Patch-clamp fluorometry-based channel counting to determine HCN channel conductance.

Counting ion channels on cell membranes is of fundamental importance for the study of channel biophysics. Channel counting has thus far been tackled by classical approaches, such as radioactive labeling of ion channels with blockers, gating current measurements, and nonstationary noise analysis. Here, we develop a counting method based on patch-clamp fluorometry (PCF), which enables simultaneous electrical and optical recordings, and apply it to EGFP-tagged, hyperpolarization-activated and cyclic nucleotide-regulated (HCN) channels. We use a well-characterized and homologous cyclic nucleotide-gated (CNG) channel to establish the relationship between macroscopic fluorescence intensity and the total number of channels. Subsequently, based on our estimate of the total number of HCN channels, we determine the single-channel conductance of HCN1 and HCN2 to be 0.46 and 1.71 pS, respectively. Such a small conductance would present a technical challenge for traditional electrophysiology. This PCF-based technique provides an alternative method for counting particles on cell membranes, which could be applied to biophysical studies of other membrane proteins.

Local and global interpretations of a disease-causing mutation near the ligand entry path in hyperpolarization-activated cAMP-gated channel.

Hyperpolarization-activated, cAMP-gated (HCN) channels sense membrane potential and intracellular cAMP levels. A mutation identified in the cAMP binding domain (CNBD) of the human HCN4 channel, S672R, severely reduces the heart rate, but the molecular mechanism has been unclear. Our biochemical binding assays on isolated CNBD and patch-clamp recordings on the functional channel show that S672R reduces cAMP binding. The crystal structure of the mutant CNBD revealed no global changes except a disordered loop on the cAMP entry path. To address this localized structural perturbation at a whole protein level, we studied the activity-dependent dynamic interaction between cAMP and the functional channel using the patch-clamp fluorometry technique. S672R reduces the binding of cAMP to the channels in the resting state and significantly increases the unbinding rate during channel deactivation. This study on a disease-causing mutation illustrates the important roles played by the structural elements on the ligand entry-exit path in stabilizing the bound ligand in the binding pocket.

Normal-mode-analysis-guided investigation of crucial intersubunit contacts in the cAMP-dependent gating in HCN channels.

Protein structures define a complex network of atomic interactions in three dimensions. Direct visualization of the structure and analysis of the interaction potential energy are not straightforward approaches to pinpoint the atomic contacts that are crucial for protein function. We used the tetrameric hyperpolarization-activated cAMP-regulated (HCN) channel as a model system to study the intersubunit contacts in cAMP-dependent gating. To obtain a systematic survey of the contacts between each pair of residues, we used normal-mode analysis, a computational approach for studying protein dynamics, and constructed the covariance matrix for C-α atoms. The significant contacts revealed by covariance analysis were further investigated by means of mutagenesis and functional assays. Among the mutant channels that show phenotypes different from those of the wild-type, we focused on two mutant channels that express opposite changes in cAMP-dependent gating. Subsequent biochemical assays on isolated C-terminal fragments, including the cAMP binding domain, revealed only minimal effects on cAMP binding, suggesting the necessity of interpreting the cAMP-dependent allosteric regulation at the whole-channel level. For this purpose, we applied the patch-clamp fluorometry technique and observed correlated changes in the dynamic, state-dependent cAMP binding in the mutant channels. This study not only provides further understanding of the intersubunit contacts in allosteric coupling in the HCN channel, it also illustrates an effective strategy for delineating important atomic contacts within a structure.

Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel.

Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide-gated (CNG) and hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand-channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand-whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs(+) and Mg(2+), effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.

State-dependent cAMP binding to functioning HCN channels studied by patch-clamp fluorometry.

One major goal of ion channel research is to delineate the molecular events from the detection of the stimuli to the movement of channel gates. For ligand-gated channels, it is challenging to separate ligand binding from channel gating. Here we studied the cyclic adenosine monophosphate (cAMP)-dependent gating in hyperpolarization-activated cAMP-regulated (HCN) channel by simultaneously recording channel opening and ligand binding, using the patch-clamp fluorometry technique with a unique fluorescent cAMP analog that fluoresces strongly in the hydrophobic binding pocket and exerts regulatory effects on HCN channels similar to those imposed by cAMP. Corresponding to voltage-dependent channel activation, we observed a robust, close-to-threefold increase in ligand binding, which was more pronounced at subsaturating ligand concentrations than higher concentrations. This observation supported the cyclic allosteric models and indicated that protein allostery can be implemented through differentiating ligand binding affinities between resting and active states. The kinetics of ligand binding largely matched channel activation. However, during channel deactivation, ligand unbinding was slower than channel closing, suggesting a delayed response to membrane potential by the ligand binding machinery. Our results provide what we believe to be new insights into the cAMP-dependent gating in HCN channel and the interpretation of protein allostery for general ligand-gated channels and receptors.

Reduced digestive vacuolar accumulation of chloroquine is not linked to resistance to chloroquine toxicity.

