The Journal of Membrane Biology

Szűcs, Attila; Szappanos, Henrietta; Batta, Tamás; Tóth, Andrea; Szigeti, Gyula; Panyi, György; Csernoch, László; Sziklai, István

doi: 10.1007/s00232-006-0045-ypmid: 17468959

Among the cells of the inner ear, the outer hair cells (OHCs) are the most important targets of noise-induced effects, being the most sensitive cell types. The aim of this study was to examine the effects of noise (50 Hz-20 kHz, 80 dB sound pressure level, 14 days) on intracellular calcium levels and on the expression pattern of purinoceptors in the membrane of the OHCs of the guinea pig and to measure the stiffness changes of the lateral membrane of these cells. In noise-exposed animals, the resting intracellular calcium concentration increased compared to nontreated animals and was slightly higher in the cells of the basal (219 ± 29 nM) than in the apical (181 ± 24 nM) turns of the cochlea. After application of 180 μM adenosine triphosphate, the intracellular calcium level rose by 60 ± 22 nM in cells from the apical and by 44 ± 10 nM in cells from the basal turns, significantly less than in nontreated animals. Expression of the P2X1, P2X2, P2X4, P2X7, P2Y1 and P2Y4 receptor subtypes was suppressed, while expression of the P2Y2 subtype did not decrease in either of the two preparations. In parallel with the increase in intracellular calcium concentration, the stiffness of the lateral wall of the OHCs was increased. Noise-induced changes in intracellular calcium homeostasis and subsequently in the calcium-dependent regulatory mechanisms may modify OHC lateral wall stiffness and may lead to reduction of the efficacy of the cochlear amplifier.

O’Connell, Robert; Yuan, Chunbo; Johnston, Linda; Rinco, Olga; Probodh, Ira; Treistman, Steven

doi: 10.1007/s00232-006-0034-1pmid: 17468961

The energy associated with a mismatch between the hydrocarbon portions of a lipid bilayer and the hydrophobic regions of a transmembrane protein requires that one or both components deform in an attempt to minimize the energy difference. Transmembrane potassium channel subunits are composed of different structural motifs, each responsible for ion-selectivity, conductance and gating capabilities. Each has an inherent degree of flexibility commensurate with its amino acid composition. It is not clear, however, how each structural motif will respond to a fixed amount of distortion applied to the whole structure. We examined the single-channel conductance (Gc) and gating (open probability, P o) of single BKCa channels (hslo α-subunits) inserted into planar lipid bilayers containing 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE) or DOPE with either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or sphingomyelin (SPM) and 1-palmitoyl-2-oleoyl-3-phosphatidylethanolamine (POPE) with SPM. These latter three binary mixtures formed stable membranes with different distributions of thickness domains as determined by atomic force microscopy. Channels placed in each composition should be exposed to different amounts of distortion. BKCa channels forced into the DOPE/SPM bilayer containing lipid domains with two different thicknesses showed two distinct levels of Gc and Po. The alterations in Gc and Po were reciprocal. A larger conductance was accompanied by a smaller value for gating and vice versa. Channels forced into the POPE/SPM bilayer containing lipid domains with different thicknesses showed more than two distinct levels of Gc and Po. Channels placed in a uniform bilayer (DOPE/DOPC) showed a uniform distribution of conductance and activation. We conclude that both the inner and outer domains of the channel where these two channel functions are localized respond to deformation and that a fixed amount of distortion results in reciprocal changes in protein function.

Wang, Runping; Su, Junda; Zhang, Xiaoli; Shi, Yun; Cui, Ningren; Onyebuchi, Vivian A.; Jiang, Chun

