A high concentration of K⁺ is maintained in the lumen of the cochlea via electrogenic secretion by the strial marginal cell epithelium 1 . One pathway of regulation is the coupling of purinergic receptors on the apical membrane of these cells to the apical potassium channels (IKs) which mediate secretion 2 . These receptors are responsive to both ATP and UTP as agonists and they were found to exert their action via the phospholipase C – protein kinase C intracellular signal pathway 3 . At the time of the original investigations, the purinergic receptor field recognized only 1 receptor responding to uracil nucleotides (P2U, now P2Y₂ receptor). The known members of the mammalian metabotropic, G protein-coupled, purinergic receptors has since grown to P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃ and P2Y₁₄ 4 . In addition to P2Y₂, both the P2Y₄ and P2Y₆ receptors also respond to uracil nucleotides. On the basis of pharmacologic agonist and antagonist profiles, P2Y₆ could be distinguished from P2Y₂ and P2Y₄ by the greater potency of UDP over UTP 5 6 . The pharmacologic distinction between P2Y₂ and P2Y₄ was more ambiguous and the accepted criteria changed rapidly 7 .

The question of the subtype of P2Y receptor mediating regulation of K⁺ secretion by strial marginal cells has been addressed by immunohistochemistry, which suggests the presence of P2Y₄ in the apical marginal cell membrane 8 . However, a functional demonstration was lacking, the time-course of activation was not investigated and an independent demonstration of gene expression was not available.

The present study was conducted with the goals of 1) obtaining dose-response profiles for potentially definitive agonists of P2Y₂, P2Y₄ and P2Y₆ receptors; 2) refining the profile by the use of antagonists, 3) testing for desensitization of the response to agonist and 4) determining the presence of transcripts for pyridine-sensitive P2Y receptors. The results support the conclusion that regulation of K⁺ secretion by strial marginal cells occurs by apical P2Y₄ receptors.

ATP perfused for 30 s at the apical side of strial marginal cell epithelium caused a monophasic decrease in I_sc (Fig. 1A), consistent with previous findings 2 . Removal of ATP led to a recovery of I_sc after an initial overshoot. Similar responses were seen from perfusion of other agonists, such as UTP and Ap4A (Fig. 1B &1C).

The P2Y₆ agonist, UDP, was tested after removal of contamination by UTP in the commercial product 6 . Contaminating UTP was digested by hexokinase in the presence of glucose. The enzymatically-purified UDP produced little effect on I_sc (Fig. 2).

Concentration-response curves for the purinergic agonists are summarized in Figure 2. The potency order was UTP ≥ ATP > Ap4A >> UDP with EC₅₀ values for UTP and ATP of about 2.3 and 1.2 × 10^-5 M. The relative potencies of the agonists are consistent with an action on P2Y₂ and/or P2Y₄ receptors.

The purinergic receptor antagonist, suramin, was tested for effectiveness in blocking the response to apical UTP. Suramin at 100 μM had no inhibitory effect on the action of the agonist, UTP (Fig. 3). UTP (1 μM) caused a decrease in I_sc by 20.6 ± 5.4% in the absence of suramin and by 21.9 ± 5.3% (P > 0.05, n = 5) in the presence of suramin.

Both P2Y₂ and P2Y₄ receptor subtypes have been reported to undergo desensitization within 5–15 min 9 10 . Surprisingly, we found that sustained exposure (15 min) of the apical membrane to ATP led to a biphasic but sustained inhibition of I_sc (Fig. 4). The recovery of I_sc after removal of agonist was much slower, incomplete and without overshoot compared to acute exposure to agonist.

Perfusion of UTP (10 μM) for 30 s led to an increase in intracellular [Ca²⁺] that had both a peak and plateau phase and was repeatable (Fig. 5, top). Both the peak and plateau were significantly reduced by 2-APB (75 μM) (to 26.5 ± 3.9% and 25.1 ± 4.6%; Fig. 5, middle). 2-APB is an inhibitor of the IP3 receptor at this concentration 11 12 and can also inhibit other transporters including store-operated Ca²⁺ channels 13 . Store operated channels, however, would not be activated under the present experimental conditions, since the Ca²⁺ stores were not emptied prior to measurement. In the absence of external Ca²⁺, both the peak and plateau responses to UTP remained but were significantly reduced (to 68.9 ± 3.0% and 60.5 ± 4.2%; Fig. 5, bottom). The reductions, however, were likely the result of a general decrease in intracellular [Ca²⁺] seen after removal of bath Ca²⁺ prior to perfusion of UTP. These findings taken together suggest that intracellular stores are the primary source of the increase in intracellular [Ca²⁺] induced by UTP.

