Supplementary Material

We evaluated the ability of icilin-induced nonselective cation current (INS) to allow passage of permeant cations. The reversal potential (Erev) of ionic currents elicited by icilin is determined by the composition of the solution on the two sides of the membrane and the relative permeabilities of the charge-carrying ions. The permeability of different ions relative to that of K+ (i.e., Cl-, K+ and N-methyl-D-glucamine+ [NMDG+]) was calculated from the Goldman-Hodgkin-Katz (GHK) equation, given below.

where PCl/PK, PNMDG/PK and PAsp/PK are the relative Cl--to-K+, NMDG+-to-K+, and aspartate--to-K+ permeability ratios, with subscript o and i referring to the values outside and inside the cell, respectively, R is the gas constant, T is the temperature in °K, and F is the Faraday constant. Therefore, based on the GHK assumption of independence and constant field (Hille 2001), Erev of iclini-induced INS can be calculated from solution composition of the relative permeabilities of ions:

It is assumed that relative permeability can be determined by steric and/or electrostatic interactions of ions and the pore. By the use of excluded-area theory, such interactions can be estimated from the effective diameters of charge carriers, the apparent diameter of the pore, and charges lining the pore (Dwyer et al., 1980; Dyachok et al., 2010; Sabovcik et al., 1995). Based on the principle that the permeability is proportional to the cross-section area of the selectivity filter (i.e., the most constricted part of the pore), the apparent pore diameter created by the presence of icilin in RAW 264.7 cells was then evaluated. The relative permeability (PX/PY) of ions X (effective diameter Dx) and Y (effective diameter DY) through a pore of diameter Dpore can be described by the excluded-area equation that relates the permeability to the surface left empty when the ion occupies the pore (Sabovcik et al., 1995). That is,

When an ion already has a radius larger than the Debye-Hückel length (lD), it is considered to be sufficiently isolated and to have an effective diameter equal to the crystal diameter. The value of lD is a function of ionic strength (I = 152 mM), absolute temperature and relative permittivity (er = 80 for bulk water and 3-20 for water in constricted pores within or around proteins) as described by the following equation:

where e0, er, kB, NA, and qe are the permittivity of vacuum, relative permittivity, Boltzmann's constant, Avogadro number and elementary charge, respectively.

Supplementary Figure 1 illustrates that, based on the calculations described above, the diameter of the cationic pore is a function of reversal potential for icilin-induced INS. The value of apparent pore diameter induced by icilin in RAW 264.7 cells that has Erev = -5.8 mV was estimated to be 29.8 Å, while PCl/PK, PAsp/PK, and PNMDG/PK were calculated to be 0.0015, 0.0015, and 0.804, respectively.

References

Dwyer TM, Adams DJ, Hille B (1980) The permeability of the endplate channel to organic cations in frog muscle. J Gen Physiol 75:469-492

Dyachok O, Zhabyeyev P, McDonald TF (2010) Electroporation-induced inward current in voltage-clamped guinea pig ventricular myocytes. J Membr Biol 238:69-80

Hill B (2001) Ion channels of excitable membranes, 3rd edn. Sinauer Associates, Sunderland, MA

Sabovcik R, Li J, Kucera P, Prod'hom B (1995) Permeation properties of a Ca2+-blockable monovalent cation channel in the ectoderm of the chick embryon: pore size and multioccupancy probed with organic cations and Ca2+. J Gen Physiol 106:149-174


Supplementary Figure 1

Supplementary Figure 1. Relationship between pore diameter and the reversal potential of INS generated by icilin. The calculation was based on the volume-excluded equation. As the reversal potential becomes less negative, the value of pore diameter is progressively elevated (red line). Straight green line shown in the plot extends to the value of pore diameter (29.8 Å) obtained from the reversal potential of icilin-induced INS in RAW 264.7 cells.

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