G-proteins modulate invertebrate synaptic calcium channel (LCa_v2) differently from the classical voltage-dependent regulation of mammalian Ca_v2.1 and Ca_v2.2 channels

Voltage-gated calcium channels in the Ca_v2 channel class are regulators of synaptic transmission and are highly modified by neurotransmitter inputs that activate synaptic G-protein-coupled receptors (GPCRs). This was first described by Dunlap and Fischbach, who showed that activating GABAB, serotonin or adrenergic receptors shortened the action potential duration by inhibiting N-type calcium channels in chick sensory neurons (Dunlap and Fischbach, 1978; Dunlap and Fischbach, 1981). Since this seminal work was published over 25 years ago, much more is now known of the molecular mechanisms of the neurotransmitter-mediated regulation of GPCRs (Tedford and Zamponi, 2006). GPCRs are membrane-associated and complexed with the Ca_v2 calcium channels [N-type (Ca_v2.2) or P/Q-type (Ca_v2.1)] at synapses (Tedford and Zamponi, 2006). Upon receptor activation, G-protein βγ subunits dissociate from Gα-GTP subunits and directly bind and inhibit calcium channels at the synaptic membrane in a membrane-delimited manner (Herlitze et al., 1996). This inhibition is pertussis-toxin-sensitive (Tedford and Zamponi, 2006) and thus involves Gαi/o βγ subunits and is more pronounced for Ca_v2.2 than for Ca_v2.1 channels (Currie and Fox, 1997). The G-protein inhibition can be artificially relieved using electrophysiological protocols such as pre-pulse facilitation (PPF) (Bean, 1989). A strong conditioning, depolarizing pre-pulse promotes facilitating or enhancing calcium currents by temporarily disassociating the inhibitory Gβγ subunits from calcium channels (Tedford and Zamponi, 2006). Relief of the G-protein inhibition by the arrival of high-frequency action potential trains in the presynaptic terminal causes a temporary enhancement of intracellular calcium levels and transmitter secretion mediated by Ca_v2 channels (Park and Dunlap, 1998). This activity-dependent G-protein regulation in mammals serves as a critical form of short-term synaptic plasticity. A major question is whether this ubiquitous and versatile form of regulation is also found in simpler nervous systems, such as invertebrates.

We have previously cloned and expressed an invertebrate calcium channel homolog, LCa_v2, from the pond snail, Lymnaea stagnalis, which is a singleton homolog in invertebrates to the mammalian Ca_v2.1 and Ca_v2.2 channels (Spafford et al., 2003a). We previously showed that, like its mammalian counterpart, LCa_v2 mediates neurotransmitter secretion at nerve synapses (Spafford et al., 2003b) and robustly expresses in HEK-293T human cell lines in the presence of accessory α2δ and β subunits (Spafford et al., 2003a). Here, we report for the first time an evaluation of mammalian G-protein regulation in an invertebrate. We show that the invertebrate LCa_v2 channel does not exhibit any of the hallmarks of mammalian voltage-dependent G-protein inhibition. Instead, in vitro expressed LCa_v2 channels are inhibited slowly by the activation of cAMP, in a manner that does not depend on Gβγ subunits. A similar G-protein and cAMP-dependent inhibition of nifedipine-insensitive LCa_v2 currents is also consistent in native and identified Lymnaea VD4 neurons. While a form of inhibition of calcium channels through activation of G-protein subunits is present in invertebrate LCa_v2 channels, it is independent of voltage, slower to develop and requires enzymatic amplification steps mediated through generating a cellular second messenger, cAMP. The slower inhibition using a cellular messenger such as cAMP may meet the modulatory needs in invertebrates while an activity-dependent regulation, evolving in vertebrates, provides a more dynamic, fine-tuning of neurosecretion by regulating the influence of neurotransmitter inputs through presynaptic GPCRs.

To study the characteristics of the LCa_v2 calcium channel, the wild-type LCa_v2 gene was cloned into pIRES2-EGFP vector from a previously modified LCa_v2 construct, dubbed 5′RatCa_v2.1-LCa_v2, in pMT2 plasmid, which bears an N-terminus from mammalian Ca_v2.1 (Spafford et al., 2003a; Spafford et al., 2004; Spafford et al., 2006). The wild-type channel was reconstructed with the N-terminus of wild-type LCa_v2 (GenBank Accession No. AF484082) using XhoI and MluI restriction enzyme sites. The MluI restriction site was previously created by silent mutation of CGC to CGT, an arginine residue at position 232 using QuikChange mutagenesis (Spafford et al., 2003a). Similar silent mutagenesis was used to introduce silent restriction enzyme sites to facilitate the generation of chimeras. BamHI (TCG to TCC, serine residue at position 1096) and SpeI (CTT to CTA, leucine residue at position 1513) sites were created, which flanked 5′ and 3′ ends of the I–II linker, respectively. A BstbI site (GAG to GAA, glutamate residue at position 307) was created to delimit the 3′ end of the N-terminal sequence, while an XhoI site flanked the 5′ position upstream of the Kozak and start codon sequence. The corresponding I–II linker and N-terminus from rat Ca_v2.2 with compatible restriction site ends were used to replace the original LCa_v2 sequences, resulting in the LCa_v2 (_L1_R2_L3_L4) chimera and the LCa_v2 (_R1_L2_L3_L4) chimera, respectively.

