Probing Structure and Function of Ion Channels Using Limited Proteolysis and Microfluidics

Even though gain, loss, or modulation of ion channel function is implicated in many diseases, both rare and common, the development of new pharmaceuticals targeting this class has been disappointing, where it has been a major problem to obtain correlated structural and functional information. Here, we present a micro­fluidic method in which the ion channel TRPV1, contained in proteo­liposomes or in excised patches, was exposed to limited trypsin proteolysis. Cleaved-off peptides were identified by MS, and electro­physiological properties were recorded by patch clamp. Thus, the structure–function relationship was evaluated by correlating changes in function with removal of structural elements. Using this approach, we pinpointed regions of TRPV1 that affect channel properties upon their removal, causing changes in current amplitude, single-channel conductance, and EC₅₀ value toward its agonist, capsaicin. We have provided a fast “shotgun” method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions

A Heating-Superfusion Platform Technology for the Investigation of Protein Function in Single Cells

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s

Cardiomyocytes from mice with the metabolic syndrome show normal β₁-AR, β₂-AR expression and distribution.

<p><b>(A)</b> Representative Western blots and mean data ± SEM (<b><i>B</i></b>; n = 6) of total expression of β₁- and β₂-ARs in left ventricles from control mice and HFD mice. Immunofluorescence staining of β₁-AR <b>(C)</b> and β₂-AR <b>(D)</b> co-stained with RyR2 in control and HFD cardiomyocytes. Merged (yellow) show the intensity overlap between β-ARs and RyR2 in the dyads. <b><i>E</i></b> and <b><i>F</i></b> show plotted intensity profiles of along the dashed lines in <i>C</i> and <i>D</i> with β-AR (red) and RyR2 (green).</p

The antioxidant NAC has no effect on the β-adrenergic stimulated SR Ca²⁺ release and contractility in cardiomyocytes from mice with the metabolic syndrome.

<p><b>(A)</b> Total body weight of mice after 8–10 weeks on control diet (Ctrl, n = 34) or high fat diet (HFD, n = 40). <b>(B)</b> Total body fat measured with DXA whole-body scan (n = 9–10). <b>(C)</b> Typical example of cross-sectional ORO staining showing fat accumulation (stained red) in left ventricles and mean data of ORO staining; eight sections per left ventricle from each group were analyzed (n = 3). Representative [Ca²⁺]_i transients from ctrl <b>(D)</b>, HFD <b>(E)</b> and <i>ob/ob</i> <b>(F)</b> cardiomyocytes obtained in the absence (full lines) and presence (dashed lines) of ISO (100 nM), and without (black lines) and with (red lines) NAC (5 mM). Average amplitude of Ca²⁺ transients <b>(G)</b>, fractional cell shortening (FS, <b><i>H</i></b>) and [Ca²⁺]_i transient decay time constant (tau) <b>(I)</b> with ISO and/or NAC as indicated from control (white bars), HFD (black bars) and <i>ob/ob</i> (grey bars) cardiomyocytes (n>16 cells from at least three mice). Representative Western blots <b>(J)</b> and mean data (n = 6) of ISO-induced PLB phosphorylation normalized to total PLB expression in left ventricles from control and HFD mice. Data are mean ± SEM; **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

The ISO-induced increases in L-type Ca²⁺ current density is ROS-independent in cardiomyocytes with the metabolic syndrome.

<p>Mean data (±SEM; n = 6–8 in each group) of <i>I-V</i> curves of peak current density in the absence and presence of ISO (100nM) and NAC (20 mM) as indicated from control <b>(A, D)</b>, HFD <b>(B, E)</b> and <i>ob/ob</i> <b>(C, F)</b> cardiomyocytes. The <i>I-V</i> relationships were obtained by giving test pulses varying from -80 mV to +50 mV from a holding potential of -80 mV. The mean basal current density (i.e. in the absence of NAC and ISO) was -6.1±0.5 pA/pF and for comparisons between groups each group were normalized its basal current to give a relative current density (relative pA/pF).</p