Chloroquine (CQ) accumulation studies in live malaria parasites are typically conducted at low nanomolar CQ concentrations, and definition of CQ resistance (CQR) has been via growth inhibition assays versus low-dose CQ (i.e., via IC(50) ratios). These data have led to the nearly universally accepted idea that reduced parasite CQ accumulation is the underlying basis of CQR. Surprisingly, when quantifying CQR via cytocidal CQ activity and examining CQ accumulation at medically relevant LD(50) doses, we find reduced CQ accumulation is not the underlying cause of CQR.

Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects.

Biochemical studies in the human malaria parasite, Plasmodium falciparum, indicated that in addition to the pathway for synthesis of phosphatidylcholine from choline (CDP-choline pathway), the parasite synthesizes this major membrane phospholipid via an alternative pathway named the serine-decarboxylase-phosphoethanolamine-methyltransferase (SDPM) pathway using host serine and ethanolamine as precursors. However, the role the transmethylation of phosphatidylethanolamine plays in the biosynthesis of phosphatidylcholine and the importance of the SDPM pathway in the parasite's growth and survival remain unknown. Here, we provide genetic evidence that knock-out of the PfPMT gene encoding the phosphoethanolamine methyltransferase enzyme completely abrogates the biosynthesis of phosphatidylcholine via the SDPM pathway. Lipid analysis in knock-out parasites revealed that unlike in mammalian and yeast cells, methylation of phosphatidylethanolamine to phosphatidylcholine does not occur in P. falciparum, thus making the SDPM and CDP-choline pathways the only routes for phosphatidylcholine biosynthesis in this organism. Interestingly, loss of PfPMT resulted in significant defects in parasite growth, multiplication, and viability, suggesting that this gene plays an important role in the pathogenesis of intraerythrocytic Plasmodium parasites.

Detection of the recombinant proteins in single transgenic microbial cell using laser tweezers and Raman spectroscopy.

Laser tweezers Raman spectroscopy (LTRS) has been used for the rapid detection of recombinant somatolactin protein produced in single Escherichia coli bacteria and Pichia pastoris yeast cell in the current study. A cDNA sequence encoding mature peptide of zebrafish somatolactin beta was inserted into two different expression vectors and transfected into E. coli or P. pastoris yeast cells. We measured Raman spectra of single E. coli cells at different culture times following the induction with isopropyl beta-d-1-thiogalactopyranoside, from which the amount of the generated somatolactin proteins was obtained by the projection of the entire cell's spectrum onto the spectrum of the pure somatolactin proteins or the dot product between these two spectral vectors. We found that the intensity of the somatolactin beta protein-associated spectra from single E. coli cells increased as the function of the culture time, which correlates with the accumulation of recombinant proteins inside the cells. This spectral observation was supported by evidence obtained by conventional methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analyses. The increased intensities of recombinant protein-associated Raman bands were also observed in another expression system, P. pastoris yeast cells. These findings demonstrate that the LTRS is a useful method for rapid sensing of recombination production in single host microorganism in vivo.

Chromosomal analysis and identification based on optical tweezers and Raman spectroscopy: reply.

We appreciate the authors' comments in their reply: "On the identification of chromosomes with Raman spectroscopy: a critical comment" [Opt. Express 15, 5997 (2007)]. Their main concern with our paper is asking if the collected spectra have shown the identification or differentiation between three human chromosomes. We think this comment is flawed because the authors misunderstood the main points of the original paper and interpreted the presented spectra data (Fig. 3 and Table 1) incorrectly.

Chromosomal analysis and identification based on optical tweezers and Raman spectroscopy.

The ability to identify specific chromosomes with certainty has been established by the development of several cytogenetic techniques based on staining. Here, we report the use of a new optical technique, laser tweezers and Raman spectroscopy (LTRS), to capture and manipulate chromosomes in order to obtain their spectral patterns for molecular analysis without the need for staining. The purpose of this study was to obtain Raman spectroscopy patterns for chromosomes number 1, 2, and 3 and to test if the Raman spectroscopy pattern could be used to distinguish these three chromosomes. In our experiment, optical tweezers were used to capture the individual chromosomes and the Raman spectral patterns were collected for the trapped chromosomes. Then, the captured chromosome was manipulated with the optical tweezers and moved to another chamber through a micro - channel, in which the chromosomes were G- banded for positive identification as chromosome number 1, 2, or 3. Generalized discriminate analysis (GDA) was used to compare the Raman signatures. This analysis revealed that chromosomes 1, 2, and 3 could be distinguished and identified based on their Raman spectra. Development of this approach will lead to more rapid automatic methods for chromosome analysis and identification without the use of prior staining. Moreover, the Raman spectral patterns may lend themselves to more detailed analysis of chromosomal structure than is currently available with standard staining protocols. Such analysis may some day be useful for rapid, automated screening and diagnosis for certain cancers.

Raman sorting and identification of single living micro-organisms with optical tweezers.

