Resting human trabecular meshwork cells experience tonic cation influx

The trabecular meshwork (TM) regulates intraocular pressure (IOP) by converting biochemical and biomechanical stimuli into intracellular signals. Recent electrophysiological studies demonstrated that this process is mediated by pressure sensing ion channels in the TM plasma membrane while the molecular and functional properties of channels that underpin ionic homeostasis in resting cells remain largely unknown. Here, we demonstrate that the TM resting potential is subserved by a powerful cationic conductance that disappears following Na+ removal and substitution with choline or NMDG+. Its insensitivity to TTX, verapamil, phenamil methanesulfonate and amiloride indicates it does not involve voltage-operated Na+, Ca2+ and epithelial Na+ (ENaC) channels or Na+/H+ exchange while a modest hyperpolarization induced by SEA-0440 indicates residual contribution from reversed Na+/Ca2+ exchange. Tonic cationic influx was inhibited by Gd3+ and Ruthenium Red but not GsMTx4, indicating involvement of TRP-like but not Piezo channels. Transcriptional analysis detected expression of most TRP genes, with the canonical transcriptome pool dominated by TRPC1 followed by the expression ofTRPV1, TRPC3 and TRPC5. TRPC3 antagonist Pyr3 and TRPC1,4,5 antagonist Pico1,4,5 did not affect the standing current, whereas the TRPC blocker SKF96365 promoted rather than suppressed, Na+ influx. TM cells thus maintain the resting membrane potential, control Na+ homeostasis, and balance K+ efflux through a novel constitutive monovalent cation leak current with properties not unlike those of TRP channels. Yet to be identified at the molecular level, this novel channel sets the homeostatic steady-state and controls the magnitude of pressure-induced transmembrane signals.


Introduction
Trabecular meshwork (TM) cells are phagocytic and contractile, embedded in a multilayered ECM architecture that serves as a biological lter and resistor system for aqueous humor (Stamer and Clark, 2014;Mao 2024).Similar to smooth muscle cells that provide structural integrity for blood vessels and regulate vascular tone, TM cells physically support the vascular-lymphatic cells that form the canal of Schlemm and control the drainage of aqueous humor (Lutjen-Drecoll, 1999; Stamer and Clark, 2017).The cells are highly mechanosensitive, responding to changes in pressure, strain, shear and osmotic gradients with changes in ionic ux which modulate the cells' shape, gene expression, contractility, and secretion of extracellular matrix (ECM) (Krizaj, 2020; Baumann et al., 2024;Mao, 2024;Redmon et al., 2024).Mechanosensitive membrane signaling is subserved by integrin-based cell-ECM contacts and mechanosensing TRPV4, Piezo1 and TREK-1 channels (Krizaj, 2020; Yarishkin et al., 2018;2021;Zhu et al., 2021;Faralli et al., 2022) that control facilitatory and suppressive effects on trabecular out ow resistance and brotic remodeling (Krizaj, 2020; Lakk and Krizaj, 2021; Yarishkin et al., 2021;Zhu et al., 2021) while ion channels that subserve the resting TM membrane remains poorly understood.
Human TM cells are highly responsive to mechanical inputs, responding to physiological stimuli (strain, shear, swelling, pressure, indentation) with a current that reverses near the resting potential (V rest ~-30 to -40 mV) (Yarishkin et al., 2018).V rest itself is set by extra-and intracellular concentrations of permeant Na + , K + , Ca 2+ and Cl-ions and the relative expression of ion channels and transporters within the TM plasma membrane.Indicating the important function of the ionic mechanisms that maintain Vrest, the TM sensitivity to pressure is likely to be affected by shifts of the membrane potential into depolarizing or hyperpolarizing directions.TM to Ca 2+ permeability modulates cytoskeletal dynamics, gene expression, secretion of ECM proteins and activity of Ca 2+ -dependent TRPM4 and BK channels (Wiederholt et al., 2000;Ryskamp et al., 2016;Lakk and Krizaj, 2021), Cl − e ux, subserved by volume-regulated anion VRAC (LTTRC), CLC2 and/or anoctamine ANO6 channels contributes to cell volume regulation (Grant et al., 2013;Gasull et al. 2019, Banerjee et al., 2016;Baumann et al., 2024) and the K + leak component, maintained by TREK-1 and auxiliary contributions from inwardly rectifying, voltage-gated K + , Ca 2+activated and TASK1 channels (Yarishkin et al., 2018;2019), subserves the hyperpolarizing component of V rest while the identity and functional properties of the leak conductance that balances TREK-1 activity under resting conditions remain unknown.In the present study we employed micro uorimetry in combination with whole cell recording and transcriptomic analyses to characterize the ionic mechanism that underpins homeostatic ionic signaling in the resting TM cell.

