, M. Arita, T. Hadama, K. Ono, The gating and conductance properties of Cav 3.2 low-voltage-activated T-type calcium channels, Japanese Journal of Physiology 53 (2003) 165–172. [12] S.H. Heinemann, F. Conti, Nonstationary noise analysis and application to patch clamp recordings, Methods in Enzymology 207 (1992) 131–148. [13] H.W. Kang, J.Y. Park, S.W. Jeong, J.A. Kim, H.J. Moon, E. Perez-Reyes, J.H. Lee, A molecular determinant of nickel inhibition in Cav 3.2 T-type calcium channels, Journal of Biological Chemistry 281 (2006) 4823–4830. [14] H.W. Kang, I. Vitko, S.S. Lee, E. Perez-Reyes, J.H. Lee, Structural determinants of the high affinity extracellular zinc binding site on Cav 3.2 T-type calcium channels, Journal of Biological Chemistry 285 (2010) 3271–3281. [15] E. Bourinet, G.W. Zamponi, A. Stea, T.W. Soong, B.A. Lewis, L.P. Jones, D.T. Yue, T.P. Snutch, The alpha 1E calcium channel exhibits permeation properties similar to low-voltage-activated calcium channels, Journal of Neuroscience 16 (1996) 4983–4993. [16] H.S. Sun, K. Hui, D.W. Lee, Z.P. Feng, Zn2+ sensitivity of high- and low-voltage activated calcium channels, Biophysical Journal 93 (2007) 1175–1183. [17] J.R. Serrano, S.R. Dashti, E. Perez-Reyes, S.W. Jones, Mg2+ block unmasks Ca2+ /Ba2+ selectivity of alpha1G T-type calcium channels, Biophysical Journal 79 (2000) 3052–3062. [18] B.P. Delisle, J. Satin, pH modification of human T-type calcium channel gating, Biophysical Journal 78 (2000) 1895–1905. [19] K. Talavera, A. Janssens, N. Klugbauer, G. Droogmans, B. Nilius, Extracellular Ca2+ modulates the effects of protons on gating and conduction properties of the T-type Ca2+ channel alpha1G (Cav 3.1), Journal of General Physiology 121 (2003) 511–528. [20] T. Cens, M. Rousset, P. Charnet, Two sets of amino acids of the domain I of Cav 2.3 Ca(2+) channels contribute to their high sensitivity to extracellular protons, Pflugers Archiv 462 (2011) 303–314.