Measuring Ca²⁺-Dependent Modulation of Voltage-Gated Ca²⁺ (Ca_v) Channels in HEK-293T cells

Abstract

Voltage-gated Ca²⁺ (Ca_v) channels regulate a variety of biological processes, such as muscle contraction, gene expression, and neurotransmitter release. Ca_v channels are subject to diverse forms of regulation, including those involving the Ca²⁺ ions that permeate the pore. High voltage-activated Ca_v channels undergo Ca²⁺-dependent inactivation (CDI) and facilitation (CDF), which can regulate processes such as cardiac rhythm and synaptic plasticity. CDI and CDF slightly differ between Ca_v1 (L-type) and Ca_v2 (P/Q-, N-, and R-type) channels. Human embryonic kidney cells transformed with SV40 large T-antigen (HEK-293T) are advantageous for studying CDI and CDF of a particular type of Ca_v channel. HEK-293T cells do not express endogenous Ca_v channels, and Ca_v channels can be exogenously expressed at high levels in these cells through transient transfection. This protocol explains how to characterize and analyze Ca²⁺-dependent modulation of recombinant Ca_v channels in HEK-293T cells.

Overview

Voltage-gated Ca_v channels mediate Ca²⁺ signals that regulate cellular excitability, muscle contraction, gene expression, and hormone/neurotransmitter release. Ca_v channels are multi-subunit complexes that consist of a pore-forming α₁ subunit and auxiliary subunits, Ca_vβ and Ca_vα₂δ (Simms and Zamponi, 2014). Ten genes encode distinct α₁ subunits that display distinct pharmacological and biophysical properties (Ca_v1.x-Ca_v3.x). Mutations in these genes have been associated with disorders such as epilepsy, migraine, deafness, and congenital stationary night blindness (Pietrobon, 2010, Striessnig et al., 2010).

The α₁ subunit mediates Ca²⁺ entry, and the auxiliary subunits regulate trafficking and other properties of the channel. In addition, the Ca_v1 and Ca_v2 α₁ subunits are constitutively associated with calmodulin (CaM), which is essential for Ca²⁺-dependent inactivation (CDI) and facilitation (CDF). The N- and C-terminal lobes of CaM each contain 2 EF-hand Ca²⁺ binding domains. Increases in global and local Ca²⁺ entry through Ca_v channels are sensed by the N- and C-terminal lobes of CaM, respectively, which play distinct roles in CDI of Ca_v1 and Ca_v2 channels. CDI of Ca_v1 channels is mediated by the C-lobe of CaM and is insensitive to strong intracellular Ca²⁺ buffering. In contrast, CDI of Ca_v2 channels is mediated by the N-lobe of CaM and is inhibited by strong intracellular Ca²⁺ buffering. Interestingly, CDF of Ca_v2.1 channels is mediated by the C-lobe of CaM, and is spared by high concentrations of intracellular EGTA. The complex regulation of Ca_v channels by CaM has been described in recent reviews (Christel and Lee, 2012, Ben-Johny and Yue, 2014).

It should be noted that factors other than Ca²⁺ can influence inactivation of Ca_v1 and Ca_v2 channels. For example, inactivation of Ca_v channels can be driven by a purely voltage-dependent mechanism (voltage-dependent inactivation, VDI), which is seen for I_Ba. “Fast” VDI occurs from the resting (closed) state (Patil et al., 1998), while “slow” VDI occurs not only from the fast-inactivated state but also the open state (Sokolov et al., 2000). Both forms of VDI are influenced by Ca_vβ subunits. Some Ca_vβ subunits, like Ca_vβ_1b promote VDI, while the membrane-associated Ca_vβ, Ca_vβ_2a, diminishes VDI (Buraei and Yang, 2010). Since CDI can be masked by strong VDI, the use of Ca_v channels containing Ca_vβ_2a is a common approach in studies of CDI (Lee et al., 2000, DeMaria et al., 2001).

The α₁ subunit of Ca_v channels is subject to alternative splicing, which increases the functional diversity of Ca_v channels (Lipscombe et al., 2013). Such splicing events can either heighten or dampen Ca²⁺-dependent regulation of Ca_v channels. For example, inclusion of the alternatively spliced exon 42A of Ca_v1.3 channels results in enhanced CDI due to a truncation of a distal C-terminal autoregulatory domain (Singh et al., 2008). In addition, alternative splicing of exon 37 in Ca_v2.1 alters CDF (Soong et al., 2002, Chaudhuri et al., 2005). These examples illustrate the capability of splicing events to serve as molecular switches for CDI and CDF of Ca_v channels.

Other regulators of CDI and CDF of Ca_v channels include a family of Ca²⁺ binding proteins (CaBPs) that are related to CaM (Haeseleer et al., 2000). Unlike CaM, CaBPs contain at least one nonfunctional EF-hand, and are almost exclusively expressed in neurons. CaBPs may compete with and/or allosterically modulate CaM interactions with Ca_v1 channels, which inhibits CDI. For Ca_v2.1 channels, one CaBP family member, CaBP1 strongly inhibits CDI and VDI, and voltage-dependent activation. The regulation of Ca_v channels by CaBPs has been recently reviewed (Christel and Lee, 2012, Lee et al., 2014).