We tested for the presence of transcripts for P2Y₂ and P2Y₄ receptors in stria vascularis. Primers proven to recognize gerbil P2Y₂ and P2Y₄ 14 were used to amplify single, gene-specific bands in stria vascularis (Fig. 6); 301 base pairs (bp) for P2Y₂ and 447 bp for P2Y₄. Controls in which the reactions were run in the absence of reverse transcriptase (-RT) demonstrated the absence of contributions from genomic DNA. PCR products were analyzed for their sequence to confirm the identity of the bands. Sequences were the same as found previously in the gerbil vestibular labyrinth 14 (GenBank accession numbers: P2Y₂, AF313448; P2Y₄, AF313447).

Purinergic agonists control strial marginal cell K⁺ secretion, a process observed as a transepithelial short circuit current, I_sc 1 2 . Strial marginal cells secrete K⁺ by a constellation of transporters previously described 1 . K⁺ is taken up across the basolateral membrane by the Na⁺, K⁺-ATPase and the Na⁺, K⁺, 2Cl^--cotransporter. Na⁺ carried into the cell on the cotransporter is removed by the Na⁺-pump and Cl^- carried into the cell on the cotransporter leaves by passive diffusion across a large Cl^- conductance in the basolateral membrane 15 16 17 . K⁺ taken up by both the Na⁺-pump and cotransporter is secreted across the apical membrane by diffusion through IKs channels 18 , consisting of KCNQ1 alpha subunits and KCNE1 beta regulatory subunits 19 20 . The epithelium regulates K⁺ secretion by a variety of signal pathways, including those initiated by apical purinergic receptors, that converge at the IKs channel complex 21 .

The previous demonstration of control of I_sc in strial marginal cell epithelium by "P2U" receptors included determination of a concentration-response to ATP and comparative activities of other nucleotides (UTP, 2-MeS-ATP and α, β-meth-ATP) at 1 μM 2 . Full concentration-response curves were obtained in the present study to better define the active subtype. The lack of response to UDP eliminated P2Y₆ as a candidate subtype 22 23 24 . The similar potency of ATP and UTP is consistent with hP2Y₂ 23 , rP2Y₂ 22 but not hP2Y₄ 6 . In fact, ATP acts as a competitive antagonist at the hP2Y₄ receptor 25 . However, these results alone do not identify the strial marginal cell apical purinergic receptor as P2Y₂ since it was subsequently found that rP2Y₄ has an agonist potency sequence similar to rat and human P2Y₂ 7 25 . Since the gerbil is expected to be more closely related to other rodents such as the rat, than to human, our finding is consistent with the contribution of P2Y₄ and/or P2Y₂.

The potency order of the agonists UTP, ATP, Ap4A and UDP in regulation of I_sc from strial marginal cells is the same as for rodent P2Y₄ receptors in other systems, including cloned mouse P2Y₄ 26 and gerbil vestibular dark cells 2 27 even though the EC₅₀ values are substantially shifted. The absolute values of EC₅₀'s are alone not indicative of receptor binding for G protein-coupled receptors, although the relative potency remains constant. Receptor density plays a large role in defining the response of downstream effectors (IKs channels in this case) and differences in density can lead to significant shifts in EC₅₀ curves.

Transcripts for both P2Y₂ and P2Y₄ were found in stria vascularis. The identity of the cell type(s) within the stria that contain these transcripts is not certain from these findings alone. Recent immunohistochemical findings show staining for the P2Y₄ receptor at the apical membrane of strial marginal cells, while the antibody for P2Y₂ stained at the basolateral region of the marginal cells and/or the intermediate cells 8 , consistent with the functional evidence for apical P2Y₄ shown here. The transcript expression confirms the immunohistochemical findings by an independent means and thereby provides an important verification of the specificity of the P2Y antibodies used in the previous study 8 .

Ap4A was found to be a potent agonist at the hP2Y₂ receptor 23 but much less potent than ATP at the hP2Y₄ 28 and rP2Y₄ receptors 24 , similar to our finding in gerbil strial marginal cells. In spite of this similarity, uncertainty arises in the identification of the apical receptor in strial marginal cells as P2Y₄ on this basis alone since there is also a conflicting report from Bogdanov et al. who found Ap4A to be equally potent as ATP at rP2Y₄ receptors 7 .

Suramin inhibits several P2 receptors, but it was found recently that at a high concentration (100 μM) it can be used to distinguish both rP2Y₂ 22 and hP2Y₂ 29 from both rP2Y₄ 7 and hP2Y₄ 29 . This criterion applied to the present results points to the apical P2Y purinergic receptor in gerbil strial marginal cells as the P2Y₄ subtype. This conclusion based on function and pharmacology is consistent with the recent report of immunohistochemical localization of P2Y₄ at the apical membrane of strial marginal cells 8 . We reported earlier that the response to ATP is increased in the absence of divalent cations 2 . This increased effect suggests that the apical receptor is preferentially activated by an uncomplexed form of ATP, as in aortic endothelial cells 30 .