All culture reagents as well as GenElute HP Endotoxin-Free Plasmid Maxiprep Kit for purification of plasmid DNA for transfections were purchased from Sigma-Aldrich Canada (Oakville, Ontario). For electrophysiology, 10 μg of calcium channel subunits [Ca_v2.2 (GenBank Accession No. NP_671482) or LCa_v2 α1 subunit, accessory subunits: α2δ1 (NP_037051), β1b (NP_059042)] and sometimes G-protein subunits Gβ1 (AAD00650) and γ2 (AAB82554) harbored in mammalian expression vectors were heterologously expressed by transfection using either calcium-phosphate or Lipofectamine (Invitrogen Canada, Burlington, Ontario) into human embryonic kidney cell line (HEK293T, M. Calos, Stanford University, USA) at 40–50% confluency. HEK-293T cells were cultured in DMEM with 10% FBS and supplemented with 0.5% (v/v) penicillin–streptomycin solution. At least 3–4 h before transfection, cells were re-plated in 60 mm (diameter) sterile Petri dishes containing 3–6 pre-sterilized poly-lysine coated glass cover slips (Circles No. 1; 0.13–0.17 mm thick; size, 12 mm; Fisher Scientific Canada, Ottawa, Ontario) used for recording. After overnight transfection, the cells were washed twice with culture media and incubated at 28°C in a humidified, 5% CO₂ chamber for three days. Cells were allowed to recover from washing at 37°C for 2 h and were then left at 28°C for at least 48 h before patching.

HEK-293T cells positively transfected with calcium channel subunits were identified by green fluorescence, emitted by EGFP on the bicistronic pIRES2–EGFP plasmid (Clonetech, Mountain View, CA, USA) containing LCa_v2 calcium channels. Electrophysiological recordings were carried out in voltage-clamp mode at room temperature, with an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA), while monitored by the epifluorescence microscope (Axiovert 40 CFL; Zeiss Canada, Toronto, Ontario). Cells were bathed in external solution containing barium as the charge carrier (20 mmol l⁻¹ BaCl₂, 1 mmol l⁻¹ MgCl₂, 10 mmol l⁻¹ Hepes, 40 mmol l⁻¹ TEA-Cl, 65 mmol l⁻¹ CsCl, 10 mmol l⁻¹ glucose, pH adjusted to 7.2 with TEA-OH, filtered through a 0.22 μm filter). Patch pipettes (25 MΩ; World Precision Instruments, Sarasota, FL, USA) were filled with internal solution (108 mmol l⁻¹ Cs-methanesulfonate, 4 mmol l⁻¹ MgCl₂, 9 mmol l⁻¹ EGTA, 9 mmol l⁻¹ Hepes, pH adjusted to 7.2 with CsOH, filtered through a 0.22 μm filter). Voltage commands were generated and data were acquired using a PC computer equipped with a Digidata 1440A interface in conjunction with pClamp10.1 software (Molecular Devices, Sunnyvale, CA, USA). Recorded currents were filtered at 10 kHz using a low-pass Bessel filter and digitized at a sampling frequency of 2 kHz. Only recordings with minimal leak (<10%) were used for analysis, and offline leak subtraction was carried out using the Clampfit 10.1 software (Molecular Devices). Series resistance was compensated to 70% (prediction and correction; 10 μs lag). Electrophysiology figures were illustrated in Origin 8 (OriginLab Corporation, Northampton, MA, USA).

Current–voltage relationships were obtained by holding cells at −100 mV before stepping to test potentials ranging from −50 to +60 mV for 150 ms. Ca²⁺ current activation curves were constructed by converting the peak current values from each current–voltage relationship data set to conductance using the equation G_Ca=I_peak/(V_command–E_Ca), where I_peak is the peak current, V_command is the command pulse potential, and E_Ca is the Ca²⁺ reversal potential as determined by linear extrapolation of the current values in the ascending portion of the current–voltage relationships. Conductance values were then normalized and individually fitted with the Boltzmann equation: G/G_max={1+[exp(−V_command−V_1/2)/k]}−1, where G is the peak conductance, G_max is the maximal peak Ca²⁺ conductance, V_command is the conditioning potential, V_1/2 is the half-maximal activation, and k is the activation slope factor.

The voltage dependence of inactivation was measured as the fraction of peak currents at a test depolarization step to +20 mV from a −100 mV holding potential, after steady-state voltage conditions, prepared with a long 4 s pre-pulse holding potential ranging from −90 to +30 mV. Normalized data were averaged and curve fit with a Boltzman equation I/I_max={1+exp[(V_inact−V_1/2/k)]}−1, where I is the peak test pulse current, I_max is the peak test pulse current when the conditioning pulse was −110 mV, V_inact and V_1/2 are the conditioning potential and the half-maximal inactivation, respectively, and k is the inactivation slope factor. Kinetics of activation, inactivation and deactivation were determined by fitting mono-exponential functions over the growing or decaying phases of each current trace using the software Clampfit 10.1.

To study the voltage-dependent G-protein facilitation, a paired pulse protocol was used to observe the pre-pulse facilitation. Facilitation was recorded by providing a +150 mV strong depolarization lasting for 50 ms, 25 ms before the 40 ms +20 mV testing potential, while cells were held at −100 mV. Facilitation was calculated by dividing the value of the peak current with a pre-pulse to the value of the peak current without a pre-pulse.

