Supplemental Material for

Interfacial polar interactions affect gramicidin channel kinetics

1Tatiana K. Rostovtseva, 2Horia I. Petrache, 1Namdar Kazemi, 1Elnaz Hassanzadeh, 1Sergey M. Bezrukov

1Laboratory of Physical and Structural Biology, NICHD, NIH, Bethesda, MD 20892; 2Department of Physics, Indiana University Purdue University Indianapolis, Indianapolis, IN 46202

1. Experimental procedures

Dioleoylphosphatidylcholine (DOPC), Dioleoylphosphatidylethanolamine (DOPE), and Dietherphosphatidylcholine (1,2-Di-O-Octadecenyl-Glycero-3-Phosphatydilcholine, DEPC), and Dilauroylphosphatidylcholine (DLPC), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL).

Gramicidin channel measurements -- Bilayer membranes were formed from monolayers across a 70 – 90 mm diameter orifices in a 15 mm thick Teflon partition that separated two chambers (1). After bilayer formation gramicidin A (a generous gift of O. S. Andersen, Cornell University Medical College) was added from 0.1 - 1 nM ethanol stock solutions to both aqueous compartments at the amount sufficient to give a single-channel activity. Aqueous solutions of KCl were buffered by 5 mM HEPES at pH 7.4. Experiments at low, 50 and 30 mM, KCl concentrations, were made in the presence of 0.1 mM EDTA. All measurements were made at room temperature, T = (23 + 1.0)0C.

The mixtures of DOPC and DOPE were prepared from aliquots of two lipid solutions in chloroform, followed by drying lipid mixtures with nitrogen and then re-dissolving them in hexane or pentane to a total lipid concentration of 5 mg/ml. The experimental Teflon chamber was sonicated for 15 min in chloroform/methanol (2:1) mixture and a new partition was used each time when lipid composition was changed in order to avoid any traces of a “foreign” lipid.

The membrane potential was maintained using Ag/AgCl electrodes with 3 M KCl and 15 % (w/v) agarose bridges. The membrane chamber and headstage were isolated from external noise sources with a double metal screen (Amuneal Manufacturing Corp., Philadelphia, PA). Conductance measurements were performed using an Axopatch 200B amplifier (Axon Instruments, Inc., Foster City, CA) in the voltage clamp mode. Data were filtered by a low-pass 8-pole Butterworth filter (Model 9002, Frequency Devices, Inc., Haverhill, MA) at 5 kHz, directly saved into the computer memory with a sampling frequency of 10 kHz, and analyzed using pClamp 9.2 software. Data reduction in the substitute average regime with sampling frequency interval of 1 or 5 ms was applied to all records, and then single channels were discriminated.

gA lifetimes were collected only from individual single-channel events and calculated by fitting logarithmic single exponentials to logarithmically binned histograms of at least 250 single-channel events (2). Nine different logarithmic probability fits were generated using different fitting procedures and the mean and standard deviation of the fitted time constants were used as mean and standard deviation for the lifetime. Each data-point in Fig. 2A and 3B is a mean lifetime of nine different log probability-fitting procedures + S.E. Mean single-channel current was calculated using “Origin” software as a Gaussian peak with standard deviation as a width of the peak.

Small-angle x-ray scattering (SAXS) -- About 10 mg of lipid powder was hydrated with purified water or high molecular weight (20 K) polyethylene glycol water solutions with known concentrations and osmotic pressures. Samples were stored at -4°C before being x-rayed for 0.5-1 hour with a fine-focus fixed copper anode x-ray source (Enraf-Nonius, Delft, The Netherlands). Sharp, uniform scattering rings were obtained indicative of sample homogeneity upon equilibration. Interlamellar repeat spacings were recorded as a function applied osmotic pressure.

Computer simulations -- A molecular dynamics simulation of a gramicidin dimer in a DOPC bilayer at 300 K was started using the CHARMM package in the NIH biowulf computer cluster. The 1JNO PDB structure was used for the gramicidin channel (3).

2. Results

Planar membranes formed from DEPC were not stable under higher than 100 mV applied voltage. Therefore, all experiments in DEPC membranes were made at 50 mV applied potential. Channel lifetime did not depend on the applied potential up to 200 mV for both DOPC and DOPE membranes (Fig. S1 A). It should be noticed that all experiments have been performed on solvent-free membranes. Therefore, applied electrical field could not induce a significant membrane compression and decrease of membrane thickness, which would result in increase of gA lifetime ((20) and see also discussion by Huang (11)). gA channel conductance was Ohmic up to 200 mV applied potential in DOPC and DOPE membranes (Fig. S1 B).

Fig. S1. gA channel lifetime (A) and conductance (B) do not depend on the magnitude of applied voltage in the “solvent-free” DOPC or DOPE membranes. Data were obtained on two individual membranes. The medium consisted of 1 M KCl. The applied potential was 150 mV.

3. References

1.  Rostovtseva, T. K., and S. M. Bezrukov. 1998. ATP transport through a single mitochondrial channel, VDAC, studied by current fluctuation analysis. Biophys. J. 74:2365-2373.

2.  Sigworth, F. J., and S. M Sine. 1987. Data transformations for improved display and fitting of single-channel dwell time histograms. Biophys. J. 52:1047-1054.

3.  Townsley, L. E.,W. A.Tucker, S.Sham, and J .F. Hinton. 2001. Structures of gramicidins A, B, and C incorporated into sodium dodecyl sulfate micelles. Biochemistry 40:11676-11686.