The methods described below present the basic framework for characterizing CDI and CDF of recombinant Ca_v channels in HEK-293T cells. These cells have proven ideal for studying the molecular determinants and biophysical mechanisms underlying CDI and CDF of Ca_v channels. Because of the presence of CDI/CDF regulatory factors such as CaBPs and alternative splicing in neurons, additional insights into the neurophysiological significance of CDI and CDF may be gained by studying these processes in various neuronal cell-types.

MATERIALS

Detailed recipes (<R>) are listed at the end of this protocol.

Reagents

• Ca_v channel cDNAs (examples shown below)

  • Ca_v2.1 (GenBank : NM023035.1) or Ca_v2.2 (GenBank: AF055477)

  • Ca_vβ_2a (GenBank: NM053581)

  • α₂δ₁ (GenBank: NM21948.1)

  • pEGFP (Life Technologies)

  • Culture medium: DMEM (GIBCO, 11965) + 10% FBS (Atlanta Biologicals, S11150)

  • DMEM (GIBCO, 11965)

  • Extracellular recording solution <R>

  • FuGene 6 Transfection Reagent (Promega, E2691)

  • HEK-293T cells (ATCC, CRL-11268)

  • Internal recording solution<R>

  • Versene (GIBCO, 15010-066)

Equipment

• Micropipettor (P1000)

• Borosilicate glass capillaries (WPI, TW 150-4)

• Cell Culture Dish (Corning, 35 mm × 10 mm)

• Data acquisition and analysis software

• Micropipette puller

• Microforge

• Patch-clamp electrophysiology setup including inverted fluorescence microscope, micromanipulators, amplifier, and perfusion setup

• Tissue culture hood Tissue culture incubator (37°C and humidified atmosphere with 5% CO₂)

METHOD

Transient transfection

• 1Plate HEK-293T cells in 35 mm culture dishes. Cells should be maintained in DMEM+ 10% FBS (2 mL) in a tissue culture incubator at 37°C with a humidified atmosphere and 5% CO₂.
• 2When cells are ~ 70% confluent, begin transient transfection with the cDNAs corresponding to the Ca_v channel of interest.
• 3About 30 minutes prior to transfection, aspirate culture medium and replace with fresh medium.
• 4In a sterile microfuge tube (1.5 mL), combine FuGene 6 transfection reagent (6 μL) with DMEM medium (100 μL). Then gently mix.

  Notes:

  For every 3 μg of DNA, use 2 μL of FuGene.

  Use DMEM without FBS for transfection mix.

• 5To the same tube, add cDNAs corresponding to the α₁ subunit (1.5 μg), Ca_vβ and α₂δ (0.5 μg each), and pEGFP (50 ng). Then gently mix.

  Note: pEGFP is used to help identify transfected cells via GFP fluorescence.

• 6Let mix sit at room temperature for 15 minutes.
• 7Next, add transfection mix to the cells in a drop wise manner covering the entire dish.
• 8Cells should be ready for recordings after incubation at 37°C for 24–72 hours. If waiting 48 hours, replace with fresh medium after 24 hours.
• 9To isolate cells for electrophysiological recordings, aspirate culture medium from transfected cells and replace with Versene (1 mL) for 1 minute. After 1 minute, aspirate the Versene and gently triturate the cell by pipetting up and down 10 times in culture medium (1 mL) with a P1000 micropipettor. Finally, for low-density plating, add cell suspension (1 to 2 drops) to culture dishes containing culture medium (2 mL).

  Notes:

  Allow cells to rest for at least 2 hours prior to recording at 37°C.

  Do not use trypsin-EDTA to isolate cells, as it may proteolyze channels

Whole-cell voltage-clamp recording

• 10Prior to recording, use a micropipette puller to produce electrodes with a resistance of 4–6 Megaohms (MΩ) in the extracellular recording solution. Polish the tips with a microforge. Electrodes can be coated with Sylgard to reduce pipette capacitance, although this is generally unnecessary.
• 11Remove culture medium and fill culture dish with extracellular recording solution (1 mL).
• 12After identifying a GFP-positive cell that has a spherical/boxlike shape, fill electrode with internal recording solution and begin electrophysiological recordings.

  Notes:

  Avoid oval and triangular shaped cells, which may yield distorted currents due to an inability to properly clamp the membrane voltage.

  The internal recording contains adenosine triphosphate (ATP) which may undergo auto-hydrolysis under some conditions. Therefore, while working solutions may be maintained at room temperature, for prolonged experiments, maintain the internal recording solution on ice.

• 13Obtain a gigaohm (GΩ) seal and change the holding voltage (V_h) to -80 mV.
• 14Apply negative pressure to rupture the membrane to gain whole-cell access. Before running voltage protocols, reduce capacitance and series resistance electronically (60–70%).

