Diminishing neuronal acidification by channelrhodopsins with low proton conduction

Abstract

Many channelrhodopsins are permeable to protons. We show that in electrically polarized cells, activation of a high-current channelrhodopsin, CheRiff, led to significant acidification. From an initial intracellular pH of 7.3, cells acidified to pH 6.9 ± 0.24 (mean ± s.d., n = 34 cells) after 60 seconds of illumination. To determine whether this acidification arose via proton transport through the opsin vs through other voltage-dependent proton channels, we performed experiments with patterned optogenetic stimulation in monolayers of HEK293 cells expressing CheRiff, an inward-rectifier potassium channel, Kir2.1, and a red-shifted pH indicator, pHoran4. Illumination with stripes of blue light led to electrical depolarization that extended beyond the illuminated spots, due to gap junctional conduction; but acidification was localized to the illuminated spots. These observations established that the acidification required proton transport through the opsin itself. We then identified and characterized two opsins which showed large photocurrents, but small proton permeability, PsCatCh2.0 and ChR2 3M. These opsins were effective at evoking neural activity, but neither induced detectable acidification. PsCatCh2.0 was also spectrally compatible with simultaneous voltage imaging with QuasAr6a. Stimulation-evoked acidification is a possible source of artifacts and disruptions to cell health in scientific and prospective therapeutic applications of optogenetics. Channelrhodopsins with low proton permeability are a promising strategy for avoiding these problems.