The great pond snail, Lymnaea stagnalis (Eukaryota; Metazoa; Mollusca; Gastropoda; Pulmonata; Basommatophora; Lymnaeoidea; Lymnaeidae; Lymnaea) were raised in 38 liter tanks linked through an 85% recirculating system with artificial freshwater at room temperature, and fed growth-chamber-raised romaine lettuce ad libitum, supplemented with spirulina pellets. Lymnaea neurons for whole-cell patch clamp recording were prepared from 2–3-month-old, juvenile snails. Outer and inner sheaths of Lymnaea brains were removed using fine forceps after trypsin exposure (2 mg ml⁻¹) and trypsin inhibitor (2 mg ml⁻¹) treatment. Isolated VD4 neurons were isolated from pinned brains in high osmolarity defined media (DMEM supplemented with 20 mmol l⁻¹d-glucose) using a fire-polished glass pipette (50 μm). Neurons were cultured onto poly-l-lysine-pretreated cover slips and recorded 18–24 h after incubation in brain conditioned media. VD4 neurons were recorded in 2 mmol l⁻¹ BaCl₂ plus (in mmol l⁻¹) 47.5 TEA-Cl, 1 MgCl₂, 10 Hepes and 2 4-aminopyridine (4-AP), pH 7.9 (adjusted with TEA-OH). VD4 neurons were recorded with 1.5–2 MΩ pipettes in calcium plus ATP/GTP-containing solution (in mmol l⁻¹): 29 CsCl, 2.3 CaCl₂, 10 ethylene glycol tetraacetic acid (EGTA), 10 Hepes, 2 ATP-Mg and 0.1 GTP-Tris, with pH 7.4 (adjusted with CsOH).

Standard HEK-293T cells tend to wash away during repeated antibody application and washing steps during an immunolabeling protocol. So, for immunolabeling work, a GripTite™ 293 MSR cell line (Invitrogen Canada Inc., Burlington, ON, Canada) was used instead, which is a genetically engineered cell line expressing a human macrophage scavenger receptor that promotes strong adherence of HEK-293T cells to culture plates. GripTite™ 293 MSR cells were transfected with calcium channel subunits and adhered onto cover slips coated with 1 μg ml⁻¹ poly-d-lysine (Sigma-Aldrich Canada) and incubated at 28°C for 5 days and fixed with 1% paraformaldehyde in PBS (preheated to 37°C) at room temperature for 2 h then at 4°C overnight. After washing, blocking was carried out by application of PBS-T 3% BSA and incubation at room temperature for 2 h. Rabbit polyclonal anti-LCa_v2 antibodies (Spafford et al., 2003a) were diluted 1:200 in PBS-T 3% BSA and applied to cells overnight at 4°C. Cells were then washed three times and then blocked with PBS-T 5% BSA for 45 min prior to application of secondary antibody. Cells were then incubated with Alexa Fluor 633 goat anti-rabbit IgG (H+L) (Molecular Probes™, Invitrogen, Eugene, OR, USA) secondary antibody diluted 1:5000 in PBS-T 3% BSA for 45 min at room temperature, then washed. Cover slips were dried at 37°C for 30 min and mounted onto glass slides using FluorSave™ Reagent (Calbiochem^® Biochemicals, EMD Chemicals, Inc., San Diego, CA, USA). Images were captured using a Zeiss LSM 510 META confocal microscope.

Invertebrates, including Lymnaea, usually possess only one gene homolog to mammalian calcium channels in each of the three families. Shown in Fig. 1A is a gene tree illustrating the relationships of human homologs to an identified set of calcium channel subunit genes from Lymnaea stagnalis, all previously expressed in human cell lines: LCa_v1 (Spafford et al., 2006), LCa_v2 (Spafford et al., 2003a) and LCa_v3 (Senatore and Spafford, 2010). Ca_v3 channels such as LCa_v3 bear transient, T-Type currents that are low voltage-activated (LVA) and open at resting membrane potentials (Senatore and Spafford, 2010). Ca_v1 and Ca_v2 channels are structurally more similar to each other and cluster together (see Fig. 1A). Opening of Ca_v1 and Ca_v2 channel gates requires strong depolarizations to a threshold significantly above the resting membrane potential, hence the term high voltage-activated (HVA). Ca_v1 channels produce L-type currents and mediate functions such as skeletal muscle and heart contraction, gene transcription and the endocrine release of hormones (Snutch et al., 2005). Ca_v2 channels, in particular Ca_v2.1 and Ca_v2.2 in mammals and invertebrate LCa_v2, are highly specialized non-L-type channels, which mediate neurotransmitter release at nerve synapses (Fig. 1A) (Spafford and Zamponi, 2003). Modulation of the activity of synaptic Ca_v2.1/Ca_v2.2 calcium channels by G-proteins is ubiquitously featured in mammals as a key mechanism of channel regulation (Tedford and Zamponi, 2006). Comparison with invertebrates provides insights into the structural and functional evolution of this mode of regulation.

