Modulation of hERG K+ Channel Deactivation by Voltage Sensor Relaxation

The hERG (human-ether-à-go-go-related gene) channel underlies the rapid delayed rectifier current, Ikr, in the heart, which is essential for normal cardiac electrical activity and rhythm. Slow deactivation is one of the hallmark features of the unusual gating characteristics of hERG channels, and plays a crucial role in providing a robust current that aids repolarization of the cardiac action potential. As such, there is significant interest in elucidating the underlying mechanistic determinants of slow hERG channel deactivation. Recent work has shown that the hERG channel S4 voltage sensor is stabilized following activation in a process termed relaxation. Voltage sensor relaxation results in energetic separation of the activation and deactivation pathways, producing a hysteresis, which modulates the kinetics of deactivation gating. Despite widespread observation of relaxation behaviour in other voltage-gated K+ channels, such as Shaker, Kv1.2 and Kv3.1, as well as the voltage-sensing phosphatase Ci-VSP, the relationship between stabilization of the activated voltage sensor by the open pore and voltage sensor relaxation in the control of deactivation has only recently begun to be explored. In this review, we discuss present knowledge and questions raised related to the voltage sensor relaxation mechanism in hERG channels and compare structure-function aspects of relaxation with those observed in related ion channels. We focus discussion, in particular, on the mechanism of coupling between voltage sensor relaxation and deactivation gating to highlight the insight that these studies provide into the control of hERG channel deactivation gating during their physiological functioning

CD44 mediates stem cell mobilization to damaged lung its novel transcriptional targets, Cortactin and Survivin

Beyond their role in bone and lung homeostasis, mesenchymal stem cells (MSCs) are becoming popular in cell therapy. Various insults may disrupt the repair mechanisms involving MSCs. One such insult is smoking, which is a major risk factor for osteoporosis and respiratory diseases. Upon cigarette smoke-induced damage, a series of reparatory mechanisms ensue; one such mechanism involves Glycosaminoglycans (GAG). One of these GAGs, namely hyaluronic acid (HA), serves as a potential therapeutic target in lung injury. However, much of its mechanisms of action through its major receptor CD44 remains unexplored. Our previous studies have identified and functionally validated that both cortactin (CTTN: marker of motility) and Survivin (BIRC5: required for cell survival) act as novel HA/CD44-downstream transcriptional targets underpinning cell motility. Here, human MSCs were treated with "" smoke to investigate the effects of cigarette smoke condensate (CSC) on these HA-CD44 novel signaling pathways. Our results show that CSC decreased the expression of both CD44 and its downstream targets CTTN and BIRC5 in MSCs, and that HA reversed these effects. Interestingly, CSC inhibited migration and invasion of MSCs upon CD44-targeted RNAi treatment. This shows the importance of CD44-HA/CTTN and CD44-HA/BIRC5 signaling pathways in MSC motility, and further suggests that these signaling pathways may provide a novel mechanism implicated in migration of MSCs during repair of lung tissue injury. These findings suggest that one should use caution before utilizing MSC from donors with history of smoking, and further pave the way towards the development of targeted therapeutic approaches against CD44-associated diseases

Differential Effects of Leptin on the Invasive Potential of Androgen-Dependent and -Independent Prostate Carcinoma Cells

Obesity has been linked with an increased risk of prostate cancer. The formation of toxic free oxygen radicals has been implicated in obesity mediated disease processes. Leptin is one of the major cytokines produced by adipocytes and controls body weight homeostasis through food intake and energy expenditure. The rationale of the study was to determine the impact of leptin on the metastatic potential of androgen-sensitive (LNCaP) cells as well as androgen-insensitive (PC-3 and DU-145) cells. At a concentration of 200 nm, LNCaP cells showed a significant increase (20% above control; P < .0001) in cellular proliferation without any effect on androgen-insensitive cells. Furthermore, exposure to leptin caused a significant (P < .01 to P < .0001) dose-dependent decrease in migration and invasion of PC3 and Du-145 prostate carcinoma cell lines. At the molecular level, exposure of androgen-independent prostate cancer cells to leptin stimulates the phosphorylation of MAPK at early time point as well as the transcription factor STAT3, suggesting the activation of the intracellular signaling cascade upon leptin binding to its cognate receptor. Taken together, these results suggest that leptin mediates the invasive potential of prostate carcinoma cells, and that this effect is dependent on their androgen sensitivity

Mechanistic Insight into Human ether-a-go-go-related Gene (hERG) K+ Channel Activation and Deactivation gating

hERG encodes the pore-forming α-subunit of the voltage-gated potassium channel that underlies the rapid delayed rectifier current, IKr, in the heart, which is essential for normal cardiac electrical activity and rhythm. Inherited mutations in, or pharmacological blockade of, hERG channels deplete the cardiac repolarization reserve, increasing the risk of life-threatening arrhythmias. The molecular bases of hERG gating events and drug binding are poorly understood. hERG channels display unique gating characteristics critical for their physiological function. They activate and deactivate slowly, yet inactivate and recover from inactivation rapidly. In addition, the promiscuous nature of drug interactions with hERG channels presents a therapeutic challenge for drug design and development. My thesis provides novel mechanistic and structural characterization of the unusual activation and deactivation gating processes of hERG. In my first study, I used a proline scan approach to define the activation gate region in hERG channels. Proximal substitutions (I655P-Q664P) impeded gate closure, trapping channels in the open state, while distal substitutions (R665P-Y667P) preserved normal gating, suggesting that Q664 marks the position of the activation gate in hERG. This is more than one helical turn lower than in related channels, which may allow for drug docking. Using two different approaches to measure voltage sensor gating in trapped open channels, I then demonstrated that slow activation is an intrinsic property of the voltage-sensing unit of hERG. In my second study, I showed that voltage-sensor stabilization slows hERG channel deactivation gating. I characterized the temporal sequence of events leading to voltage-sensor stabilization upon membrane depolarization. I showed that this occurs via two separable mechanisms, one derived from pore-gate-opening and the other from the voltage-sensing unit itself. In addition, I show that voltage sensor return in hERG channels is less energetically favourable than pore closure during repolarization and thus is what limits deactivation. Finally, I characterize the use of voltage clamp fluorimetry as a technique to track conformational rearrangements of the hERG voltage sensor associated with gating. These findings provide novel and in depth understanding regarding how hERG channels function and foundational knowledge relevant to finding targets for the treatment and management of cardiac arrhythmias