Small-conductance Ca2+-activated K+ (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca2+ microdomains necessary for SK channel activation. SK currents coupled with Ca2+ influx via L-type Ca2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca2+ store, suggesting that LTCCs provide the immediate Ca2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Cav1.2 Ca2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Cav1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca2+ signaling.
The mammalian cochlea relies on active electromotility of outer hair cells (OHCs) to resolve sound frequencies. OHCs use ionic channels and somatic electromotility to achieve the process. It is unclear, though, how the kinetics of voltage-gated ionic channels operate to overcome extrinsic viscous drag on OHCs at high frequency. Here, we report ultrafast electromechanical gating of clustered Kv7.4 in OHCs. Increases in kinetics and sensitivity resulting from cooperativity among clustered-Kv7.4 were revealed, using optogenetics strategies. Upon clustering, the half-activation voltage shifted negative, and the speed of activation increased relative to solitary channels. Clustering also rendered Kv7.4 channels mechanically sensitive, confirmed in consolidated Kv7.4 channels at the base of OHCs. Kv7.4 clusters provide OHCs with ultrafast electromechanical channel gating, varying in magnitude and speed along the cochlea axis. Ultrafast Kv7.4 gating provides OHCs with a feedback mechanism that enables the cochlea to overcome viscous drag and resolve sounds at auditory frequencies.
The normal function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane. Small-conductance, Ca -activated K channels (SK, K 2) are expressed in human atrial myocytes and are responsible for shaping atrial action potentials. Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes. We have previously demonstrated that the C- and N-termini of SK2 channels interact with the actin-binding proteins α-actinin2 and filamin A, respectively. However, the roles of the interacting proteins on SK2 channel trafficking remain incompletely understood. Using total internal reflection fluorescence (TIRF) microscopy, we studied the mechanisms of surface membrane localization of SK2 (K 2.2) channels. When SK2 channels were co-expressed with filamin A or α-actinin2, the membrane fluorescence intensity of SK2 channels increased significantly. We next tested the effects of primaquine and dynasore on SK2 channels expression. Treatment with primaquine significantly reduced the membrane expression of SK2 channels. In contrast, treatment with dynasore failed to alter the surface membrane expression of SK2 channels. Further investigations using constitutively active or dominant-negative forms of Rab GTPases provided additional insights into the distinct roles of the two cytoskeletal proteins on the recycling processes of SK2 channels from endosomes. α-Actinin2 facilitated recycling of SK2 channels from both early and recycling endosomes while filamin A probably aids the recycling of SK2 channels from recycling endosomes.
Highlights d Prestin is considered to be expressed exclusively in outer hair cells of the inner ear d The current study shows that prestin is expressed in cardiomyocytes d Prestin serves to amplify actin-myosin force generation in cardiomyocytes d Prestin serves a broader cellular motor function
Background Intracellular pH (pHi) is critical to cardiac excitation and contraction; uncompensated changes in pHi impair cardiac function and trigger arrhythmia. Several ion transporters participate in cardiac pHi regulation. Our previous studies identified several isoforms of a solute carrier Slc26a6 to be highly expressed in cardiomyocytes. We show that Slc26a6 mediates electrogenic Cl−/HCO3− exchange activities in cardiomyocytes, suggesting the potential role of Slc26a6 in regulation of not only pHi, but also cardiac excitability. Methods and Results To test the mechanistic role of Slc26a6 in the heart, we took advantage of Slc26a6 knockout (Slc26a6−/−) mice using both in vivo and in vitro analyses. Consistent with our prediction of its electrogenic activities, ablation of Slc26a6 results in action potential (AP) shortening. There are reduced Ca2+ transient and sarcoplasmic reticulum Ca2+ load, together with decreased sarcomere shortening in Slc26a6−/− cardiomyocytes. These abnormalities translate into reduced fractional shortening and cardiac contractility at the in vivo level. Additionally, pHi is elevated in Slc26a6−/− cardiomyocytes with slower recovery kinetics from intracellular alkalization, consistent with the Cl−/HCO3− exchange activities of Slc26a6. Moreover, Slc26a6−/− mice show evidence of sinus bradycardia and fragmented QRS complex, supporting the critical role of Slc26a6 in cardiac conduction system. Conclusions Our study provides mechanistic insights into Slc26a6, a unique cardiac electrogenic Cl−/HCO3− transporter in ventricular myocytes, linking the critical roles of Slc26a6 in regulation of pHi, excitability, and contractility. pHi is a critical regulator of other membrane and contractile proteins. Future studies are needed to investigate possible changes in these proteins in Slc26a6−/− mice.
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