We report on a novel technique for sorting and identification of single biological cells and food-borne bacteria based on laser tweezers and Raman spectroscopy (LTRS). With this technique, biological cells of different physiological states in a sample chamber were identified by their Raman spectral signatures and then they were selectively manipulated into a clean collection chamber with optical tweezers through a microchannel. As an example, we sorted the live and dead yeast cells into the collection chamber and validated this with a standard staining technique. We also demonstrated that bacteria existing in spoiled foods could be discriminated from a variety of food particles based on their characteristic Raman spectra and then isolated with laser manipulation. This label-free LTRS sorting technique may find broad applications in microbiology and rapid examination of food-borne diseases.

Spectroscopic analysis of Kaposi's sarcoma-associated herpesvirus infected cells by Raman tweezers.

Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus-8 (HHV-8), is a tumor causing virus. KSHV is the cause of several disease conditions known as Kaposi's sarcoma, multicentric Castleman disease, and primary effusion lymphoma. Cell culture supernatants from KSHV infected hematopoietic cells induced angiogenic tubule formation to a significantly greater extent than uninfected hematopoietic cells. Raman spectrum profiles were generated to differentiate the uninfected from KSHV infected cells. In general, profiles from all the hematopoietic cells shared similar peaks; however, the relative abundance of specific components varied significantly between the cells. Subsequent use of the multivariate analysis of the Raman spectra revealed significant differences between the uninfected and the KSHV infected cells. Taken together, this study reports the use of Raman tweezers to distinguish and analyze the biological relevance of KSHV infected cell signaling.

Activation-dependent phases of T cells distinguished by use of optical tweezers and near infrared Raman spectroscopy.

Near-infrared Raman spectroscopy may provide a highly sensitive, noninvasive means to identify activation status of leukocytes. The purpose of the current study was to establish Raman spectroscopic characteristics of T cell activation. Activation of the RsL.11 T cell clone in vitro with Con A resulted in specific decrements in band intensities at 785, 1048, 1093, and 1376 cm(-1) but did not alter a majority of other band intensities including those at 1004 cm(-1) (phenylalanine) and 1660 cm(-1) (amide bonds). Activation-dependent decrements in these band intensities occurred subsequent to IL-2 production and correlated closely with T cell blastogenesis. Activation-dependent decrements in these band intensities were not strictly a function of cell size because the same observations were noted in size-controlled comparisons of resting and activated T cells. Like the RsL.11 clone, freshly isolated thymocytes that were activated by Con A or IL-2 showed decrements in particular emissions. These findings indicate that near-infrared Raman spectroscopy can be used as a noninvasive technique to reveal the activation status of single living T cells.

Observation of asymmetrically dynamic motion of single colloidal particles in a polarized optical trap.

In this study we report on the dynamic motion of a nano-sized colloidal particle captured in a polarized optical trap. A polystyrene sphere (300nm-diameter) that is electrically charged in solution was trapped with an optical tweezers formed by a linearly polarized TEM00 Gaussian beam, while the Brownian displacements of the trapped particle in x and y directions were measured so that the position of the particle's mass center can be mapped on the transverse plane and the corss-correlation between x and y displacements can be calculated. We found that the position's fluctuation of the trapped nano-sized particle in the parallel direction to the laser polarization is significantly larger than that in the normal direction, which suggests that there exists an additional random electric force parallel to the laser polarization direction exerting on the charged particle beside the known radiation forces on the dielectric particle. This asymmetry in dynamic motion is significant when the particle size is well less than the wavelength of the trapping laser. However, in an optical trap formed by a circularly polarized beam, this asymmetry in dynamic motion was observed to disappear. We present both the experimental results and a theoretical analysis.

Real-time Raman spectroscopy of optically trapped living cells and organelles.

We report on real-time Raman spectroscopic studies of optically trapped living cells and organelles using an inverted confocal laser-tweezers-Raman-spectroscopy (LTRS) system. The LTRS system was used to hold a single living cell in a physiological solution or to hold a functional organelle within a living cell and consequently measured its Raman spectra. We have measured the changes in Raman spectra of a trapped yeast cell as the function of the temperature of the bathing solution and studied the irreversible cell degeneration during the heat denaturation. In addition, we measured the in-vitro Raman spectra of the nuclei within living pine cells and B. sporeformer, Strep. salivarius, and E. coli bacteria suspended in solution and showed the possibility of using LTRS system as a sensor for rapid identification of microbes in a fluid.

Near-infrared Raman spectroscopy of single optically trapped biological cells.

We report on the development and testing of a compact laser tweezers Raman spectroscopy (LTRS) system. The system combines optical trapping and near-infrared Raman spectroscopy for manipulation and identification of single biological cells in solution. A low-power diode laser at 785 nm was used for both trapping and excitation for Raman spectroscopy of the suspended microscopic particles. The design of the LTRS system provides high sensitivity and permits real-time spectroscopic measurements of the biological sample. The system was calibrated by use of polystyrene microbeads and tested on living blood cells and on both living and dead yeast cells. As expected, different images and Raman spectra were observed for the different cells. The LTRS system may provide a valuable tool for the study of fundamental cellular processes and the diagnosis of cellular disorders.