Characterization of CDI

For Ca_v2 channels, the internal recording solution should contain a relatively low concentration of EGTA (0.5 mM), as higher concentrations inhibit CDI (Fig. 1A). By contrast, CDI of Ca_v1 channels can be recorded with internal recording solutions containing a higher concentration of EGTA (5 – 10 mM).

Figure 1. CDI of Ca_v2.2 channels.

A,B) Top, Voltage protocols and representative current traces. Currents were leak-subtracted using the P/4 method. Bottom, I_res/I_peak represents residual current amplitude at the end of the pulse normalized to the peak current amplitude, and is plotted against test voltage. In A, internal recording solution contained 0.5 mM or 10 mM EGTA. In B, internal recording solution contained 0.5 mM and extracellular solution contained 10 mM Ca²⁺ (I_Ca ) or Ba²⁺ (I_Ba ). CDI = I_res/I_peak for I_Ba - I_res/I_peak for I_Ca, where I_res/I_peak for I_Ba = 0.69 ± 0.02 and I_Ca = 0.19 ± 0.07 for test pulse to 0 mV. Currents and averaged data for I_Ca (n=10) and I_Ba (n=8) are from different cells.

• 15Ca²⁺ currents (I_Ca) can be evoked by a long (1 second) depolarizing test pulse from V_h (-80 mV) to voltages ranging from -20 mV to +20 mV (Fig. 1A). Inactivation can be measured as the residual current amplitude at the end of the pulse normalized to the peak current amplitude (I_res/I_peak). A U-shaped curve is observed when I_res/I_peak is plotted against test voltages (Fig. 1), with maximal inactivation at the test voltage evoking maximal inward I_Ca.
• 16With Ba²⁺ as the permeant ion, Ca_v channels undergo inactivation that is voltage-rather than Ca²⁺ -dependent. To measure voltage-dependent inactivation (VDI), repeat step 16 with extracellular solution containing Ba²⁺ instead of Ca²⁺ (Fig. 1B). Ba²⁺-containing solution can be exchanged for the Ca²⁺-containing solution via a gravity-driven or pressurized perfusion system. Alternatively, population averages of cells recorded in Ca²⁺- or Ba²⁺-containing solution are often used, since the differences in CDI and VDI are robust.

  Note: Unlike Ca²⁺, Ba²⁺ binds poorly to calmodulin (CaM), an essential mediator of CDI.

• 17A convenient metric for CDI is the difference between I_res/I_peak for I_Ca and I_Ba (Fig. 1B).

Characterization of CDF for Ca_v2.1 channels

• 18To characterize CDF of Ca_v2.1 channels, the internal recording solution should contain a relatively high concentration of EGTA (10 mM), which will minimize CDI.
• 19To measure CDF, a test pulse from V_h (-80 mV) to 0 mV (P1) is given 1 s before, and 5 ms after (P2), a 50-ms prepulse to various voltages (Fig. 2A).
• 20Repeat step 21 with Ba²⁺ as the charge carrier (Fig. 2B).

  Note: The test pulse for P1 and P2 should be set about -10 mV for I_Ba as compared to that used for I_Ca. Due to surface charge screening effects, Ba²⁺ causes a negative shift in the voltage-dependence of activation, which should be compensated for in channel modulation protocols.

• 21Facilitation (F) is measured as the ratio of P2 to P1 and plotted against prepulse voltage. If P2/P1 is greater than 1, then the current is facili0ated.
• 22A convenient metric for CDF is the difference between facilitation of I_Ca and I_Ba at the prepulse voltage eliciting maximal facilitation of I_Ca (Fig. 2B).

Figure 2. CDF of Ca_v2.1 channels.

A) Top, voltage protocol and representative I_Ca and I_Ba evoked by 10-ms test pulses to 0 mV and -10 mV, respectively, before (grey) and after (red) a 50-ms prepulse to +30 mV. Currents were leak-subtracted using the P/4 method. B) The ratio of P2/P1 current amplitudes is plotted against prepulse voltage. CDF = the difference in P2/P1 for I_Ca and I_Ba using a +30-mV prepulse, where P2/P1 for I_Ca =1.4 ± 0.03 (n=4) and for I_Ba =1.2 ± 0.05 (n=4) for a +30-mV prepulse. Currents were recorded in 10 mM extracellular Ca²⁺ or Ba²⁺ and 10 mM intracellular EGTA. Currents and averaged data for I_Ca and I_Ba are from different cells.

RECIPES

Extracellular recording solution

Reagent	Final concentration (in mM)
CaCl2 or BaCl2	10
Tris	150
MgCl2	1

pH to 7.3 with methanesulfonic acid. Osmolarity should be around 290–310. Filter and store at at 4 °C.

Intracellular recording solution

Reagent	Final concentration (in mM)
EGTA	0.5 or 10
N-methyl-D-glucamine (NMDG)	140
HEPES	10
MgCl2	2
Mg-ATP	2

pH to 7.3 with methanesulfonic acid. Osmolarity should be around 290–310. Filter and store at Sterile filter and store in aliquots at -20°C.