Cell cultures and transfection.
Immortalized cells, isolated from the juxtacanalicular region of the human eye (hTM cells), were procured from ScienCell Research Laboratories (Carlsbad, CA), and primary cultures of TM (pTM) cells were dissected from 3 human donors with no history of eye disease (healthy donors: 76-year-old female, 76-year-old male, 78-year-old male; POAG donors two 80-year old males and a 77-year old female; Utah Lions Eye Bank) following the consensus recommendations (Keller et al., 2018), and following the standards set by the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.The phenotype was periodically validated by pro ling for TM markers AQP, TIMP3, MYOC and MGP, and by dexamethasone-induced upregulation of myocilin expression, as described (Lakk et al., 2021;Baumann et al., 2024;Redmon et al., 2024).Passage 2-6 (or up to 4 in case of pTM) cells were seeded onto Collagen I-seeded coverslips and grown in Trabecular Meshwork Cell Medium (ScienCell, Catalog#6591) at 37 0 C and 5% CO 2 .

Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction (Q-RT-PCR)
Total RNA was isolated using Arcturus PicoPure RNA Isolation Kit according to the manufacturer instructions (Applied Biosystems).One microgram of total RNA was used for reverse transcription.Firststrand cDNA synthesis and PCR ampli cation of cDNA were performed using qScript™ XLT cDNA Supermix (Quanta Biosciences).SYBRGreen based real-time PCR was performed using Apex qPCR GREEN Master Mix (Genesee Scienti c).The experiments were performed as triplicates of at least three independent experiments and are expressed as a ~ fold change compared to the control.The C T method (ΔΔC T ) was used to measure relative gene expression where the ~ fold enrichment was calculated as: 2 − [ΔCT (sample) − ΔCT (calibrator)] after normalization.GAPDH and β-tubulin were utilized as endogenous controls to normalize the uorescence.The primer sequences are listed in Table 1.Results are presented as the mean ± S.E.M.

TM cells possess a constitutively active cation in ux pathway
The resting membrane potential (V rest ) of TM cells is ~ -40 mV, with its deviation from the Nernst potential for K + (~ -100 mV), Na + (~+61 mV), Ca 2+ ( + 137 mV) and Cl -(~-64 mV) ions indicating concurrent activation of multiple ion channels.We recently characterized the properties of the K + channel that mediates the principal hyperpolarizing component of TM V rest (Yarishkin et al., 2018(Yarishkin et al., , 2019) ) while the properties of the cationic channel that provides the depolarizing component remain unknown.
An insight into its permeation properties was sought by substituting extracellular Na + ([Na + ] o ) with nonpermeant quaternary ammonium choline and N-methyl-d-glucamine (NMDG + ), as well as Li + or Cs + ions.