To functionally characterize the G-protein regulation of the synaptic LCa_v2 channel in vitro, the full-length 6426 bp sequence coding for the 2144 amino acid LCa_v2 channel was placed in bicistronic vector pIRES2–EGFP, where enhanced GFP (EGFP) and LCa_v2 channel are generated from the same mRNA via transcription from a CMV promoter. Using EGFP fluorescence on the pIRES2-EGFP plasmid as a marker, cells containing adequately sized LCa_v2 currents could be readily identified for whole-cell patch clamp recording (see Fig. 1C). A minimum threshold of 100 nA amplitude was chosen for all electrophysiological recordings used in this study, which bore a mean ± s.e.m. for mammalian Ca_v2.2 of 287±38.91 nA (range=100.5–1197.44 nA, N=34) and a mean ± s.e.m. for Lymnaea LCa_v2 of 246±28.34 nA (range=100.2–567.7 nA, N=42) (Fig. 1B). Our first experiments were to measure parameters that are modulated by G-proteins such as voltage sensitivities (Fig. 2) and channel kinetics (Fig. 3).

LCa_v2 and mammalian Ca_v2.2 (Ca_v2.2) calcium currents (Fig. 2A) were generated in 10 mV voltage steps from a holding potential of −100 mV in whole-cell voltage clamp and were plotted as a peak current versus test voltage (Fig. 2B). Both LCa_v2 and Ca_v2.2 channels respond to depolarization as expected for HVA channels, with a threshold of current generation above resting potentials at approximately −20 mV and rising to a peak at 20 mV in the presence of 20 mmol l⁻¹ extracellular Ba²⁺ as the charge carrier (see Fig. 2B). The half-activation potential determined from the Boltzmann transformations of individual activation curves was nearly identical for LCa_v2 channels (8.4±0.708 mV, N=7) and Ca_v2.2 channels (8.3±0.573 mV, N=6) (Fig. 2C). Steady-state availability curves were generated by measuring the fraction of maximal current during a test pulse after a long 4 s pre-pulse at different voltages. The half-inactivation potential generated from Boltzmann curve fits of individual availability curves corresponds to voltages at which there were 50% available and non-inactivated channels during steady-state conditions. Both LCa_v2 and Ca_v2.2 have voltage sensitivities of inactivation in the range expected for HVA channels, although the half-inactivation potential for LCa_v2 (−39.3±0.453 mV, N=9) was ~10 mV more positive than for Ca_v2.2 (−49.2±0.422 mV, N=10) (Fig. 2D).

Dramatic differences are apparent for the gating kinetics between LCa_v2 and Ca_v2.2 channels. This is illustrated in Fig. 3A, plotted as averaged, normalized current ± s.e.m. of N=6 current traces for LCa_v2 and Ca_v2.2 channels elicited by a single voltage step to +40 mV for 150 ms from a holding potential of −100 mV. Tau values extrapolated from the single exponential curve fits are illustrated for kinetics of activation (Fig. 3B) and kinetics of inactivation decay (Fig. 3C). At every potential, LCa_v2 had significantly faster activation kinetics than Ca_v2.2 and also faster kinetics of inactivation decay at potentials above +20 mV. Evaluation of the effects of G-proteins on biophysical parameters such as activation kinetics for LCa_v2 and Ca_v2.2 channels is illustrated in Fig. 4.

Pre-pulse relief of G-protein inhibition was generated with a strong depolarizing pre-pulse (150 mV for 50 ms duration), 25 ms preceding the current generating test pulse (+PP), and the degree of facilitation was measured as the ratio of current change in the presence (+PP) and absence (−PP) of a G-protein relieving pre-pulse. Ca_v2.2 currents were facilitated dramatically, almost doubling the maximal peak current size (1.95±0.11 +PP/−PP ratio, N=8; see Fig. 4B), in response to a strong depolarizing pre-pulse when Ca_v2.2 channels were co-expressed with G-protein β1and γ2 subunits. Comparing sample traces, the voltage-dependent relief of G-protein inhibition for Ca_v2.2 (Fig. 4A; sample trace, right) was not evident for LCa_v2 (see Fig. 4A; sample trace, left). Background expression of endogenous G-protein subunits in HEK-293T cells provides a reduced (1.37±0.07 +PP/−PP ratio, N=9), but still statistically significant, pre-pulse facilitation of Ca_v2.2, while there was no enhancement of current with a depolarizing pre-pulse for LCa_v2 in the absence (1.01±0.03 +PP/−PP ratio, N=8) or presence (0.95±0.03 +PP/−PP ratio, N=7) of exogenous G-protein subunits. The LCa_v2 +PP/−PP ratio near 1.0 reflects a lack of voltage-dependent G-protein modulation for LCa_v2 channels.

Hallmark features of G-protein modulation, such as changes in activation kinetics and voltage sensitivity (Tedford and Zamponi, 2006), are also absent for LCa_v2. Gβγ bound to Ca_v2.2 channels characteristically slows the onset of channel opening, leading to a delayed rate of rise to the generation of peak current (−PP; see Fig. 4A, right), while activation kinetics becomes faster after pre-pulse relief of the Gβγ inhibition (+PP; see Fig. 4A, right). The faster activation kinetics for Ca_v2.2 in the presence of pre-pulse relief of G-protein inhibition is reflected in the shorter tau constants derived from exponential fits of the rate of activation [2.600±0.161 (+PP) versus 2.951±0.268 (−PP), N=9]. Faster activation kinetics and shorter tau constants during pre-pulse relief of G-protein inhibition were even greater when exogenous Gβγ was cotransfected with Ca_v2.2 [2.165±0.187 (+PP) versus 2.860±0.302 (−PP), N=8] (Fig. 4C).

