cDNA for NaV1.5 (hH1a) was kindly provided by H. Hartmann and A. Brown (Hartmann et al. 1994). Four-primer PCR (Higuchi et al. 1988; Ho et al. 1989) was used to substitute a cysteine for the native residue at one or more sites in the channel as follows (using the numbering system of hH1; Makielski et al. 2003): (1) the phenylalanine in the IFM motif at amino acid position 1485 (ICM), (2) the third outermost charged residue (an arginine) in DIII at position 1306 (R3C-DIII), and/or (3) the second outermost arginine at position 1626 in DIV (R2C-DIV). These were used to construct WT-ICM, R3C-DIII, R3C-DIII + ICM, R2C-DIV, R2C-DIV + ICM, R3C-DIII + R2C-DIV, and R3C-DIII + R2C-DIV + ICM. The entire inserts containing the mutated sites were confirmed by sequencing. In addition, in all constructs a tyrosine was substituted for the cysteine within the external vestibule of the pore at position 373 (C373Y) to increase the sensitivity to block by tetrodotoxin (TTX) (Chen et al. 1996) and to prohibit its reaction with extracellular MTS reagents. For expression in mammalian cells, the cDNAs were subcloned directionally into the mammalian expression vector pRcCMV (Invitrogen, Carlsbad, CA, USA).
Single tsA201 cells (SV40 transformed HEK293 cells) cells were fused as previously described (Sheets et al. 1996) or by the following procedure. tsA201 cells were grown on 100 mm Petri dishes until approximately 95% confluent. Four hours after the addition of fresh standard medium (Dulbecco's modified Eagle's medium (DMEM) from Sigma Corp. supplemented with 10% fetal bovine serum and 1% pencillin–streptomycin), the plate was then washed with medium containing only DMEM. Two millilitres of fusion solution containing 50% polyethylene glycol (Mr 1450, ATCC, Manassas, VA, USA) was added dropwise to the cells before incubating for 10 min at 37°C for 10 min. The fusion solution was then aspirated, 10 ml of standard medium was added, and the cells were placed back in the incubator at 37°C overnight. The next day the fused tsA201 cells were separated from single cells using a 20 μm mesh filter as previously described (Sheets et al. 1996).
In both cases cells were transiently transfected using a calcium phosphate precipitation method (Invitrogen, Carlsbad, CA, USA) either before or after fusion. TTX (600 nm) and/or lidocaine (100 μm) were added to the standard culture medium of cells expressing ICM mutations because anecdotal evidence suggested that expression levels of inactivation-impaired Na+ channel mutants may be increased. Three to six days after transfection fused cells were detached from culture dishes with trypsin-EDTA solution (Gibco, Grand Island, NY, USA) and studied electrophysiologically.
Recording technique, solutions, experimental protocols and analysis
Recordings were made using a large bore, double-barrelled glass suction pipette for both voltage clamp and internal perfusion as previously described (Sheets et al. 1996). INa and Ig were measured with a virtual ground amplifier (Burr-Brown OPA-101) using a 2.5 MΩ feedback resistor. Voltage protocols were imposed from a 16-bit DA converter (Masscomp 5450, Concurrent Computer, Tinton Falls, NJ, USA or National Instruments, Austin, TX, USA) over a 30/1 voltage divider. Data were filtered by the inherent response of the voltage-clamp circuit (corner frequency near 125 kHz) and recorded with a 16-bit AD converter at 200 kHz. A fraction of the current was fed back to compensate for series resistance. Temperature was controlled using a Sensortek (Physiotemp Instruments, Inc., Clifton, NJ, USA) TS-4 thermoelectric stage mounted beneath the bath chambers and varied less than 0.5°C. Cells were studied at 13°C.
A cell was placed in the aperture of the pipette, and after a high resistance seal had formed the cell membrane inside the pipette was disrupted with a manipulator-controlled platinum wire. Voltage control was assessed by evaluating the time course of the capacitive current and the steepness of the negative slope region of the peak current–voltage relationship as per criteria previously established (Hanck & Sheets, 1992). The holding membrane potential (Vhp) was typically set to −150 mV except for cells expressing Na+ channels with the ICM mutation, where it was −120 mV unless otherwise specified. Ig protocols contained four repetitions at each test voltage that were one-quarter of a 60 Hz cycle out of phase to maximize rejection of this frequency thereby improving the signal to noise ratio.