Constitutive depolarizing in ux is nonselective for monovalent cations
To ascertain whether the tonic cation in ux mechanism discriminates between monovalent cations, [Na + ] o was substituted for [Cs + ] o and [Li + ] o .The Cs + -based saline induced a modest but signi cant (P < 0.01) depolarization from -29.1 ± 1.2 mV to -20.6 ± 1.8 mV and a negative I hold shift from -4.9 ± 7.6 pA to -19.4 ± 4.9 pA (Fig. 3) (n = 6) whereas Li + -based extracellular solution was associated with a hyperpolarization, from -28.8 ± 3.2 to -34.7 ± 3.4 mV (P < 0.01) and V hold shift from -4.9 ± 3.4 pA to 2.3 ± 3.2 pA (n = 6).Both cations are thus able to substitute for Na + in maintaining V rest , with neither reproducing the dramatic effects of choline/NMDG substitutions.Their effect on V rest might re ect differential channel permeability to monovalent cations with reduced permeability for cations having larger hydrated radius (Cs + > Na + > Li + ).
---Figure 3 near here---Constitutive Na + in ux is not mediated by ubiquitous transporters, exchangers or voltage-operated channels To ascertain the molecular identity of the mechanism mediating tonic in ux of Na another inhibitor of DEG/E Na C channels, evoked modest depolarizations (Fig. 4B).The Na + /Ca 2+ exchange inhibitor SEA-0440 hyperpolarized the cells by ~11 mV, but the tachyphylaxis associated with NCX inhibition argues against a central role in constitutive cation in ux (Fig. 4A).The tonic pathway thus does not involve TTX-sensitive Na + conductances, epithelial Na + channels, Na + /H + exchange and reversed NCX exchange.
---Figure 5 near here---Molecular expression of TRP channels in TM cells.
We pro led the TM transcriptome to identify TRP candidates for tonic cation in ux.The expression pro le was dominated by TRPC1 (Fig. 6A), followed by the expression of TRPM4>TRPA1>TRPV1>TRPV2>TRPC5>TRPV4 mRNAs.Expression of the TRPC1 gene in gTM samples derived from 3 different POAG donors was not different from healthy donor samples (Fig. 6B).
---Figure 6 near here--- The standing cationic in ux is not mediated by TRPC1 homomers or heteromers To assess the involvement of TRPC1 channels in maintaining the standing cationic inward current, we exposed the cells to SKF96365, a general inhibitor of TRPC channels (Szikra et al., 2009;Molnar et al., 2016).Unexpectedly, Na + Green -loaded cells responded to the inhibitor (25 mM) with large increases in [Na + ]i.While substituting [Na + ]o with choline obviated this effect (n =3) to show it is mediated by a Na +permeable channel, the SKF96365 -independence of tonic Na + in ux argues against the involvement of TRPC1 (and TRPC isoforms in general).Consistent with this, Pico-145 (5 mM), an antagonist of TRPC4, TRPC5 homomers and TRPC1/4,5 heteromeric channels (Rubaiy et al., 2017) did not induce obvious changes in TM membrane potential or current (n=3).The TRPC3 inhibitor pyrazole 3 (Pyr3; 5 mM), which does not affect other DAG-sensitive canonical isoforms but blocks Orai channels (Schleifer et al., 2012), had a modest facilitatory effect whereas the TRPV1 channel capsazepine (5 mM) (Jo et al., 2017) had no effect (Fig. 5C).