As expected for channels that are not regulated by G-proteins in a voltage-dependent manner, LCa_v2 did not bear any kinetic differences in activation (+PP or −PP; see Fig. 4A, left), which was reflected in unchanging activation tau constants in the absence [0.845±0.0902 (+PP) versus 0.853±0.0631 (−PP), N=7] or presence of exogenous G-protein βγ subunits [0.686±0.125 (+PP) versus 0.630±0.118 (−PP), N=8]. Normally, G-proteins also shift the activation voltage to more depolarizing potentials but this was also not apparent for LCa_v2 (Fig. 4D).

The lack of G-protein modulation of LCa_v2 channels under the identical conditions for Ca_v2.2 channels, such as the presence of accessory α2δ1 and Ca_vβ1 subunits and G-protein β1 and γ2, suggests that there are structural elements lacking for mammalian G-protein modulation in the LCa_v2 channel. Voltage-dependent G-protein modulation requires pre-association of a Ca_vβ subunit to the Ca_v2 α1 subunit, so the determinants for Ca_vβ subunit binding are also critical for understanding voltage-dependent G-protein modulation (Zhang et al., 2008). Key structures in the calcium channel for Ca_vβ subunit association include the N-terminal end of the cytopasmic I–II linker of the Ca_v2 channel, which can be divided into a rigid α-helical IS6-AID linker sequence (20 amino acids) (Vitko et al., 2008) followed by an α-helical, alpha-interacting domain (AID) (18 amino acids) (Opatowsky et al., 2004) (see Fig. 5A,C). Primary sequences for G-protein βγ binding with Ca_v2–Cavβ complexes are reported to primarily include sequences in the I–II linker at a Gβγ-1 site overlapping with Ca_vβ and a downstream Gβγ-2 site in the I–II linker (Fig. 5C) (Tedford and Zamponi, 2006). The N-terminus of Ca_v2.2, labeled NTB (Fig. 5B), has also been identified as significant in G-protein βγ binding (Agler et al., 2005).

The I–II linker or the N-terminus of Ca_v2.2 was swapped into LCa_v2 to test whether either of these regions would endow LCa_v2 channels with mammalian voltage-dependent G-protein modulation. Unique restriction sites spanning the N-terminus (XhoI–BstBI) and I–II linker (BamHI–SpeI) were created by site-directed mutagenesis for insertion of PCR-based synthetic DNA fragments of the Ca_v2.2 sequence with appropriate restriction site ends. G-protein modulation was evaluated with the pre-pulse facilitation protocol. Absence of enhancement of current resulting from the depolarizing pre-pulse suggests that neither LCa_v2 harboring the I–II linker (L1R2L3L4LCa_v2, N=5) nor the N-terminus (R1L2L3L4-LCa_v2, N=6) of Ca_v2.2 create voltage-dependent G-protein modulation in LCa_v2, even in the presence of exogenous G-protein βγ subunits (Fig. 6).

Preliminary investigation suggests that invertebrate synaptic Ca_v2 channels are inhibited by G-proteins but that it does not involve Gβγ subunits (Spafford et al., 2003a). Treatment of LCa_v2 channels with a non-hydrolysable GTP analog, GTP-γS, leads to a constitutive activation of G-proteins and a voltage-independent modulation, where peak currents run down over time without relief of the inhibition by depolarization pre-pulses (Spafford et al., 2003a). A possible downstream target for this G-protein activation is a cytoplasmic second messenger, which may mediate the inhibition of LCa_v2 channels. Micro perfusion of a cell-permeable cAMP analog, 8-bromo-cAMP (8Br-cAMP), caused a slowly developing inhibition of LCa_v2 channel activity in vitro that required 1–3 min to develop (see Fig. 7A, sample time course) and reached an average peak of inhibition of 33.7±9.4% (N=6) for LCa_v2 currents (Fig. 7B). The possible involvement of G-protein βγ subunits was specifically addressed by co-transfection of βark-ct, the C-terminus of the beta adrenergic receptor kinase. βark-ct binds G-protein βγ subunits with high affinity and thus serves as a scavenger of G-protein βγ subunits. 8Br-cAMP did inhibit LCa_v2 channels in the presence of βark-ct, suggesting that GPCR activation elevates cAMP levels through activation of G-protein α but not βγ subunits.

