Supporting information for:

Supporting information for: Cyclodextrin Ion Channels

Jonathan K.W. Chui and Thomas M. Fyles* Voltage Clamp Data Acquisition

A model BC-525A bilayer clamp (Warner Instrument Corp.) was used for planar bilayer experiments. The analogue output was filtered with an 8-pole Bessel filter (Frequency Devices, model 902) and digitized with a 330 kHz digitizer (Axon Instruments, Digidata 1200A). Data acquisition was controlled by the pClamp8 software package (Axon Instruments). Data were collected at 10 Hz, analogue filtered at 1 Hz, and digitally filtered at 50 Hz. The headstage and the bilayer chamber (3 mL polystyrene cuvette with 250 µm diameter aperture held in a 5 mL PVC holder) were placed on a floating table and electrically shielded by a grounded aluminum Faraday cage. Agar salt bridges (2 M KNO3 in 1% Agar) were used to stabilize junction potentials and were employed between the electrolyte in each well of the cell and Ag/AgCl electrodes. Electrolyte solutions were prepared from high purity salts and nanopure water. A stock solution of diphytanoyl phosphatidylcholine (diPhyPC) in chloroform (Avanti Polar Lipids; shipped on dry ice) was divided into sealed glass vials under an argon atmosphere and stored at -12 C. For use in an experiment, a stream of dry nitrogen was passed through the vial for 1 hour. The dried lipid was diluted with decane to give a solution concentration of 25 mg/mL in lipid.

Bilayers were formed by either brushing or dipping: after lipid in decane had been introduced by brushing, a lipid/ decane film formed on the surface of the electrolyte, and bilayers could then be formed by withdrawal of 2-3 mL of electrolyte from the cell holder by syringe to expose one face of the aperture to the air-water interface held in the cell holder, followed by reintroduction of the electrolyte to oppose monolayers across the aperture in the cuvette. Bilayer quality was monitored via the

capacitance and stability under applied potential, using the criteria previously described1. The measured voltage was applied with respect to the trans (cuvette) side of the bilayer, making the trans side the relative ground. Digitized data files were analyzed using the pClamp10 suite of programs.

The compounds are introduced to the membrane in two ways, depending on the solvent in which the compound can be dissolved:

Direct injection - all injection experiments utilized bilayers that were apparently stable at 100 mV for periods of 20 minutes or more. Aliquots (1-5 µL of transporter solutions in MeOH were injected with a microliter syringe as close as possible to the bilayer in the free well of the cuvette holder (cis side), and gently stirred with a stream of nitrogen for 5 minutes.

Pre-mixed into lipid - in this method, 1mol% of compound (in CDCl3 or MeOH-d4) was added to the

diPhyPC/CHCl3 solution, and solvent removed with a stream of N2, and bilayer membrane prepared by

brushing/dipping as described above. Most of the bilayers formed with this method gave bilayers with good quality.

Of the two methods, direct injection is preferable, as it allows monitoring of pristine bilayer prior to compound introduction. Following direct injection, channel behaviour typically appears within 20

the pClamp suite. A customized threshold search was used to generate the list of events. The

threshold was set across the fluctuating section of the trace to maximize the number of events. Within that segment,  is insensitive to the choice of threshold. A minimum duration was fixed at 50ms. The threshold search automatically logs event start and event end fromwhich the duration can be calculated. The resultant values were exported to the fitting program.

Power Law Fitting The list of opening durations, obtained above as a plain-text file, can then be fitted using the method of Clauset et al2, implemented in python3. The code performs the Maximum Likelihood Estimate fit, and provides , xmin, n, and p-value as outputs.

Summary of bilayer activity

Annotated activity grids, as well as full conductance records (and expansions where appropriate), are provided below for every compound studied. The activity grids were prepared as previously described4. The summaries are arranged first by compound, then individual experiments. Within each experiment, the first page(s) summarizes the experimental conditions as well as activity grids charted; subsequent pages shows the full conductance record as the top panel, with expansions indicated by corresponding letters.

1. Fyles, T. M.; Knoy, R.; Müllen, K.; Sieffert, M., Membrane activity of isophthalic acid derivatives: ion channel formation by a low molecular weight compound. Langmuir 2001, 17, 6669-6674.

2. Clauset, A.; Shalizi, C. R.; Newman, M. E. J., Power-law distributions in empirical data. SIAM Rev.

2009, 51, 661-703.

3. Ginsberg, A. <http://code.google.com/p/agpy/wiki/PowerLaw> (accessed November 7). 4. (a) Chui, J. K. W. A New Paradigm for Voltage-Clamp Studies of Synthetic Ion Channels.

University of Victoria, Victoria, 2011; (b) Chui, J. K. W.; Fyles, T. M.; Luong, H., Planar bilayer activities of linear oligoester bolaamphiphiles. Beilstein J. Org. Chem. 2011, 7, 1562-1569.

electrolyte 10uM 30uM lipid contact injection brush transfer br oken bila yer

IV

IV

A

62-0001

A

A

B

B

C

C

D

D

blue, or yellow?

fractal?

