ICOR channel

The increase in the open probability of the ICOR channel induced by hormones in cell attached patches is moderate at best. 

The macroscopic CP-conductance induced by such hormones does not reflect the properties of the ICOR channel [12]. 

CP-channels with even smaller conductance have been described for the lacrimal and other exocrine glands [76,77]. These channels have a conductance of 1-2 pS. Unlike the ICOR-channel they appear to be blocked by millimolar concentrations of furosemide [77]. Most recent and only partially published data from my own laboratory obtained with the above modified nystatin technique [50,133,134] indicate that the respiratory epithelial cells and colonic carcinoma cells possess these types of small CP channels, and that these channels are involved in hormonal regulation of CP-conductance (cf. section 5). These CP-channels are regulated by cytosolic Ca. Hormonally induced increases in cytosolic Ca lead to an abrupt increase in the probability of these small CP-channels being open, yet they have no effect on the ICOR-channel. Data of this kind reinforce that the physiological importance of these small CP-channels may have been grossly underestimated. 

Fig. 2. Agents controlling the opening of Cl -channels. The structural formula, the name, the respeetive Cl -channel, and an appropriate reference are provided. (A) Note the similarity of GABA, taurine, and P-alanine. Note also that DPC and its analogues such as NPPB contain a -alanine backbone. (B) The structure of torasemide comes close to both DPC and furosemide or buraetanide. It blocks the Na ZCPK" -cotransporter with very high affinity and with lesser affinity also the TAL-Cl -channel. Note that IAA-94 is related to phenoxyaeetic acids (e.g., ethacrynic acid). Amidine is related to IAA-94 but it has a positive net charge. Amidine as well as IAA-94 block the ICOR channel at around lO mol/1 [63].

In a recent study on the ICOR-channel we have compared the effect of NPPB to that of the disulphonate stilbenes (Fig. 2), IAA-94 (Fig. 2) and amidine (Fig. 2). We found that all these compounds induced a flicker-type block. NPPB showed the highest affinity [63]. The results of this study were perplexing inasmuch as compounds which appear to be chemically different, and even possess opposite net charges, exert comparable effects. In further studies on the ICOR channel others and we were able to show that unsaturated fatty acids, bumetanide, the buffer HEPES and even Ca -antagonists such as verapamil inhibited the ICOR channel... 

In respiratory epithelial (RE) cells the Cl -conductance was attributed to the ICOR channel. In fact, it was reported by Frizzell et al. and Welsh s laboratories that catecholamines increased the incidence of ICOR channels in cell attached patches of normal RE cells but failed to do so in CF cells [110,111], Later both laboratories presented data on excised membrane patches of RE cells in which the protein kinase A which was added to the cytosolic side produced ICOR channel activity in the normal cells but not in the CF tissues [19,20]. This finding was reproduced by Guggino and coworkers [22] for RE cells and by others for lymphocytes [46]. Protein kinase C at physiological Ca -activities had a comparable effect in normal cells but also failed to function in CF cells [22,112]. 

Our own laboratory obtained different results. Not only were we unable to see a clear cut correlation between the incidence of ICOR channels in cell attached patches and the degree of hormonal stimulation [57], we were also unable to reproduce the activation studies in excised patches. In our hands, the activation of ICOR channels occurred simply by the excision, and this was equally true for the normal as for the CF cells [57]. We did note, however, that the other laboratories worked at room temperature whereas we always work at 37°C. Welsh s laboratory has shown meanwhile that excision activation of ICOR Cl -channels is a temperature-dependent process [113]. At low temperature, excision activation is largely delayed [113] but it is immediate in our experiments at 37°C [57]. We concluded that the activation of the ICOR channels has probably little to do with phosphorylation but is rather due to the fact that the excised patch faces a new environment on the cytosolic side [57,72],... 

Meanwhile we have shown that the excision activation of ICOR channels is due to disinhibition [72]. The respective inhibitor, operationally named cytosolic inhibitor (Cl), is present in the cytosol of placenta trophoblast cells HT29- and Tg4-colonic carcinoma cells and RE cells of normal and CF patients. The molecule has an apparent molecular weight of 700-1 500 Da it is amphiphilic heat stable and not digested by trypsin, proteases, nucleotidases, lipases or amylase [72]. Burc-khardt, Fromter and their collaborators [114] have confirmed our results and extracted a similar or identical Cl from kidney cortex. 

In our patch clamp studies in excised membrane patches in which we attempted to characterize the Cl we have noted that Cl did not only inhibit the probability of the ICOR channel being open but we also found that the input conductance of the patch was reduced at the same time and with the same time course [72]. We have followed up on this observation and we were able to show that this reduction in input conductance is caused by an inhibition of small ( lOpS) Cl -channels. Hence, we postulate that the same patches containing ICOR channels also contain small (unresolved, cf. section 2.4) Cl -channels which are inhibited reversibly by CL It cannot be excluded at this stage that these small Cl -channels are responsible for the defect in CF. 

Frizzell and coworker s laboratory has examined the properties of the whole-cell currents generated by cAMP, increases in cytosolic Ca ", and increases in cell volume [12,124], They found that the properties of all three Cl -currents are different. The cAMP induced Cl -current is linear and conducts Cl better than I. These findings do not fit with the properties of the ICOR channel, which is outwardly rectifying and conducts 1 better than CP (cf. above). On the other hand, the CP-current induced by hypoosmolar solutions had characteristics similar to the ICOR channel. Comparable results have meanwhile been obtained in another laboratory (Fromter, personal communication). 

The physiological role of the ICOR is not clear and may be heterogeneous in the various tissues. In the thick ascending limb of the loop of Henle this channel appears to serve as the exit for CP at the basal cell pole [16,65,66], This conductive mechanism, therefore, is required for the reabsorption of Na and CP by this segment of the nephron [16]. In the rectal gland of Squalus acanthias a very similar channel is utilized for Na" and CP secretion. In these latter cells the CP-channel is present in the luminal membrane and is controlled by cytosolic cAMP [15,56,71]. It has been claimed that this kind of channel is also responsible for the secretion of CP in the colonic crypt cell, in colonic carcinoma cells and in respiratory epithelial cells [17,19,20,22]. Recent data have cast some doubt on this concept ... 

CP-channels with smaller conductance have first been noted in the rectal gland of Squalus acanthias by ourselves and in the colonic carcinoma cell line HT29 [61,73]. Later these types of 5-15 pS CP-channels were also found in pancreatic ducts, A6-cells and many other cells [74,75]. It is now claimed that this kind of channel is much more relevant than the ICOR for the pathophysiology of cystic fibrosis [12]. 

The receptor-operated Cl -channels of the central nervous system (CNS) are gated by the respective agonists GABA and glycine. Most Cl -channels can be inhibited by disulphonate stilbenes. Muscle Cl -channels can be inhibited by anthracene-9-carboxylate (A9C) and probably by IAA-94. The ICOR Cl -channel is fairly sensitive to NPPB. It should be noted, however, that none of these probes, except for the GABA- and glycine-receptor Cl -channels, is of sufficient affinity and selectivity to permit the channel identification by its use. This dilemma is one of the reasons why the purification of epithelial Cl -channels lags behind that of the CNS Cl -channels. 

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