The inhibition of LCa_v2 channels via a cAMP pathway was confirmed in Lymnaea VD4 neurons. VD4 neurons bear two HVA currents, including an L-type (or LCa_v1) current and an LCa_v2 current, and they operate in the same voltage range. It was previously shown that LCa_v2 channels can be separated from LCa_v1 channels with 10 μmol l⁻¹ nifedipine, which completely blocks LCa_v1 channels without affecting LCa_v2 channels in vitro (Spafford et al., 2006). LCa_v1 current generally comprises ~20% of the total calcium current in VD4 neurons, but this value may vary between 10% and 35% depending on the cell (Spafford et al., 2006). The nifedipine-insensitive calcium current is likely to be conducted by LCa_v2 channels, since it can be blocked with 100 μmol l⁻¹ cadmium, which completely blocks LCa_v2 in vitro (Spafford et al., 2006). Perfusion of 8Br-cAMP onto the nifedipine-insensitive currents in VD4 neurons caused a slowly developing inhibition (2–5 min) (Fig. 7C) that peaked at 26±11% inhibition (N=4; Fig. 7D). This response to cAMP corresponds to the in vitro results of a cAMP-mediated inhibition of LCa_v2 via a voltage-independent activation of G-proteins, through Gα and not Gβγ subunits.

Calcium ions passed through voltage-gated calcium channels service a diverse array of functions, including the activation of highly sensitive intracellular signaling pathways that respond to slight changes in cytosolic Ca²⁺ levels. For this reason, even slight changes in channel gating characteristics can cause dramatic responses such as at nerve synapses, where the secretory capacity is strongly dependent on the gating behavior of Ca_v2 channels. G-protein βγ subunits participate in a form of short-term synaptic plasticity because of the voltage-sensitive nature of the G-protein binding to Ca_v2 channels (Tedford and Zamponi, 2006). Relief of the G-protein βγ inhibition by action potential trains provides temporary rises in intracellular calcium and corresponding increases in neurosecretion (Park and Dunlap, 1998). The inhibition and disinhibition of Ca_v2 channels is highly responsive to changes in the pool of activated Gβγ subunits from neurotransmitter inputs that secrete onto GPCRs of the presynaptic membrane (Tedford and Zamponi, 2006). Variety in types of GPCRs and G-proteins and distinctly different regulation between Ca_v2.1 and Ca_v2.2 channels provide enormous variety in neurocomputational possibilities (Tedford and Zamponi, 2006). It is particularly important to evaluate whether this ubiquitous and versatile mechanism in mammals is also present in Ca_v2 channels of the invertebrate nervous system.

The relationship between Ca_v2 and other calcium channels is reflected in the gene tree of homologs in Fig. 1. A likely genomic duplication event led to speciation of genes within the three calcium channel classes (Ca_v1, Ca_v2 and Ca_v3), whilst the invertebrates mostly retained single gene homologs in the three classes. Ca_v3 channels operate at the resting membrane potential and thus are more associated with shaping the firing behavior of action potentials and providing calcium at resting membrane potentials (Senatore and Spafford, 2010). Ca_v1 and Ca_v2 channels require a strong depolarization to a firing threshold and thus are recruited by nerve activity (Snutch et al., 2005). Ca_v1 and Ca_v2 channels serve as electromechanical response units transducing action potentials to functions. Examples for Ca_v1 channels include skeletal muscle contraction (Ca_v1.1) and heart contraction (Ca_v1.2), gene transcription (Ca_v1.2), endocrine release of hormones (Ca_v1.3) and vision (Ca_v1.4) (Snutch et al., 2005). Mammalian Ca_v2.1 and Ca_v2.2 channels and invertebrate LCa_v2 are specialized channels for synaptic junctions, where they are closely embedded with the release machinery associated with the release of transmitter at the presynaptic membrane (Spafford and Zamponi, 2003).

Side-by-side comparison of invertebrate LCa_v2 and mammalian Ca_v2.2 channels in vitro indicates that the voltage sensitivity of both activation and inactivation is similar, although LCa_v2 bears a ~10 mV shift in the steady-state inactivation compared with Ca_v2.2 (Fig. 2).

Similar voltage sensitivities between vertebrate and invertebrate orthologs are indicative of similar responsiveness to calcium channel gating during action potential volleys served to the presynaptic terminal. Constraints on the voltage range of channel activity reflect the shared responsibility of LCa_v2 and Ca_v2.2 in gating the precise rises in intracellular calcium for mediating transmitter release at nerve synapses (Dodge et al., 1967).

More variable between calcium channels is the rate of change of gating modes, reflected in activation and inactivation kinetics and interpreted as tau curve fits of the descending and rising slopes, respectively, of the whole-cell currents. LCa_v2 has dramatically faster activation and inactivation kinetics and shorter tau constants for activation and for inactivation in the voltage range greater than +20 mV (Fig. 3). Activation and inactivation rates are highly variable amongst Ca_v1 and Ca_v2 calcium channels and can be modified to a tremendous extent by accessory subunits, especially the cytoplasmic beta subunit that it is paired with (Snutch et al., 2005). Mammals have four different beta subunit genes (β1, β2, β3 and β4), each possessing tremendous variability in alternative splicing, which serves to fine-tune biophysical parameters such as gating kinetics of the Ca_v1 and Ca_v2 channels (Snutch et al., 2005). Although invertebrates, such as Lymnaea, have only a single β subunit gene (Spafford et al., 2004), we have observed parallel alternatively spliced patterns of similar exons in the N-terminus and HOOK region, which can create diversity in gating kinetics of invertebrate Ca_v1 or Ca_v2 channels (T.F.D., S. Harel, A.S., A. Boone and J.D.S., unpublished observations).