Discussion
In this study we identify show that a novel monovalent cation leak pathway that maintains the resting membrane potential in unstimulated human TM cells.The constitutive cation in ux mediated by the leak channels was largely unaffected by substituting Na + with Li + and Cs + , resisted Piezo1, epithelial Na + and voltage-gated Ca 2+ or Na + channel inhibitors but was blocked by nonselective inhibitors of TRP channels.The new conductance, which remains to be identi ed at the molecular level, plays a fundamental function as a main regulator of the cells' V rest that balances mechanosensitive TREK-1 activity (Yarishkin et al., 2018), controls the driving forces for K + , Na + , Ca 2+ and Cl -uxes, and modulates the amplitude of the pressure-activated current.
The rapid sustained hyperpolarization and [Na + ] i decrease observed following substitution of extracellular Na + with choline or NMDG + , quaternary amines that cannot cross the plasma membrane or pass through Na + channels due to their xed charge, indicate the presence of constitutive cationic in ux in hTM/pTM cells.Our observation that the depolarizing leak channel is permeable to Na Because E Na C and Ca 2+ channel inhibition should hyperpolarize the cells we thus cannot explain the small depolarizing effects of verapamil and phenamil methanesulfonate; more studies are required to understand these effects, which we tentatively ascribe to nonselective action.Amiloride insensitivity argues against involvement of E Na Cs and Na + /H + exchange and excludes a major role for proton homeostasis (Yarishkin et al., 2019a), and insensitivity to amiloride and Cs + excludes a role for the HCN current that might follow TREK-1 mediated tonic hyperpolarization (Yarishkin et al., 2018;Yarishkin et al., 2019a, b).The modest (~11 mV) yet consistent hyperpolarization induced by SEA-0440 is, however, of interest as indicates the possibility that resting TM cells experience calcium in ux via reversed Na + /Ca 2+ exchange.
Na + substitution with Cs + and Li + induced modest (~ 5 mV) Vrest changes in opposite directions, depolarizing (Cs + ) and hyperpolarizing (Li + ), with neither replicating the powerful hyperpolarizations induced by nonpermeable cations.This indicates that the constitutive in ux pathway is permeable to small monovalent cations, with the differential effects on V rest and I hold potentially re ecting the effects of different radii, differential effects on the Na + /K + ATPase and/or Cs + /Li + -sensitive channels and transporters.
In contrast to the lack of the effect of voltage-gated channel and exchange inhibitors, the block of tonic in ux by broad-spectrum TRP inhibitors Ruthenium Red and Gd 3+ accords with potential involvement of TRP or other non-selective cation channels.Of the seven members (TRPC1-7) of the canonical family we found expression to be by far the most prominent for the TRPC1 gene, with additional TRPC3, TRPC5 and In conclusion, we characterized the properties of a powerful novel ionic conductance in human TM cells with potentially signi cant functions in out ow regulation, brotic remodeling and glaucoma.From a physiological perspective, this constitutively activated channel controls the driving forces for Na + , K + Cland Ca 2+ ions and thus determines the reversal potential for the pressure-activated current.Following its identi cation, this channel could be targeted to regulate the magnitude of the TM pressure response mediated by Piezo1, TRPV4 and TREK-1 channels.
Constitutive    The expression pattern for principal TRP isoforms in pTM cells, normalized to TRPV1.(A) TRPC1 expression was the most abundant, followed by TRPM4, TRPA1 and TRPV1 mRNAs.(B) TRPC1 expression in pTM vs gTM cells (N = 3).
cation in ux in resting human TM cells.Whole cell recording, current-clamp.(A) Substitution of external Na + with nonpermeable quaternary cations hyperpolarizes the TM resting potential and induces a positive shift in I hold .Choline substitutions result in rapid, reversible and reproducible hyperpolarizations.(B) Averaged data for [Na + ]o substitution with external choline and NMDG.The response of pTM cells was identical to the hTM response (C & D) Voltage clamp.[Na + ]o substitution with choline and NMDG is associated with a positive shift in the holding current, indicating loss of tonic cation in ux.*, P <0.05; ** P<0.01; *** P < 0.005.

Figure 4 Na
Figure 4
(Lu et al., 2007;Monteil et al., 2024)24)24)n et al., 2024)ch has been implicated in regulation of TM contractility and conventional out ow(Luo et al., 2014;Ryskamp et al., 2016;Redmon et al., 2024)and TRPM4, a Na + channel downstream from TRPV4(Yarishkin et al., 2022), our understanding of the expression and function of the other 26 TRP isoforms is minimal for any mammalian TM preparation.The expanded roster of expressed TRP genes will hopefully encourage further study of these polymodal cation channels.In particular, we would like to highlight the potential signi cance of the TRPC1 channel, which dominates the pTM TRP transcriptomic pro le.This vertebrateYarishkin et al., 2022;Baumann et al., 2024).Future studies may look into the involvement of NALCN, a Na + leak channel that subserve non-selective voltage-independent leak currents in subsets of neurons, heart and secretory organs(Lu et al., 2007;Monteil et al., 2024).Similar to the tonic current in TM cells, NALCN is permeable to Cs + and Li + , inhibited by replacing Na + with NMDG + , insensitive to TTX, inhibited by Gd 3+ and resistant to SKF96365(Lutas et al., 2016;Monteil et al., 2024).NALCN but not the TM standing current, however, is inhibited by verapamil(Lu et al., 2007;Monteil et al., 2024).