# Spatially Resolved Mapping of Voltage‐Gated Proton Channel Activity Reveals Delayed Proton Transport in Local Microenvironments

## TL;DR

A new imaging method reveals how proton channels create local pH changes, which could be important for understanding neurological diseases.

## Contribution

A novel single-molecule imaging platform enables high-throughput spatial mapping of Hv1 proton channel activity and dynamics in living bacteria.

## Key findings

- Hv1 channels cause local pH fluctuations and do not close sharply when voltage is removed.
- Proton transport through Hv1 is delayed in hypertonic environments.
- The imaging method can detect as few as seven protons and maps ≈2300 channels in a large field.

## Abstract

Voltage‐gated proton channel Hv1 is a proton‐selective channel that lacks the pore domain typically found in other ion channels, and it is thought to participate in signal transduction by inducing transient, localized changes in proton concentration. However, the contribution of Hv1 channel distribution and activity to the overall pH homeostatic process is still not well understood. Here a single‐molecule photobleaching‐based quantitative imaging method is developed with an Hv1‐fused ratiometric pHluorin probe to study the distribution and dynamics of the proton channel in living bacteria. It is shown that the Hv1 channel does not close sharply when voltage is removed and can directly cause local fluctuations of intracellular pH gradient. Especially, the proton transport through Hv1 is significantly delayed in hypertonic environments, offering novel perspectives at cellular level for neuropathological research. This spatially localized pH microenvironment created by the Hv1 channel may play a crucial role in signal transduction in neuron cells.

A revolutionary imaging platform integrating quantitative single‐molecule photobleaching with ratiometric fluorescence probes enables high‐throughput (≈2300 individually resolvable channels) mapping of individual proton channels in a large field (≈3000 µm2), overcoming the spatial and temporal limitations of conventional electrophysiology. This approach has a high sensitivity threshold capable of 7 protons, revealing Hv1 channel kinetics delayed about seven‐folds under pathomimetic hyperosmotic stress.