A

C

D

D

B

B

A

62-0003

A

B

C

C

B

A

fractal?

electrolyte 10uM lipid contact injection brush transfer

-45->45mV

5mV steps

62b-0000

A

A

B

B

fractal?

A

B

C

D

D

C

B

23 pS

53 sec

42 pS

2 sec

10 pS

62b-0003

A

B

C

C

A

38 pS

3 sec

38 pS

12 pS

22 pS

8.3 sec

_{6 sec}

A

B

C

C

B

A

30 pS

electrolyte 2uM 4uM

lipid

contact injection brush transfer

63-0006

63-0007

no activity

no activity

63-0008

63b-0000

63b-0001

A

B

B - {

63b-0003

A

B

8.5 pS

8.5 pS

C

C

63b-0006

8 pS

34 sec

A

A

6 pS

A

A

electrolyte lipid 1% contact injection brush transfer

0002

0003

0004

0005

apparent potential/history dependence?

IV steps

80 -> -80mV

4mV steps

A

B

B

C

C-{

65-0001

A

A

B

C

A

65-0003

A

A

A

A

B

C

B

D

65-0006

A

B

B

A

Fractal?

A

B

65-0009

A

B

C

B

A

C - { }

contact injection brush transfer

70-0000

A

B

B - {

}

A

27 pS

5 pS

A

B

C

D

D

A

Not clear what is being looked at here.

538pS

70-0002

A

B

C

D

E

B

A

C

D

E

A

B

C

electrolyte 24/16uM

lipid

contact injection brush transfer

78-0001

A

A

B

C

C

B

14 pS

22 pS

60 pS

14 pS

A

B

B

C

C

D

150 pS

135 pS

44 pS

135 pS

35 pS

117 pS

Note the lack of downward

deflection at the true baseline

electrolyte lipid contact injection brush transfer

0003

0005

0004

A

164pS

164pS

18pS 6 pS

23pS

23pS

18 pS

91-0003

135pS

135pS

48pS

14pS

48pS

A

A

144pS

A

91-0005

135pS

A

B

C

C

B

A

135pS

A

electrolyte 17.8/26.6uM lipid contact injection brush transfer br oken bila yer

A

92-0001

A

C

C

B

B

A

A

92-0003

A

electrolyte 2uM lipid contact injection brush transfer electrolyte lipid Adamantyl guest br oken bila yer

electrolyte 4uM 8uM 12uM 22/17uM lipid contact injection brush transfer electrolyte lipid Adamantyl guest

0001

0002

0003

0004

IV steps

IV steps

IV steps

-100 -> 100mV

10mV steps

IV steps

-150 -> 150mV

10mV steps

What are the mechanistic implications of an asymmetric/rectifying distribution?

(i) The compound must not equilibrate between leaflets readily (readily = on the analysis timescale = minutes). Otherwise the compound would be symmetrically distributed.

(ii) The active structure cannot be symmetric about the midplane of bilayer. This is weird to think about, because the compound seems very much to need participation from both leaflets.

I'm not sure what to think about this now. Can it be that the potential modifies the penetration of the cyclodextrin ring (or the coformation of the triazoles)... I don't know. I should note that seemingly the open probability, as well as lifetimes were both affected.

0001

0002

0003

electrolyte 19.4uM (holder) 19.4/19.4uM

lipid contact injection brush transfer electrolyte lipid Adamantyl guest

IV steps

-160 -> 160mV

10mV steps

A

B

64.2.0000

A

64.2.0002

br

eak

age?

14 pS

31 pS

A

A

B

B

C

35 pS

41 pS

41 pS

41 pS

6 sec

79 sec

A

B

C

C

B

64.2.0004

A

5 pS

44pS

32pS

A

A

C

C

64.2.0006

B

B

44pS

_44pS

40pS

A

A

Note this interesting yellow;

64.2.0008 - IV

170pS

64.3.0000

A

A

64.3.0002

64.3.0004

A

B

B

A

44 pS

44 pS

300ms

4.4 sec

64.3.0006

41pS

12pS

A

B

C

C

B

A

A

B

C

C

42pS

42pS

46pS

12pS

electrolyte 97uM (holder) 97/97uM

lipid (1%) contact injection brush transfer

electrolyt 20ul 0.25mg / mle

lipid Pyrene guest br oken bila yer br oken bila yer no activity 0006 0008

66-0005

A

B

C

D

D

C

B

A

35 pS

38 pS

14 ps

36 ps

24 ps

25 ps

A

B

C

D

D

34 pS

38 pS

17.6 pS

10 pS

36 pS

66-0007

0mV

A

B

C

D

D

C

B

A

A

B

C

22pS

36pS

12pS

electrolyte 117uM lipid (4%) contact injection brush transfer electrolyte lipid Adamantyl guest br oken bila yer br oken bila yer

