Abstract
During accommodation, the lens changes focus by altering its shape following contraction and relaxation of the ciliary muscle. At the cellular level, these changes in shape may be accompanied by fluid flow in and out of individual lens cells. We tested the hypothesis that some of this flow might be directly modulated by pressure-activated channels. In particular, we used the whole cell patch clamp technique to test whether calcium-activated-chloride channels (CaCCs) expressed in differentiating lens cells are activated by mechanical stimulation. Our results show that mechanical stress, produced by focally perfusing the lens cell at a constant rate, caused a significant increase in a chloride current that could be fully reversed by stopping perfusion. The time course of activation and recovery from activation of the flow-induced current occurred rapidly over a time frame similar to that of accommodation. The flow-induced current could be inhibited by the TMEM16A specific CaCC blocker, Ani9, suggesting that the affected current was predominantly due to TMEM16A chloride channels. The mechanism of action of mechanical stress did not appear to involve calcium influx through other mechanosensitive ion channels since removal of calcium from the bath solution failed to block the flow-induced chloride current. In conclusion, our results suggest that CaCCs in the lens can be rapidly and reversibly modulated by mechanical stress, consistent with their participation in regulation of volume in this organ.
Highlights
The lens is an avascular, transparent organ whose primary function is to focus light on the retina
To investigate the possible role of calcium influx through mechanosensitive channels in the generation of flow-induced calcium-activated chloride currents, we examined the effect of flow on the calcium-activated chloride current using a paired ramp protocol either in the nominal absence of external calcium or following application of gadolinium (Gd3+), a non-specific cation channel blocker which has been previously reported to block piezo channels (Coste et al, 2010), gap junctional hemichannels (Eskandari et al, 2002), L-type calcium channels (Biagi and Enyeart, 1990; Lacampagne et al, 1994), and some TRP channels (Yang and Sachs, 1989; Gunthorpe et al, 2002; Hamill, 2006)
We show that mechanical stress created by focal perfusion resulted in an increase in an outwardly rectifying membrane current in differentiating lens epithelial cells and newly elongating fiber cells
Summary
The lens is an avascular, transparent organ whose primary function is to focus light on the retina. One of the main advantages of using this preparation to perform electrophysiological experiments is that the isolation of fiber cells from lens of these KO mice can be performed using milder dissociation conditions than those used in past attempts to dissociate fiber cells, possibly due to alterations in cell adhesion properties (Wang et al, 2016; Hu et al, 2017) Another advantage is that the double KO fiber cells lack large, calcium-sensitive connexin hemichannel currents and can tolerate exposure to calcium-containing solutions without requiring the addition of non-specific cation channel blockers such as 1 mM Gadolinium to prevent fiber cell vesiculation and death (Ebihara et al, 2011; Tong et al, 2019). Our results demonstrate that CaCCs in dissociated lens can be rapidly and reversibly modulated by mechanical stress, consistent with their participation in regulation of volume in this organ
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