Kir2.1 and K2P1 channels reconstitute two levels of resting membrane potential in cardiomyocytes

Abstract

Inward rectifier K channel subfamily 2 (Kir2) channels primarily maintain the normal resting membrane potential of cardiomyocytes. At subphysiological extracellular K concentrations or pathological hypokalaemia, human cardiomyocytes show both hyperpolarized and depolarized resting membrane potentials; these depolarized potentials cause cardiac arrhythmia; however, the underlying mechanism is unknown. In the present study, we show that inward rectifier K channel subfamily 2 isoform 1 (Kir2.1) currents non-linearly counterbalance hypokalaemia-induced two pore-domain K channel isoform 1 (K2P1) leak cation currents, reconstituting two levels of resting membrane potential in cardiomyocytes. Under hypokalaemic conditions, both human cardiomyocytes derived from induced pluripotent stem cells with enhanced Kir2.1 expression and mouse HL-1 cardiomyocytes with ectopic expression of K2P1 channels recapitulate two levels of resting membrane potential. These cardiomyocytes display N-shaped current-voltage relationships that cross the voltage axis three times and the first and third zero-current potentials match the two levels of resting membrane potential. Inhibition of K2P1 expression eliminates the phenomenon, indicating contributions of Kir2.1 and K2P1 channels to two levels of resting membrane potential. Second, in Chinese hamster ovary cells that heterologously express the channels, Kir2.1 currents non-linearly counterbalance hypokalaemia-induced K2P1 leak cation currents, yielding the N-shaped current-voltage relationships, causing the resting membrane potential to spontaneously jump from hyperpolarization at the first zero-current potential to depolarization at the third zero-current potential, again recapitulating two levels of resting membrane potential. These findings reveal ionic mechanisms of the two levels of resting membrane potential, demonstrating a previously unknown mechanism for the regulation of excitability, and support the hypothesis that Kir2 currents non-linearly balance inward background cation currents, accounting for two levels of resting membrane potential of human cardiomyocytes.

Kir2 inward rectification–controlled precise and dynamic balances between Kir2 and HCN currents initiate pacemaking activity

“…Recordings of membrane potentials and whole‐cell currents were performed at a sampling rate of 2 kHz and filtered at 5 kHz with the EPC‐10 USB amplifier (HEKA Elektronik, Lambrecht, Germany) and a Dell 745 computer running PatchMaster software (HEKA Elektronik) as previously described (22, 23). Briefly, borosilicate glass pipettes were prepared with a P‐97 puller (Sutter Instrument, Novato, CA, USA) to produce a tip with 2.0–2.5 MV resistance when filled with the pipette solution.…”

Kir2.1 channels set two levels of resting membrane potential with inward rectification

“…Adenovirus construction, purification, and titration were performed by Vector Biolabs. Viral particles of Ad-mCherry-hKir2.1 and Ad-GFP-hK2P1 shRNA #1 were prepared as described previously [(39)]. Kir2.1•E224G mutant was generated using a QuikChange kit (Stratagene).…”

“…Culture of human iPSC-derived cardiomyocytes (CMC-100-110-001, Cell Dynamic International) was described previously in detail [(39)]. Briefly, human iPSC-derived cardiomyocytes were cultured in a 37 °C, 5% CO 2 incubator according to the vendor’s instructions, and transduced on days 8 to 20 of culture.…”

“…We recently provided evidence supporting that Kir2.1 channels contribute to the two levels of resting membrane potential of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) [(39)]. Human iPSC-derived cardiomyocytes conduct small Kir2 currents and have a resting membrane potential of around −60 mV, which is significantly depolarized compared to the approximately −80 mV of human adult cardiomyocytes [(6, 15, 19)].…”

“…We found that 18 of 24 of the iPSC-derived cardiomyocytes that showed the phenomenon had relatively large K2P1-like inward Na + currents in 0 mM [K + ] e ; the other six cells did not conduct K2P1-like inward Na + currents. We also demonstrated that inhibition of K2P1 expression reduced the percentage of the cardiomyocytes that had two levels of resting potential in 2 mM [K + ] e from 35% to 8.8% [(39)].…”