Use of nonelectrolytes reveals the channel size and oligomeric constitution of the Borrelia burgdorferi P66 porin

Abstract

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than 'normal' Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be ≤1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80-90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.

Figure 1. Distribution of the single-channel conductance of P66 in the presence of nonelectrolytes.

Histograms were constructed from the evaluation of at least 100 insertional events into a PC membrane in the presence of 20% (w/v) maltose (A), PEG 400 (B), PEG 600 (C) and PEG 1,000 (D) in the bathing solution 1 M KCl. The insets show original recordings of the single-channel current vs. time. The base lines of these recordings represent the zero current level. V_m = 20 mV; T = 20°C.

Figure 2. Dependence of the single-channel conductance of P66 on the molecular mass (A) and the hydrodynamic radius (B) of the nonelectrolytes.

G_(+NE)/G_(−NE) is the ratio of the mean single-channel channel conductance in the presence of NEs (taken from table 1) to that in the absence of NEs (11.0 nS [1]). Molecular masses and hydrodynamic radii of the nonelectrolytes were taken from table 1. The bars indicate absolute errors.

Figure 3. Dependence of the channel filling on the hydrodynamic radii of nonelectrolytes.

for each nonelectrolyte was calculated according to Eq. 3. Lines are best fits to the experimental points. The channel filling of maltose, PEG 400 and PEG 600 was not included in this diagram, because the calculated values of and were unreasonably high and could not be used due to possible interactions of these compounds with the channel interior (for details see text). The horizontal lines connect the points derived from measurements in the presence of PEG 1,000, PEG 3,000 and PEG 6,000. The other line regression was used to describe the points for the nonelectrolytes with radii ranging from 0.26 nm to 0.6 nm (ethylenglycol, glycerol, arabinose, sorbitol, PEG200, PEG300). Hydrodynamic radii of the nonelectrolytes were taken from table 2.

Figure 4. Titration of the P66-induced membrane conductance with PEG 600 (A) and maltohexaose (B).

The membrane was formed with PC/n-decane. The aqueous phase contained ∼100 ng ml⁻¹ P66, 1 M KCl and respective nonelectrolytes in the concentration as indicated; temperature = 20°C; applied voltage = 20 mV.

Figure 5. PEG 400-induced blockage of P66 on the single-channel level.

Small amounts of highly diluted P66 (1∶1,000 in 1% Genapol) was added to both sides of a diphytanoyl PC membrane. After reconstitution of one single 11 nS P66 unit, 90 mM PEG 400 was added to both sides of the membrane. The P66 conductance was blocked stepwise exhibiting subconductance steps of approximately 1.5 nS; temperature = 20°C; applied voltage = 20 mV.

Figure 6. Power density spectrum of PEG 600-induced current noise of 61 P66 channels.

Trace 1 shows the control, the aqueous phase contained 1- and maltohexaose-induced current noises resulted in similar power density spectra (data not shown).

Figure 7. Blue native (BN) PAGE, SDS PAGE and WB analysis of the P66 complex.

The complete outer membrane fraction of B. burgdorferi B31 was applied to a 4–16% BN PAGE. A 460 kDa band that reacted to the P66 antibody was extracted from the gel and loaded again in a 4–16% BN PAGE (BN/BN PAGE). This gel was stained with silver nitrate (SN) and subjected to a western blot (WB) against P66 (A). The 460 kDa band was extracted from the gel using detergents and resolved again under denaturing conditions in glycine and tricine SDS PAGE (B). A whole lane from the BN/BN PAGE containing the P66 complex was resolved in a second dimension SDS PAGE (C). The elution of the 460 kDa band from the BN/BN PAGE was tested in planar bilayers for pore-forming activity. Step-like increases in the conductance of the membrane were observed after adding the sample to the salt solution (D, left panel) and at least 100 insertional events were summarized in a histogram (D, right panel).