The overshoot of I_sc after removal of purinergic agonists from the apical perfusate (Fig. 1) is most likely due to a release of K⁺ accumulated in the epithelial cells during the inhibition of secretion across the apical membrane. The basolateral K⁺ uptake mechanisms continue to operate for a time after inhibition of apical IKs channel complexes by activation of purinergic receptors, bringing the cytosolic K⁺ concentration to a level higher in electrochemical potential above the apical bath than in the absence of agonist. Upon removal of agonist this "extra pool" of K⁺ is suddenly released, resulting in the observed overshoot of I_sc. The same phenomenon was reported previously 31 when K⁺ secretion was first diminished by raising the apical K⁺ concentration, thereby reducing the outward gradient across the apical cell membrane. Suddenly returning the apical perfusate K⁺ concentration to the original low level led to an overshoot of I_sc, as observed with the purinergic agonists.

The decrease in I_sc observed in response to purinergic agonists could be due a priori to a reduction of electrogenic K⁺ secretion but could also be accounted for by a stimulation of secretion of Cl^- or absorption of Na⁺. However, it was found in a previous study that the decrease in I_sc could be completely accounted for by a decrease in K⁺ secretion. Apical perfusion of 100 μM ATP lead to a decrease of I_sc by 37.1 ± 3.7% and of K⁺ secretory flux by 22.2 ± 5.5% 3 .

The peak and plateau increases in intracellular [Ca²⁺] during perfusion of agonist in the presence and absence of bath Ca²⁺, as well as the reduction by 2-APB, are consistent with a release of Ca²⁺ from intracellular stores. Ikeda et al. found a small monophasic increase in intracellular [Ca²⁺] of the entire stria vascularis that could not be analyzed further 32 . The same laboratory evaluated the purinergic response of cultured marginal cells and found agonist-induced increases in intracellular [Ca²⁺] that were not reduced upon removal of Ca²⁺ from the bath 33 . Those cells were derived from guinea pig, the responses were monophasic and UTP was not tested as agonist.

The apparent absence of desensitization (Fig. 4) was a surprising finding in view of reports of rapid desensitization of both cloned P2Y₂ and cloned P2Y₄ purinergic receptors 9 10 . Desensitization has typically been observed as a decrease of inositol phosphate production and/or cytosolic Ca²⁺ increase, either directly 34 35 or via the effect of Ca²⁺ on transepithelial anion secretion 36 . Since the sustained response to UTP (Figure 4) is biphasic, it therefore may represent the summation of at least 2 processes.

The question arises as to the basis for the observed sustained decrease in IKs. Possible explanations include: 1) The P2Y₄ receptor desensitizes several minutes after the onset of agonist, but the initial activation of P2Y₄ leads to phosphorylation of KCNE1 via PKC 3 and the channel subunit remains phosphorylated due to inhibition of phosphatase activity. 2) Activation of P2Y₄ leads to localized depletion of phosphatidylinositol-4,5-bisphosphate (PIP₂) in the plasma membrane and to the consequent deactivation of the PIP₂-dependent IKs channel.

I_sc is controlled by apical P2Y₄ receptors via the protein kinase C pathway by phosphorylation of the beta subunit of the IKs channel complex 3 [vida infra]. The sustained reduction in I_sc and the incomplete recovery of I_sc after prolonged exposure to agonist can both be explained if there is a decrease of phosphatase activity at the phosphorylation site. The channel subunit would then be expected to remain phosphorylated and the effect on I_sc would be sustained even with a desensitization of the receptor. However, dephosphorylation rates have been reported to increase with elevated intracellular [Ca²⁺] in olfactory sensory neurons 37 , arguing against this hypothesis.

The second explanation is more consistent with our findings. It has been found that activation of receptors (such as P2Y₄) coupled to G_q/11 deplete PIP₂ 38 and that IKs (KCNQ1/KCNE1) activity is controlled by PIP₂ 39 . In fact, the time course of desensitization of another K⁺ channel by phenylephrine induced depletion of PIP₂ via α₁-adrenergic receptors is similar to the secondary effects seen here 38 .

The response to purinergic agonists at the apical side of strial marginal cells was shown here to be functionally mediated by P2Y4 receptors, previously shown to be present by immunostaining. The decrease in electrogenic K⁺ secretion evoked by agonists was a) initially accompanied by a biphasic increase in intracellular Ca²⁺ and b) surprisingly sustained in the continued presence of agonist. The sustained decrease in K⁺ secretion was best explained by rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP₂. Impaired PIP₂ and IKs interaction was recently shown to be the basis of several mutations causing long QT syndrome, a genetic disease characterized by cardiac arrhythmias and deafness 45 .

The author(s) declare that they have no competing interests.

DCM conceived, designed and coordinated the study and drafted the manuscript. JL acquired the short-circuit current data with the Ussing chamber. JHL acquired the short-circuit current data with the vibrating probe. EQS acquired and analyzed the intracellular calcium data. MAS designed, performed and carried out the RT-PCR experiments. PW designed, interpreted and supported the intracellular calcium experiments.