A voltage-dependent G-protein modulation is ubiquitously featured in mammalian Ca_v2.1 and Ca_v2.2 channels (Tedford and Zamponi, 2006). Gβγ subunits are considered to stabilize a closed state conformation of channels in a ‘reluctant’ gating mode (Bean, 1989). The transition from a reluctant to ‘willing’ gating mode can be simulated artificially using stimulation protocols such as pre-pulse facilitation (PPF) (Tedford and Zamponi, 2006). As observed for Ca_v2.2 currents, G-protein inhibition was associated with a delay in activation kinetics (measured as longer activation time constants) (Bean, 1989). Strong depolarizing pre-pulses were facilitating as reluctant calcium channels unbind from Gβγ subunits and transition to an opening willing mode (Fig. 4) (Bean, 1989). LCa_v2 completely lacked any of the features of voltage-dependent G-protein modulation such as kinetic slowing of inactivation, pre-pulse facilitation or change in voltage dependence of activation (Fig. 4). The lack of voltage-dependent G-protein modulation suggests that the mammalian Ca_v2.2 sequence that renders the channels sensitive to voltage-dependent G-protein inhibition is missing in LCa_v2.

A key feature of mammalian G-protein modulation is a necessary pre-associated accessory beta subunit to Ca_v2.2 channels, but the key binding surfaces between invertebrate LCa_v2–Ca_vβ subunits are highly conserved (Spafford et al., 2004). Thus, it is not likely that invertebrate calcium channel beta subunit interactions are responsible for the absence of mammalian G-protein modulation. Ca_v1/Ca_v2 channels associate via a highly conserved AID peptide (alpha-interacting domain) sequence in the cytoplasmic I–II linker, which forms an alpha helix that is deeply embedded in the guanylate kinase (GK) domain of Ca_vβ (Fig. 5B) (Opatowsky et al., 2004). Also important upstream of the AID sequence is a highly conserved IS6–AID sequence, which also contributes to a rigid secondary structure for beta subunit binding and is considered to be important for voltage-dependent charge movements of IS6 (Vitko et al., 2008) and to drive the G-protein βγ unbinding (Fig. 5B) (Zhang et al., 2008). Disruption of the rigid α-helical structure in the IS6–AID sequence or prevention of Ca_vβ subunits from binding to the AID sequence completely eliminates voltage dependence of G-protein regulation (Zhang et al., 2008).

G-proteins closely associate with the region of the I–II linker that associates with β subunits, and indeed one putative Gβγ binding site (Gβγ-1), with a signature QQIER motif, is not likely to be a key determinant because the sequence is buried when Ca_vβ is complexed with Ca_v2 channels (Fig. 5) (Opatowsky et al., 2004). Gβγ-2 serves as a second identified Gβγ binding site downstream of Gβγ-1 in the I–II linker (Tedford and Zamponi, 2006), and its sequence is not conserved between LCa_v2 and Ca_v2.1/Ca_v2.2 channels (Fig. 5B). Gβγ subunits also associate with an NTB sequence in the N-terminus of Ca_v2.2 (Fig. 5B) (Agler et al., 2005) that is not well conserved in LCa_v2 channels. However, neither the replacement of the I–II linker nor the N-terminus of Ca_v2.2 into LCa_v2 was sufficient (Fig. 6) to endow LCa_v2 with mammalian G-protein modulation. One possibility reported by Yue and colleagues is that the N-terminus and I–II linker unite together and form a platform for G-protein modulation (Agler et al., 2005), but testing this option has not been possible since double replacement of the N-terminus and I–II linker of Ca_v2.2 into LCa_v2 did not produce channels with sufficiently resolvable currents expressed in vitro.

Despite lacking determinants for voltage-dependent regulation, LCa_v2 is modulated by G-proteins in a voltage-independent manner that does not involve Gβγ subunits. In particular, activation of G-proteins inhibits calcium channel activity through a cytosolic messenger cascade involving cAMP that progresses over a slow time course (minutes). It is likely that the downstream target of cAMP is the LCa_v2 channel, since the degree of cAMP inhibition of LCa_v2 alone, transfected in HEK-293T cells (Fig. 7B, 26±11% inhibition, N=4), was similar to the degree of cAMP inhibition (Fig. 7D; 33.7±9.4%, N=6) of the HVA, nifedipine-insensitive current in VD4 neurons. Ca_v2 channel inhibition through a cAMP-mediated pathway provides a means of regulating transmitter release by activation of dopamine (Barnes et al., 1994) or serotonin (McCamphill et al., 2008) GPCRs on presynaptic Lymnaea VD4 neurons. Voltage-independent regulation of Ca_v2.2 channels has also been reported in select mammalian neurons, utilizing a number of different G-protein-dependent second messenger pathways (Tedford and Zamponi, 2006).

All Ca_v2.2 channels in mammalian neurons are inhibited by means of the voltage-dependent pathway mediated by direct interaction of βγ subunit heterodimers (Tedford and Zamponi, 2006). While the only form of G-protein regulation for LCa_v2 is voltage independent, this form of regulation is only observed in select neuron types in mammals (Tedford and Zamponi, 2006). Interestingly, just the selective inclusion of a short C-terminal exon (exon 37a) in Ca_v2.2 channels creates a tyrosine kinase phosphorylation site for voltage-independent G-protein modulation in mammalian nociceptive neurons (Raingo et al., 2007).