doi: 10.1007/s00232-006-0038-xpmid: 17468960

The adenosine triphosphate-sensitive K+ (KATP) channels are gated by several metabolites, whereas the gating mechanism remains unclear. Kir6.2, a pore-forming subunit of the KATP channels, has all machineries for ligand binding and channel gating. In Kir6.2, His175 is the protonation site and Thr71 and Cys166 are involved in channel gating. Here, we show how individual subunits act in proton binding and channel gating by selectively disrupting functional subunits using these residues. All homomeric dimers and tetramers showed pH sensitivity similar to the monomeric channels. Concatenated construction of wild type with disrupted subunits revealed that none of these residues had a dominant-negative effect on the proton-dependent channel gating. Subunit action in proton binding was almost identical to that for channel gating involving Cys166, suggesting a one-to-one coupling from the C terminus to the M2 helix. This was significantly different from the effect of T71Y heteromultimers, suggesting distinct contributions of M1 and M2 helices to channel gating. Subunits underwent concerted rather than independent action. Two wild-type subunits appeared to act as a functional dimer in both cis and trans configurations. The understanding of KATP channel gating by intracellular pH has a profound impact on cellular responses to metabolic stress as a significant drop in intracellular pH is more frequently seen under a number of physiological and pathophysiological conditions than a sole decrease in intracellular ATP levels.

Duta, Valentin; Duta, Florentina; Puttagunta, Lakshmi; Befus, A.; Duszyk, Marek

doi: 10.1007/s00232-006-0062-xpmid: 17468957

The presence of basolateral Cl− channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl− conductance sensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl− channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl− channels via a cGMP-dependent pathway. Anion replacement and 36Cl− flux studies showed that NO affected both Cl− and HCO 3 − secretion. Two different types of Cl− channels are known to be present in the basolateral membrane of epithelial cells: Zn2+-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl− conductance in the presence of Zn2+ ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl− conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl− channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.

Wang, Ya-Jean; Sung, Ruey; Lin, Ming-Wei; Wu, Sheng-Nan

doi: 10.1007/s00232-007-0027-8pmid: 17483867

Cardiac fibroblasts are involved in the maintenance of myocardial tissue structure. However, little is known about ion currents in human cardiac fibroblasts. It has been recently reported that cardiac fibroblasts can interact electrically with cardiomyocytes through gap junctions. Ca2+-activated K+ currents (I K[Ca]) of cultured human cardiac fibroblasts were characterized in this study. In whole-cell configuration, depolarizing pulses evoked I K(Ca) in an outward rectification in these cells, the amplitude of which was suppressed by paxilline (1 μM) or iberiotoxin (200 nM). A large-conductance, Ca2+-activated K+ (BKCa) channel with single-channel conductance of 162 ± 8 pS was also observed in human cardiac fibroblasts. Western blot analysis revealed the presence of α-subunit of BKCa channels. The dynamic Luo-Rudy model was applied to predict cell behavior during direct electrical coupling of cardiomyocytes and cardiac fibroblasts. In the simulation, electrically coupled cardiac fibroblasts also exhibited action potential; however, they were electrically inert with no gap-junctional coupling. The simulation predicts that changes in gap junction coupling conductance can influence the configuration of cardiac action potential and cardiomyocyte excitability. I k(Ca) can be elicited by simulated action potential waveforms of cardiac fibroblasts when they are electrically coupled to cardiomyocytes. This study demonstrates that a BKCa channel is functionally expressed in human cardiac fibroblasts. The activity of these BKCa channels present in human cardiac fibroblasts may contribute to the functional activities of heart cells through transfer of electrical signals between these two cell types.

Pearson, Wade; Skatchkov, Serguei; Eaton, Misty; Nichols, Colin

doi: 10.1007/s00232-006-0058-6pmid: 17468958

Inward rectifier potassium (Kir) channels serve important functional and modulatory roles in a wide variety of cells. While the activity of several members of this channel family are tightly regulated by intracellular messengers such as adenosine triphosphate, G proteins, protein kinases and pH, other members are tonically active and activity is controlled only by the expression level of the protein. In a number of Kir channels, sequence motifs have been identified which determine how effectively the channel is trafficked to and from the plasma membrane. In this report, we identify a number of trafficking determinants in the Kir4.2 channel. Using mutational analysis, we found that truncation of the C terminus of the protein increased current density in Xenopus oocytes, although multiple mutations of the C terminus had no effect on current density. Instead, mutation of a unique region of the channel significantly increased current density. Selective mutation of a putative tyrosine phosphorylation site within this region mimicked the increase in current, suggesting that tyrosine phosphorylation of the protein increases channel retrieval from the membrane (or prevents trafficking to the membrane). Mutation of a previously identified trafficking determinant, K110N, also caused an increase in current density, and combining these mutations caused a multiplicative increase in current, suggesting that these two mutations increase current by independent mechanisms. These data demonstrate novel determinants of Kir4.2 channel expression.