A

A

67-0002

electrolyte 15/10uM

lipid

contact injection brush transfer

80-0000

A

A

B

B

electrolyte 17/24uM

lipid

contact injection brush transfer

A

electrolyte 0 / 1mM

lipid

simultaneous openings seen,

both different and indifferent

93-0001

A

B

C

C

B

38.1 pS

_{33.2 pS}

_{39.3 pS}

A

A

B

C

D

9 pS

19 pS

93-0003

A

A

br

oken bila

yer

Spontaneously became silent over an hour

94-0001

pot

en

tial change

pot

en

tial change

pot

en

tial change

A

20 pS

A

94-0003

A

B

C

C

B

A

20 pS

15 pS

total

electrolyte 278uM (holder)

potential

dependent?

example of

suddenly

onset noise

(or purple)

prolonged

partial

closure

95-0000

pot

en

tial changes

A

B

C

C

D

D

B

A

mean = 265 pS

_{mean = 381 pS}

_{mean = 228 pS}

39 pS

2 pS

A

B

pot

en

tial changes

A

B

B

A

39 pS

33 pS

95-0002

prolonged partial closure

C

A

B

C

Drifting conductances

in a square top

19 pS

95-0004

A

B

B

A

34 pS

42 pS

A

B

C

Note the absence of flicker in

the true baseline;

compare with expansion D

D

A

B

B

95-0006

electrolyte 66uM lipid contact injection brush transfer electrolyte 2.5mM (cup) lipid Adamantyl COOH electrolyte 40mg bCD

nothing with AdCOOH alone

br

oken bila

96-0008

A

A

B

42 pS

22 pS

A

A

B

B

96-0010

A

B

B

765 pS 820 pS

950 pS

electrolyte 30/20uM 60/40uM 83/59uM lipid contact injection brush transfer electrolyte 30/20uM lipid Adamantyl COOH br oken bila yer

97-0003

A

A

B

B

C

C

D

E

E

D

97-0005

A

B

D

D

C

B

A

C

28pS

14pS

97-0007

A

A

B

C

C

D

D

E

15pS

B

60pS

long-living regular flicker

(small)

97-0009

A

97-0011

A

B

C

C

B

A

30pS

177pS

323pS

15pS

D

D

A

A

97-0013

A

B

B

A

15pS

6pS

electrolyte

lipid

contact injection 66uM brush transfer

100-0011

electrolyte 20uM lipid contact injection brush transfer

asymmetric electrolyte

A

A

35 sec

A

A

101-0004

no flickering like 101-0003 seen

3.5 pS

23 pS

47 pS

A

A

B

C

C

B

classified as all of blue, green and yellow

D

electrolyte 30uM lipid contact injection brush transfer electrolyte lipid Adamantyl guest

Asymmetric electrolyte

IV

-100 -> 100mV,

10mV steps

reversal potential 22.6mV

102-0003

21 pS 30 pS

43 pS

flickering absent in baseline sans opening A

A

9pS 100ms

brush transfer electrolyte 2mM lipid Adamantyl COOH electrolyte 2mM lipid bCD broken bilayer

103-0000 A B B A 10pS Blue-yellow

A

5 pS

A B 103-0004 A B 22pS

105-0000

17pS

105-0002

105-0004

electrolyte 82uM 148uM

lipid

contact injection brush transfer

electrolyte 0.1 pmol (holder)

lipid gramicidin br oken bila yer br oken bila yer nothing seen

112-0001

A

B

B

electrolyte 30/20uM lipid contact injection brush transfer br oken bila yer br oken bila yer br oken bila yer br oken bila yer

0mV

electrolyte 11uM 66uM lipid contact injection brush transfer electrolyte lipid CuSO4 br oken bila yer br oken bila yer br oken bila yer no activity

68-0002

A B C 40 pS 3 pS 16 pS

68-0004

A

A

A B B C 40 sec 4 pS D

electrolyte lipid 2% contact injection brush transfer electrolyte lipid Adamantyl guest

0000

0001

0002

0003

0004

0005

0006

0007

br

oken bila

yer

no particular changes observed

20pS baseline opening

69-0001

great leakage

3 pS A

A

B

C

B B C C A 69-0003

Brief closing of the long-lived opening from 0000

15 pS 15 pS

15 pS A

14 pS

B A

A B B A 69-0005 10 pS

A

69-0007 A B C C B A 39 pS 26 sec

A C C B B 6 pS 115 sec

69-0009 A A B B C D D 131 pS 27 pS 42 pS 42 pS 27 pS 3 sec

81-0002 A B C C D B A 180pS 330 pS 252 pS 80pS

See separate discussion on “assignment”

490pS 305pS

150 pS

F

G

0003

85-0002

85-0003

A B 24 pS 24 pS 230msec 5 sec 42 pS

electrolyte 21/14uM

lipid

contact injection 77/48uM total

brush transfer

0003

0006

0009

87-0002

87-0004

A

B

B A

A

87-0006

87-0007

A

A

B

87-0009 A A B B yellow?

A A {

electrolyte 28/19uM

lipid

contact injection 77/48uM total brush transfer

H G H F 1530pS 72 pS E

see separate “assignment”