A question arises as to why some mammalian neurons selectively maintain voltage-independent forms of G-protein regulation while simultaneously maintaining a ubiquitous, voltage-dependent form (Tedford and Zamponi, 2006). Voltage-dependent G-protein modulation exerts an inhibition and relief of inhibition on Ca_v2.1/Ca_v2.2 channels in a manner proportional to a changing pool of activated Gβγ subunits. This allows for rapid and dynamic regulation of intracellular calcium and neurosecretion by transmitters that secrete onto appropriate presynaptic GPCRs in the presynaptic terminal. Changes in calcium channel activity occur rapidly and with little amplification and take place in conditions where the ligands for GPCRs are in abundance. Also, the voltage-dependent unbinding of Gβγ subunits operates like a high-pass or low-cut filter. Brief stimuli do not dislodge Gβγ subunit complexed to Ca_v2 channels, but stronger stimuli overcome the inhibition, with a relief of Gβγ subunit inhibition that is in proportion to the firing of action potential trains. Gβγ subunit inhibition thus serves as a critical form of short-term synaptic plasticity that causes a temporary enhancement of neurotransmitter release with the arrival of high-frequency action potential trains in the presynaptic terminal.

Voltage-independent inhibition lacks the dynamic aspects of the Gβγ inhibition but uniquely provides a persistent inhibition independent of cellular activity through activation of Gα subunits. The inhibition has a slower onset and recovery and requires amplification through cell signaling cascades. Many different intracellular pathways may converge into the cell signaling cascade, providing a highly modifiable response. Another advantage is a consistency in the inhibition even if the GPCR ligand is not in great abundance. Both forms of G-protein inhibition thus provide qualitatively different responses. The combination of both pathways simultaneously utilizes both Gα and Gβγ of the heterotrimeric G-protein in the inhibition of calcium channels to maximize the fine-tuning of calcium influx in mammals.

Lack of the voltage-dependent G-protein modulation in LCa_v2 suggests that unique determinants, such as in the N- and C-terminus in mammalian synaptic calcium channels, evolved as a vertebrate specialization for G-protein modulation. While it is not possible to discount other factors that could account for the lack of voltage-dependent modulation of LCa_v2, such as structural differences in the invertebrate Ca_vβ subunits or the G-proteins themselves, as being incompatible with mammalian G-protein modulation, these are not likely to be of importance due to the high conservation of Ca_vβ subunits and G-protein subunits in invertebrates. Lymnaea G-protein β1 (GenBank Accession # CAA80652) is 89% similar and 84% identical to mammalian G-protein β1 subunit, and all the critical determinants circumscribed for G-protein modulation (Tedford et al., 2006) are also conserved in Lymnaea G-protein β1. Each of the four Ca_vβ subunit types (β1, β2, β3, β4) influences the degree of G-protein modulation, but only a minimal and highly conserved core GK domain invariant in invertebrate beta subunits is required for mammalian voltage-dependent G-protein modulation (Zhang et al., 2008).

Other modulatory structures, such as the synprint region, are also lacking in invertebrate Ca_v2 channels, suggestive of a primitive condition for invertebrate synaptic calcium channels (Spafford et al., 2003b). Synprint is a large 245 amino acid platform in the cytoplasmic II–III linker of Ca_v2 channels for binding of synaptic proteins such as syntaxin1, SNAP-25, cysteine string protein (CSP) and synaptotagmin (see Fig. 5B) (Spafford and Zamponi, 2003) and serves as a highly integrated center for modulation. For example, syntaxin1A binding to the synprint region promotes voltage-dependent G-protein inhibition of Ca_v2.2 channels (Jarvis et al., 2002), while PKC-dependent phosphorylation of the channel antagonizes the G-protein inhibition via βγ subunit dimers (Viard et al., 2004). Mammalian sequences have likely been adapted to embed Ca_v2 channels into the synaptic vesicle fusion apparatus and couple with GPCRs. These form part of a presynaptic complex in mammals that contains a web of regulatory and scaffolding proteins in the active zone (Schoch and Gundelfinger, 2006). Invertebrates exhibit a synaptic organization lacking key structural proteins such as Bassoon and CAST present in mammalian synapses and bear a synaptic substructure, like the Drosophila T-bar, which is unlike the mammalian presynaptic density (Atwood, 2006). The lack of activity-dependent G-protein regulation is one of these features lacking in invertebrates. A slower inhibition using a cellular messenger like cAMP may meet the modulatory needs in invertebrates while an activity-dependent regulation, evolving in vertebrates, provides a more dynamic, fine-tuning of neurosecretion.

This research was supported by NSERC of Canada, and a NSERC CGS-D graduate scholarship to A.S. We thank Adrienne Boone for editing of the manuscript.

 
• 8Br-cAMP

  8-bromo cyclic AMP

 
• CMV

  cauliflower mosaic virus

 
• EGFP

  enhanced green fluorescent protein

 
• G

  conductance

 
• GPCR

  G-protein-coupled receptor

 
• HEK-293T

  human embryonic kidney cells 293T

 
• HVA

  high voltage-activated channel

 
• I

  current

 
• LVA

  low voltage-activated channel

 
• PP

  pre-pulse

 
• V_command

  conditioning potential

 
• βark-ct

  the C-terminus of the beta adrenergic receptor kinase