Nanopores: maltoporin channel as a sensor for maltodextrin and lambda-phage

Abstract

BACKGROUND: To harvest nutrition from the outside bacteria e.g. E. coli developed in the outer cell wall a number of sophisticated channels called porins. One of them, maltoporin, is a passive specific channel for the maltodextrin uptake. This channel was also named LamB as the bacterial virus phage Lambda mis-uses this channel to recognise the bacteria. The first step is a reversible binding followed after a lag phase by DNA injection. To date little is known about the binding capacity and less on the DNA injection mechanism. To elucidate the mechanism and to show the sensitivity of our method we reconstituted maltoporin in planar lipid membranes. Application of an external transmembrane electric field causes an ion current across the channel. Maltoporin channel diameter is around a few Angstroem. At this size the ion current is extremely sensitive to any modification of the channels surface. Protein conformational changes, substrate binding etc will cause fluctuations reflecting the molecular interactions with the channel wall. The recent improvement in ion current fluctuation analysis allows now studying the interaction of solutes with the channel on a single molecular level. RESULTS: We could demonstrate the asymmetry of the bacterial phage Lambda binding to its natural receptor maltoporin. CONCLUSION: We suggest that this type of measurement can be used as a new type of biosensors.

Figure 1

Schematic representation of a typical planar bilayer set-up for ion current recording. 1.a) Two half cells made of Delrine separated by a 25 μm Teflon foil with a hole in the center. Both parts are clamped together. 1.b) Below a microscope picture of the Teflon septum containing a hole. 1.c) Schema of a lipid bilayer with a reconstituted trimeric porin. The Cl^- ions are attracted to the positive electrode and K⁺ to the negative one. Ions are permeating the channel in the MHz range which is beyond the current time resolution. 1.d) The insertion of a single channel will give raise to a jump in conductance. Any objects diffusing in the channel may reduce the permeation time of ions and may be detected either in conductance fluctuations or an averaged reduced conductance.

Figure 2

Typical recordings of ion current through a single Maltoporin trimer in presence of modified maltohexaose (see [16] for details). (A) Shows the unmodified maltohexaose and on the right hand side the modified sugar molecule. We designed this molecule according the crystal structure to guarantee the low penetration ability from one side. (B) M6-ANDS was added to trans (left) and then to cis (right). Sugar analogue modulates ion current only to the cis-side, the side of Maltoporin addition. The average residence time is 5.0 ms. (C) First, M6-ANDS was injected to the trans-side and no variation in the ion current occurs. As control, maltohexaose was added to the same side (left). The natural substrate is translocated demonstrating that it enters the channel from trans with the reducing end first. Then, M6-ANDS was added additionally to the cis-side (right) generating long current interruptions superimposed to maltohexaose blockade events seen in the figure of the left side. The dashed lines corresponding to zero current. Membrane bathing solution was 1 M KCl, 10 mM Tris, 1 mM CaCl₂, pH 7.4, the applied voltage was + 150 mV.

Figure 3

Here we show the ability to recognize bacterial phage Lambda by blocking the ion conductance through the natural receptor Maltoporin. We first reconstituted about 300 Maltoporin channel in a solvent containing planar lipid bilayer. This leads to a stable conductance after about 30 min with no further protein insertion. Titration of 7 and 42 nM of the fusion protein MBP-gpJ from the bacterial virus Lambda to the compartment corresponding the intracellular side of the channel showed no effect. However, titration to the opposite side corresponding to the extracellular side caused a significant reduction of the ion conductance. Membrane bathing solution was unbuffered 1 M KCl giving a pH of about 6. The applied voltage was + 20 mV.