Calmodulin Antagonist W-7 Enhances Intermediate Conductance Ca2+- Sensitive Basolateral Potassium Channel (IKCa) Activity in Human Colonic Crypts

Intermediate conductance potassium (IKCa) channels are exquisitively Ca2+ sensitive, intracellular Ca2+ regulating channel activity by complexing with calmodulin (CaM), which is bound to the cytosolic carboxyl tail. Although CaM antagonists might be expected to decrease IKCa channel activity, the effect of W-7 in human T lymphocytes are conflicting. We therefore evaluated the effect of W-7 on basolateral IKCa channels in human colonic crypt cells. Intact crypts obtained from normal human colonic biopsies by Ca2+ chelation were used for patch clamp studies of basolateral IKCa channels in the cell-attached configuration. IKCa channel activity was studied when the bath Ca2+ concentration was changed from 1.2 mmol/L to 100 μmol/L and back to 1.2 mmol/L, as well as from 100 μmol/L to 1.2 mmol/L and back to 100 μmol/L, both in the absence and presence of 25 μmol/L W-7. Decreasing bath Ca2+ from 1.2 mmol/L to 100 μmol/L decreased IKCa channel activity reversibly in the absence of W-7, whereas there was a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. In separate experiments, increasing bath Ca2+ from 100 μmol/L to 1.2 mmol/L increased IKCa channel activity reversibly in the absence of W-7, whereas there was again a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. We, therefore, propose that W-7 has a specific stimulatory effect on basolateral IKCa channel activity, despite its ability to inhibit Ca2+/CaM-mediated, IKCa channel-dependent Cl− secretion in human colonic epithelial cells. Graphic Abstract

CaV channels reject signaling from a second CaM in eliciting Ca2+-dependent feedback regulation

Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca2+ feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca2+ regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaMs to the CaV nanodomain by tethering either WT or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary β2A subunit. We observed that a single CaM tethered to either the α or β2A subunit tunes Ca2+ regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit–tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca2+ feedback of CaV channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-dependent feedback of channel gating but may support alternate regulatory functions.

PENGOLAHAN GONDORUKEM MENJADI BAHAN PENDARIHAN SEBAGAI ADITIF PADA PEMBUATAN KERTAS

Gondorukem (Resina colophium) is produced by steam distillation of pine resin (oleoresin). It is yellow-brown solid containing mainly tricyclic alkyl organic acids (abietat acid and pimarate acid). The two acids have ampliphilic characteristics due to their hydrophobic carboxyl tail and hydrophilic tricyclic molecules. The purpose of this research was to prepare emulsion rosin as a sizing agent. Two types of gondorukem (WW and X grades) were saponified by adding a sodium hydroxide solution at a temperature below 100oC. For improving the quality of saponified rosins, the rosins were fortified by adding some maleic anhydride. Some tests were conducted to measure saponified and acidic numbers of a known rosin grade. The tests show that the acidic and saponified numbers of an X grade rosin were 179.92 and 199.74, respectively. Those of a WW grade were 176.58 and 195.84 for acidic and saponified numbers, respectively. Therefore, it can be concluded that the X grade gondorukem has a better quality as a rosin sizing agent than the WW grade.Keywords: gondorukem, saponification, sizing agent, fortified rosin ABSTRAK Gondorukem (Resina colophium) adalah hasil olahan destilasi uap dari getah sadapan pada batang pinus (oleoresin). Gondorukem berbentuk padatan berwarna kuning kecoklatan dengan komponen kimia utamanya terdiri dari asam organik alkyl tricyclic tak jenuh yaitu asam abietat dan asam pimarat. Kedua molekul asam ini memiliki sifat amfipatik yaitu mempunyai gugus karboksil yang bersifat hidrofilik dan molekul tricyclic yang hidrofobik. Penelitian ini bertujuan untuk pembuatan darih rosin emulsi dari gondorukem serbuk sebagai aditif pada pembuatan kertas. Gondorukem dengan kualitas WW dan X dilakukan reaksi saponifikasi menggunakan soda pada suhu di bawah 100oC. Peningkatan kualitas dari rosin tersabunkan yang terbentuk dilakukan dengan reaksi fortifikasi menjadi rosin terfortifikasi dengan penambahan senyawa anhidrida maleat. Parameter uji berupa bilangan penyabunan dan bilangan asam dilakukan untuk mengetahui kualitas darih rosin yang terbentuk. Dari penelitian ini didapatkan bilangan asam dan penyabunan kualitas X adalah 179,92 dan 199,74 sedangkan untuk kualitas WW 176,58 dan 195,84. Dengan hasil pengujian kualitas bahan baku gondorukem X mempunyai kualitas yang lebih baik untuk dijadikan darih rosin pada kertas dibandingkan dengan WW.Kata kunci: gondorukem, saponifikasi, bahan pendarihan, rosin terfortifikasi