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Key points: Ex vivo proliferated c‐Kit+ endogenous cardiac progenitor cells (eCPCs) obtained from mouse and human cardiac tissues have been reported to express a wide range of functional ion channels. In contrast to previous reports in cultured c‐Kit+ eCPCs, we found that ion currents were minimal in freshly isolated cells. However, inclusion of free Ca2+ intracellularly revealed a prominent inwardly rectifying current identified as the intermediate conductance Ca2+‐activated K+ current (KCa3.1) Electrical function of both c‐Kit+ eCPCs and bone marrow‐derived mesenchymal stem cells is critically governed by KCa3.1 calcium‐dependent potassium channels. Ca2+‐induced increases in KCa3.1 conductance are necessary to optimize membrane potential during Ca2+ entry. Membrane hyperpolarization due to KCa3.1 activation maintains the driving force for Ca2+ entry that activates stem cell proliferation. Cardiac disease downregulates KCa3.1 channels in resident cardiac progenitor cells. Alterations in KCa3.1 may have pathophysiological and therapeutic significance in regenerative medicine. Abstract: Endogenous c‐Kit+ cardiac progenitor cells (eCPCs) and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) are being developed for cardiac regenerative therapy, but a better understanding of their physiology is needed. Here, we addressed the unknown functional role of ion channels in freshly isolated eCPCs and expanded BM‐MSCs using patch‐clamp, microfluorometry and confocal microscopy. Isolated c‐Kit+ eCPCs were purified from dog hearts by immunomagnetic selection. Ion currents were barely detectable in freshly isolated c‐Kit+ eCPCs with buffering of intracellular calcium (Ca2+i). Under conditions allowing free intracellular Ca2+, freshly isolated c‐Kit+ eCPCs and ex vivo proliferated BM‐MSCs showed prominent voltage‐independent conductances that were sensitive to intermediate‐conductance K+‐channel (KCa3.1 current, IKCa3.1) blockers and corresponding gene (KCNN4)‐expression knockdown. Depletion of Ca2+i induced membrane‐potential (Vmem) depolarization, while store‐operated Ca2+ entry (SOCE) hyperpolarized Vmem in both cell types. The hyperpolarizing SOCE effect was substantially reduced by IKCa3.1 or SOCE blockade (TRAM‐34, 2‐APB), and IKCa3.1 blockade (TRAM‐34) or KCNN4‐knockdown decreased the Ca2+ entry resulting from SOCE. IKCa3.1 suppression reduced c‐Kit+ eCPC and BM‐MSC proliferation, while significantly altering the profile of cyclin expression. IKCa3.1 was reduced in c‐Kit+ eCPCs isolated from dogs with congestive heart failure (CHF), along with corresponding KCNN4 mRNA. Under perforated‐patch conditions to maintain physiological [Ca2+]i, c‐Kit+ eCPCs from CHF dogs had less negative resting membrane potentials (−58 ± 7 mV) versus c‐Kit+ eCPCs from control dogs (−73 ± 3 mV, P < 0.05), along with slower proliferation. Our study suggests that Ca2+‐induced increases in IKCa3.1 are necessary to optimize membrane potential during the Ca2+ entry that activates progenitor cell proliferation, and that alterations in KCa3.1 may have pathophysiological and therapeutic significance in regenerative medicine. [ABSTRACT FROM AUTHOR]

The rapidly delayed rectifier current (IKr) has been described in ventricular myocytes isolated from many species, as well as from neonatal mice. However, whether IKr is present in the adult mouse heart remains controversial. We used cell-attached patch-clamp recording in symmetrical K+ solutions to assess the presence and behaviour of single IKr channels in adult mouse cardiomyocytes (mIKr). Of 314 patches, 158 (50.1%) demonstrated mIKr currents as compared with 131 (42.3%) for the IK1 channel. Single mIKr channel activity was rarely observed at potentials positive to -10 mV. The slope conductance at negative potentials was 12 pS. Upon repolarization, ensemble-averaged mIKr showed slow deactivation with a biexponential time course. A selective IKr blocker, E-4031 (1 µm), completely blocked mIKr channel activity. Extracellular Ca2+ and Mg2+ at physiological concentrations shifted the activation by ∼30 mV, accelerated deactivation kinetics, prolonged long-closed time, and reduced open probability without affecting single-channel conductance, suggesting a direct channel-blocking effect in addition to well-recognized voltage shifts. HERG subunits expressed in Chinese hamster ovary cells produced channels with properties similar to those of mIKr, except for the more-negative activation of the HERG channels. Despite the abundant expression of mIKr, single-channel events were rarely observed during action-potential clamp and 5 µm E-4031 had no detectable effect on the action potential parameters, confirming that mIKr plays at best a minor role in repolarization of adult mouse cardiomyocytes, probably because the modulatory effects of divalent cations prevent significant mIKr opening under physiological conditions. [ABSTRACT FROM AUTHOR]

Heteromeric channel assembly is a potential source of physiological variability. The potential significance of Kir2 subunit heterotetramerization has been controversial, but recent findings suggest that heteromultimerization of Kir2.1-3 may be significant. This study was designed to investigate whether the recently described Kir2.4 subunit can form heterotetramers with the important subunit Kir2.1, and if so, to investigate whether the resulting heterotetrameric channels are functional. Co-expression of either dominant negative Kir2.1 or Kir2.4 subunits in Xenopus oocytes with either wild-type Kir2.1 or 2.4 strongly decreased resulting current amplitude. To examine physical association between Kir2.1 and Kir2.4, Cos-7 cells were co-transfected with a His(6)-tagged Kir2.1 subunit (Kir2.1-His(6)) and a FLAG-tagged Kir2.4 subunit (Kir2.4-FLAG). After pulldown with a His(6)-binding resin, Kir2.4-FLAG could be detected in the eluted cell lysate by Western blotting, indicating co-assembly of Kir2.1-His(6) and Kir2.4-FLAG. Expression of a tandem construct containing covalently linked Kir2.1 and 2.4 subunits led to robust current expression. Kir2.1-Kir2.4 tandem subunit expression, as well as co-injection of Kir2.1 and Kir2.4 cRNA into Xenopus oocytes, produced currents with barium sensitivity greater than that of Kir2.1 or Kir2.4 subunit expression alone. These results show that Kir2.4 subunits can co-assemble with Kir2.1 subunits, and that co-assembled channels are functional, with properties different from those of Kir2.4 or Kir2.1 alone. Since Kir2.1 and Kir2.4 mRNAs have been shown to co-localize in the CNS, Kir2.1 and Kir2.4 heteromultimers might play a role in the heterogeneity of native inward rectifier currents.