The control extracellular solution for INa measurements contained (mm): 15 Na+ (or 100), 185 TMA+ (or 100), 2 Ca2+, 200 Mes− and 10 Hepes (pH 7.2), and the intracellular solution contained 200 TMA+, 75 F−, 125 Mes−, 10 EGTA, and 10 Hepes (pH 7.2). For measurement of Ig the intracellular solution remained unchanged while the extracellular solution had Na+ replaced with TMA+ and 1 μm saxitoxin was added (Calbiochem Corp., San Diego, CA, USA). For modification by external N-biotinoylaminoethylmethanethiosulphonate (MTSEA-biotin) or by N-biotinoylcaproylaminoethyl methanethiosulphonate (MTSEA-biotincap) a 1.0 mm solution was made by initially dissolving the agent in 20: l dimethyl sulfoxide (DMSO) before adding the mixture to 10 ml of extracellular solution (making a 1: 500 ratio of DMSO to solution). The cell was then perfused with the biotin solution for about 20 min while the membrane potential was stepped from −120 mV to 0 mV for 100 ms at 1 Hz. Control studies on WT-ICM exposed to extracellular MTSEA-biotin showed no effect on INa or peak current–voltage (I–V) relationships (n= 2 cells, data not shown). 2-Trimethylammonium ethylmethanothiosulphonate (MTSET) was dissolved in the perfusate solutions just prior to its use. For intracellular application of MTSET, 2.5 mm was added to the intracellular solution for 8–10 min before switching back to control solutions. While pulsing to −30 mV at 0.5 Hz, the additional slowing of INa decay in cells expressing the ICM mutation was readily noticeable after 3 min and was typically complete by 10 min. Previous studies have shown that perfusion with MTSETi under similar conditions did not affect wild-type NaV1.5 (Sheets et al. 2000). Cells were first exposed to extracellular MTSEA-biotin before exposure to intracellular MTSET because R2C-DIV is accessible by both extracellular and intracellular sulfhydryl agents (Yang et al. 1996). All MTS reagents were from Toronto Chemical Corp., Toronto, Canada. Avidin (Pierce Biotechnology, Rockford, IL, USA) was dissolved in extracellular solutions containing Cl− because solutions containing MES− precipitated avidin. For the determination of IC50 values, cumulative dose–response curves were constructed by starting with the lowest concentration of lidocaine and incrementing to the next highest concentration. All cells were perfused with lidocaine for at least 3 min before voltage-clamp protocols were started.
Leak resistance was calculated as the reciprocal of the linear conductance (G) between −180 mV and −110 mV, and cell capacitance was measured from the integral of the current responses to voltage steps between −150 mV and −180 mV. For determination of peak INa voltage relationships, Vhp was −150 mV and step potentials were from −130 to 20 mV for 50 ms at 1 Hz. Data were capacity corrected using 4–8 scaled current responses recorded from voltage steps typically between −150 mV and −180 mV. Peak INa was taken as the mean of four data samples clustered around the maximal value of data digitally filtered at 5 kHz and leak corrected by the amount of the calculated time-independent linear leak. Peak INa as a function of potential was fitted with a Boltzmann distribution:
where INa is the peak current in response to a step depolarization, and Vt is the test potential. The fitted parameters were V1/2, the half-point of the relationship, s, the slope factor in millivolts and Vrev, the reversal potential, and G=INa/(Vt−Vrev). For comparison between cells, data were normalized to the maximum peak conductance (Gmax) for each cell.
For Ig measurements the membrane potential was held at −150 mV and stepped to various test potentials for 25 (or 26.5) ms at 1 Hz. All Ig values were leak corrected by the mean of 2–4 ms of data usually beginning 8 ms after the change in test potential and capacity corrected using four to eight scaled current responses to steps between −150 mV and −180 mV taken immediately before and after the test step. To determine time constants of Ig decay, current traces were trimmed until the decay phase was clearly apparent, and then fitted by a sum of up to two exponentials with DISCRETE (Provencher, 1976), a program that provides a modified F statistic in order to evaluate the number of exponential components that best describe the data. Because two exponential fits were better only 28% of the time for R3C-DIII and only 12% of the time for R2C-DIV, single exponential fits were selected for plotting.
Q–V relationships were fitted with a simple Boltzmann distribution:
where Q is the charge during depolarizing step and Vt is the test potential, and the fitted parameters are Qmax, the maximum charge, V1/2, the half-point of the relationship, and s, the slope factor in millivolts. Fractional Q was calculated as Q/Qmax.
Steady-state voltage-dependent Na+ channel availability was evaluated from peak INa in voltage steps to 0 mV after conditioning for 500 ms over a range of potentials. Cycle frequency was 0.5 Hz, and Vhp between conditioning steps was −150 mV. Peak INa as a function of conditioning potential was fitted with a Boltzmann distribution:
where INa is the peak current after the conditioning pulse, Imax is the maximal INa, Vc is the conditioning potential, Ir is the residual current if INa did not fully inactivate at the most positive Vc (for channels with the ICM mutation), V1/2 is the half-point of the relationship, and s is the slope factor in millivolts.
Single-site binding curves were fitted by:
where fractional Gmax was normalized to Gmax in control, dose was the concentration of lidocaine, and the IC50 was the effective concentration at one-half the reduction in Gmax.
Interactions between lidocaine and Na+ channel mutations were calculated according to thermodynamic mutant cycle analysis (Horovitz & Fersht, 1990; Hidalgo & MacKinnon, 1995) where the coupling coefficient, Ω, was calculated by:
where IC50(WT-ICM) is the IC50 for block of WT-ICM by lidocaine, etc. Coupling energy was calculated as
where R is the gas constant of 1.98 cal K−1 mol−1 and T is the absolute temperature.
Data were analysed and graphed on a SUN Sparcstation using SAS (Statistical Analysis System, Cary, NC, USA) or Matlab (The Mathworks, Natick, MA, USA) and Origin (OriginLab Corp., Northhampton, MA, USA). Unless otherwise specified summary statistics are expressed as means ± 1 standard deviation (s.d.). Figures show means ± standard error of the mean (s.e.m.). Student's t test for paired or non-paired data was used to determine statistical significance at the P < 0.05 level.