TMEM16 (transmembrane protein 16) proteins, which possess eight putative transmembrane domains with intracellular NH₂- and COOH-terminal tails, are thought to comprise a Cl⁻ channel family. The function of TMEM16F, a member of the TMEM16 family, has been greatly controversial. In the present study, we performed whole cell patch-clamp recordings to investigate the function of human TMEM16F. In TMEM16F-transfected HEK293T cells but not TMEM16K- and mock-transfected cells, activation of membrane currents with strong outward rectification was found to be induced by application of a Ca²⁺ ionophore, ionomycin, or by an increase in the intracellular free Ca²⁺ concentration. The free Ca²⁺ concentration for half-maximal activation of TMEM16F currents was 9.6 μM, which is distinctly higher than that for TMEM16A/B currents. The outwardly rectifying current-voltage relationship for TMEM16F currents was not changed by an increase in the intracellular Ca²⁺ level, in contrast to TMEM16A/B currents. The Ca²⁺-activated TMEM16F currents were anion selective, because replacing Cl⁻ with aspartate⁻ in the bathing solution without changing cation concentrations caused a positive shift of the reversal potential. The anion selectivity sequence of the TMEM16F channel was I⁻ > Br⁻ > Cl⁻ > F⁻ > aspartate⁻. Niflumic acid, a Ca²⁺-activated Cl⁻ channel blocker, inhibited the TMEM16F-dependent Cl⁻ currents. Neither overexpression nor knockdown of TMEM16F affected volume-sensitive outwardly rectifying Cl⁻ channel (VSOR) currents activated by osmotic swelling or apoptotic stimulation. These results demonstrate that human TMEM16F is an essential component of a Ca²⁺-activated Cl⁻ channel with a Ca²⁺ sensitivity that is distinct from that of TMEM16A/B and that it is not related to VSOR activity.

Cisplatin [cis-diamminedichloroplatinum (II)] is a platinum-based anticancer drug widely used for the treatment of various cancers. It forms interstrand and intrastrand cross-linking with DNA and block DNA replication, resulting in apoptosis. On the other hand, intrinsic and acquired cisplatin resistance restricts its therapeutic effects. Although some studies suggest that dramatic epigenetic alternations are involved in the resistance triggered by cisplatin, the mechanism is complicated and remains poorly understood. Recent studies reported that cytoskeletal structures regulate cisplatin sensitivity and that activities of membrane transporters contribute to the development of resistance to cisplatin. Therefore, we focus on the roles of actin filaments and membrane transporters in cisplatin-induced apoptosis. In this review, we summarize the relationship between actin cytoskeleton and membrane transporters in the cisplatin resistance of cancer cells.

Persistent cell shrinkage, called apoptotic volume decrease (AVD), is a pivotal event of apoptosis. Activation of the volume-sensitive outwardly rectifying Cl⁻ channel (VSOR) is involved in the AVD induction. On the other hand, activation of the MAP kinase (MAPK) cascade is also known to play a critical role in apoptosis. In the present study, we investigated the relationship between the AVD induction and the stress-responsive MAPK cascade activation during the apoptosis process induced by staurosporine (STS) in HeLa cells. STS was found to induce AVD within 2–5 min and phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK after over 20–30 min. VSOR blockers suppressed not only STS-induced AVD but also phosphorylation of JNK and p38 as well as activation of caspase-3/7. Moreover, a p38 inhibitor, SB203580, and a JNK inhibitor, SP600125, failed to affect STS-induced AVD, whereas these compounds reduced STS-induced activation of caspase-3/7. Also, treatment with ASK1-specific siRNA suppressed STS-induced caspase-3/7 activation without affecting the AVD induction. Furthermore, sustained osmotic cell shrinkage per se was found to trigger phosphorylation of JNK and p38, caspase activation, and cell death. Thus, it is suggested that activation of p38 and JNK is a downstream event of AVD for the STS-induced apoptosis of HeLa cells.

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of the inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.