Nanoscale dielectric spectroscopy of ion channels

Many physiological processes down to the cellular and sub-cellular level are based on electrostatic interactions and the transport of ionic charges. Particularly interesting aspects are conduction properties of ion channels as essential components of the cell membrane, which are typically investigated using patch-clamp techniques. Research in this field has important applications in pharmacology etc. However, for patch-clamp measurements in the time domain the electrical bandwidth is limited such that electrical currents faster than 1s can hardly be measured. To access also faster processes, ion channel electric conduction properties can be also measured in the frequency domain using dielectric spectroscopy. Recent instrumentation in this field allows electrical characterization at GHz and even higher frequencies which is a range where relaxation processes are associated to nanometer structures like proteins including ion channels. This scale is similarly accessible by electrical Scanning Probe Microscopy (SPM) which has the capability to study local phenomena in Biophysics. In this project wide band dielectric spectroscopy is combined with electrical SPM, in order to merge the strength of both complementary techniques and to open a new field of Nanoscale Dielectric Spectroscopy in Biophysics. The developed technique is used to characterize the high-frequency ion conductance of the well known KcsA potassium channel, reconstituted in lipid membranes. To couple effectively electric fields into ion channels and gain the required sensitivity for the channel electric properties the nanoscopic SPM probe is approached close to the membrane. The conduction properties are probed by acquiring dielectric spectra in a wide frequency range from 0.5MHz up to 20GHz. This will give the possibility to measure channel conductance at frequencies corresponding to the typical time scale for an ion passing through a channel (around 1-100ns or 10MHz-1GHz), such that resonance effects as predicted in literature could get visible. It is expected that measurements under different external conditions (including pH, drugs/inhibitors) will give new insights into the mechanisms of ion channel conductance at a completely new time scale. Further investigations on the gating process of voltage dependent ion channels KvAP are carried out in order to measure fast structural gate movements of the alpha helices not accessible with patch clamp techniques. In a second part of the project, ion channel activity is investigated directly in single cells like in standard patch-clamp, but here on small bacterial cells and at high frequencies. Dielectric spectra are acquired to extract selectively signal contributions from cytosol and cell membrane. Channel activity of KcsA is controlled again by adjusting the pH. The advantages of direct cell measurements like simpler sample preparation etc. are evident. In future projects the developed single cell spectroscopy techniques could be also applied to study working mechanisms of drugs through associated changes in the bacterial dielectric signature.

The response of biological matter to electric fields is important for many fundamental electrophysiological functions like the selective transport in ion channels. This process occurs at the cellular level - specifically in the cell membrane and thus at the micrometer or nanometer scale. To carry out local electrical measurements at this length scale is very challenging and especially fast electrical processes are therefore difficult to observe and study. In this project two new methods have been developed that allow for detection of electrochemical currents and electrical movements o charges within a cellular membrane with nano-metre resolution. The first newly developed technique, that is termed broad band electrostatic force microscopy, can measure movement of charges and dipoles at a wide frequency range (from static to GHz frequencies) and can be used to locally characterize these so called "dielectric" properties of biological and other micro-and nanoscale objects, in order to investigate related (bio)-physical processes. From the functional perspective the frequency dependency of the dielectric permittivity plays an essential role, since it determines how fast the dipoles associated with the integral molecular protein structures change the conformation according to an external electrical field. It is supposed that the mobility of the dipoles is strongly coupled with the presence of bound water which enables dipole movements and the function of the protein itself. By experiments on bacterial protein membranes it could be shown in this project that the dielectric response is indeed strongly dependent on the water surrounding of the protein molecule and dipole movements characteristic for protein subunits could be observed. Despite the importance of the dipole dynamics for the biophysical function of the protein, the intrinsic time constants in which structural changes in proteins occur can be used also as a dielectric fingerprint. At THz and higher frequencies, the identification of specific materials by their "dielectric" or "chemical" fingerprint has been proven. In this project it was shown that dielectric fingerprinting and investigation of nanoscale dielectric relaxation and resonant processes at lower time scales is also accessible. The second newly developed technique is the electrochemical scanning microwave microscope (ec-SMM), which allows for local measurement of ionic and electrochemical currents at GHz frequencies. As shown in this project, the SMM itself can measure the nanoscale dielectric and conductive properties of a wide range of materials from bacteria over semiconductors and has the capability to also identify atomically small electrical features in the subsurface of the sample under study. For the measurement of the small ionic currents through ion channels careful adaptation of the technique was required. Beyond this project, the technique will be a valuable tool for the study of local electrochemistry in energy materials like lithium ion batteries.