Noncontact Nanoscale Imaging of Cells

The reduction in ion current as a fine pipette approaches a cell surface allows the cell surface topography to be imaged, with nanoscale resolution, without contact with the delicate cell surface. A variety of different methods have been developed and refined to scan the topography of the dynamic cell surface at high resolution and speed. Measurement of cell topography can be complemented by performing local probing or mapping of the cell surface using the same pipette. This can be done by performing single-channel recording, applying force, delivering agonists, using pipettes fabricated to contain an electrochemical probe, or combining with fluorescence imaging. These methods in combination have great potential to image and map the surface of live cells at the nanoscale.

Single-molecule imaging of cholesterol-dependent cytolysin assembly

We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway of the Cholesterol-Dependent Cytolysin, Perfringolysin O. This enables quantification of the stoichiometry of PFO complexes during assembly with millisecond temporal resolution and 20 nanometre spatial precision. Our results support a model of overall stepwise irreversible assembly, dominated by monomer addition, but with infrequent assembly from larger partial complexes. Furthermore, our results suggest a dominant proportion of inserted, but non-conductive intermediates in assembly.

Deletion of renal Nedd4-2 abolishes the effect of high K+ intake (HK) on Kir4.1/Kir5.1 and NCC activity in the distal convoluted tubule.

High-dietary K+ (HK) intake inhibits the basolateral Kir4.1/Kir5.1 activity in the distal convoluted tubule (DCT) and HK-induced inhibition of Kir4.1/Kir5.1 is essential for HK-induced inhibition of Na-Cl cotransporter (NCC). We now examine whether Nedd4-2-deletion compromises the effect of HK on basolateral Kir4.1/Kir5.1 and NCC in the DCT. Single- channel-recording and whole-cell-recording showed that neither HK decreased nor low-dietary-K+ (LK) increased the basolateral Kir4.1/Kir5.1 activity of the DCT in kidney-tubule-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. In contrast, HK inhibited and LK increased Kir4.1/Kir5.1 activity in the control mice (Nedd4lflox/flox). Also, HK-intake decreased the negativity of K+-current (IK) reversal potential in the DCT (depolarization) only in the control mice but not in Ks-Nedd4-2 KO mice. Renal clearance experiments showed that HK-intake decreased while LK intake increased hydrochlorothiazide (HCTZ)-induced renal Na+ excretion only in the control mice but this effect was absent in Ks-Nedd4-2 KO mice. Western blot also demonstrated that HK-induced inhibition of phosphor-NCC (pNCC at Thr53) and total NCC (tNCC) was observed only in the control but not in Ks-Nedd4-2 KO mice. Furthermore, the expression of all three subunits of epithelia-Na+-channel (ENaC) in the Ks-Nedd4-2 KO mice on HK was higher than in the control mice. Thus, plasma K+ concentrations were similar between Nedd4lflox/flox and Ks-Nedd4-2 KO mice on HK for 7 days despite high NCC expression. We conclude that Nedd4-2 plays a role in regulating HK-induced inhibition of Kir4.1/Kir5.1 and NCC in the DCT.

Homomeric GluA2(R) AMPA receptors can conduct when desensitized

Desensitization is a canonical property of ligand-gated ion channels, causing progressive current decline in the continued presence of agonist. AMPA-type glutamate receptors (AMPARs), which mediate fast excitatory signaling throughout the brain, exhibit profound desensitization. Recent cryo-EM studies of AMPAR assemblies show their ion channels to be closed in the desensitized state. Here we present evidence that homomeric Q/R-edited AMPARs still allow ions to flow when the receptors are desensitized. GluA2(R) expressed alone, or with auxiliary subunits (γ-2, γ-8 or GSG1L), generates large fractional steady-state currents and anomalous current-variance relationships. Our results from fluctuation analysis, single-channel recording, and kinetic modeling, suggest that the steady-state current is mediated predominantly by conducting desensitized receptors. When combined with crystallography this unique functional readout of a hitherto silent state enabled us to examine cross-linked cysteine mutants to probe the conformation of the desensitized ligand binding domain of functioning AMPAR complexes.

Homomeric Q/R edited AMPA receptors conduct when desensitized

Desensitization is a canonical property of ligand-gated ion channels, causing progressive current decline in the continued presence of agonist. AMPA-type glutamate receptors, which mediate fast excitatory signaling throughout the brain, exhibit profound desensitization. Recent cryo-EM studies of AMPAR assemblies show their ion channels to be closed in the desensitized state. Here we report the surprising finding that homomeric Q/R edited AMPARs still allow ions to flow when the receptors are desensitized. GluA2(R) expressed alone, or with auxiliary subunits (γ-2, γ-8 or GSG1L), generates large steady-state currents and anomalous current-variance relationships. Using fluctuation analysis, single-channel recording, and kinetic modeling we demonstrate that the steady-state current is mediated predominantly by ‘conducting desensitized’ receptors. When combined with crystallography this unique functional readout of a hith-erto silent state enabled us to examine cross-linked cysteine mutants to probe the conformation of the desensitized ligand binding domain of functioning AMPAR complexes within the plasma membrane.