Journal of General Physiology最新文献

JGP study (Si et al. https://doi.org/10.1085/jgp.202413578) reveals that, although they are present at low levels and only generate small currents in the sinoatrial node, Kv1.1 channels have a significant impact on cardiac pacemaking.

Cholinergic signaling in the retina is mediated by acetylcholine (ACh) released from starburst amacrine cells (SACs), which are key neurons for motion detection. SACs comprise ON and OFF subtypes, which morphologically show mirror symmetry to each other. Although many physiological studies on SACs have targeted ON cells only, the synaptic computation of ON and OFF SACs is assumed to be similar. Recent studies demonstrated that gene expression patterns and receptor types differed between ON and OFF SACs, suggesting differences in their functions. Here, we compared cholinergic signaling pathways between ON and OFF SACs in the mouse retina using the patch clamp technique. The application of ACh increased GABAergic feedback, observed as postsynaptic currents to SACs, in both ON and OFF SACs; however, the mode of GABAergic feedback differed. Nicotinic receptors mediated GABAergic feedback in both ON and OFF SACs, while muscarinic receptors mediated GABAergic feedback in ON SACs only in adults. Neither tetrodotoxin, which blocked action potentials, nor LY354740, which blocked neurotransmitter release from SACs, eliminated ACh-induced GABAergic feedback in SACs. These results suggest that ACh-induced GABAergic feedback in ON and OFF SACs is regulated by different feedback mechanisms in adults and mediated by non-spiking amacrine cells other than SACs.

We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins.

The release of neurotransmitter from a single synaptic vesicle generates a quantal response, which at excitatory synapses in voltage-clamped neurons is referred to as a miniature excitatory postsynaptic current (mEPSC). We analyzed mEPSCs in cultured mouse hippocampal neurons and in HEK cells expressing postsynaptic proteins enabling them to receive synaptic inputs from cocultured neurons. mEPSC amplitudes and rise-times varied widely within and between cells. In neurons, mEPSCs with larger amplitudes had longer rise-times, and this correlation was stronger in neurons with longer mean rise-times. In HEK cells, this correlation was weak and unclear. Standard mechanisms thought to govern mEPSCs cannot account for these results. We therefore developed models to simulate mEPSCs and assess their dependence on different factors. Modeling indicated that longer diffusion times for transmitters released by larger vesicles to reach more distal receptors cannot account for the correlation between rise-time and amplitude. By contrast, incorporating the vesicle size dependence of fusion pore expulsion time recapitulated experimental results well. Larger vesicles produce mEPSCs with larger amplitudes and also take more time to lose their content. Thus, fusion pore flux directly contributes to mEPSC rise-time. Variations in fusion pores account for differences among neurons, between neurons and HEK cells, and the correlation between rise-time and the slope of rise-time versus amplitude plots. Plots of mEPSC amplitude versus rise-time are sensitive to otherwise inaccessible properties of a synapse and offer investigators a means of assessing the role of fusion pores in synaptic release.

The presence of impermeant molecules within a cell can lead to an increase in cell volume through the influx of water driven by osmosis. This phenomenon is known as the Donnan (or Gibbs-Donnan) effect. Animal cells actively transport ions to counteract the Donnan effect and regulate their volume, actively pumping Na+ out and K+ into their cytosol using the Na+/K+ ATPase (NKA) pump. The pump-leak equations (PLEs) are a system of algebraic-differential equations to model the membrane potential, ion (Na+, K+, and Cl-), and water flux across the cell membrane, which provide insight into how the combination of passive ions fluxes and active transport contribute to stabilizing cell volume. Our broad objective is to provide analytical insight into the PLEs through three lines of investigation: (1) we show that the provision of impermeant extracellular molecules can stabilize the volume of a passive cell; (2) we demonstrate that the mathematical form of the NKA pump is not as important as the stoichiometry for cell stabilization; and (3) we investigate the interaction between the NKA pump and cation-chloride co-transporters (CCCs) on cell stabilization, showing that NCC can destabilize a cell while NKCC and KCC can stabilize it. We incorporate extracellular impermeant molecules, NKA pump, and CCCs into the PLEs and derive the exact formula for the steady states in terms of all the parameters. This analytical expression enables us to easily explore the effect of each of the system parameters on the existence and stability of the steady states.

The voltage-sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage-sensing proteins, the VSDs do not interact with one another, and the S1-S3 helices are considered mainly scaffolding, except in the voltage-sensing phosphatase (VSP) and the proton channel (Hv). To investigate its contribution to VSP function, we mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134, and L137), individually or in combination. Most of these mutations shifted the voltage dependence of activity to higher voltages; however, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered, with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions was consistently shifted to lower voltages and indicated a second voltage-dependent motion. Additionally, none of the mutations broke the VSP dimer, indicating that the S1 impact could stem from intra- and/or intersubunit interactions. Lastly, when the same mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme's conformational response to membrane potential transients and influencing the function of the VSD.

Voltage-gated ion channels are responsible for the electrical excitability of neurons and cardiomyocytes. Thus, they are obvious targets for pharmaceuticals aimed to modulate excitability. Compounds activating voltage-gated potassium (KV) channels are expected to reduce excitability. To search for new KV-channel activators, we performed a high-throughput screen of 10,000 compounds on a specially designed Shaker KV channel. Here, we report on a large family of channel-activating compounds with a carboxyl (COOH) group as the common motif. The most potent COOH activators are lipophilic (4 < LogP <7) and are suggested to bind at the interface between the lipid bilayer and the channel's positively charged voltage sensor. The negatively charged form of the COOH-group compounds is suggested to open the channel by electrostatically pulling the voltage sensor to an activated state. Several of the COOH-group compounds also activate the therapeutically important KV7.2/7.3 channel and can thus potentially be developed into antiseizure drugs. The COOH-group compounds identified in this study are suggested to act via the same site and mechanism of action as previously studied COOH-group compounds, such as polyunsaturated fatty acids and resin acids, but distinct from sites for several other types of potassium channel-activating compounds.

JGP study (Rayaprolu et al. https://doi.org/10.1085/jgp.202313467) shows that hydrophobic residues in the S1 transmembrane domain modulate the voltage sensor movements and enzymatic activity of voltage-sensing phosphatase.

The TMEM16A calcium-activated chloride channel is a promising therapeutic target for various diseases. Niclosamide, an anthelmintic medication, has been considered a TMEM16A inhibitor for treating asthma and chronic obstructive pulmonary disease (COPD) but was recently found to possess broad-spectrum off-target effects. Here, we show that, under physiological Ca2+ (200-500 nM) and voltages, niclosamide acutely potentiates TMEM16A. Our computational and functional characterizations pinpoint a putative niclosamide binding site on the extracellular side of TMEM16A. Mutations in this site attenuate the potentiation. Moreover, niclosamide potentiates endogenous TMEM16A in vascular smooth muscle cells, triggers intracellular calcium increase, and constricts the murine mesenteric artery. Our findings advise caution when considering clinical applications of niclosamide as a TMEM16A inhibitor. The identification of the putative niclosamide binding site provides insights into the mechanism of TMEM16A pharmacological modulation and provides insights into developing specific TMEM16A modulators to treat human diseases.