Human-induced pluripotent stem cell (hiPSC) and stem cell (hSC) derived cardiomyocytes (CM) are gaining popularity as in vitro model for cardiology and pharmacology studies. A remaining flaw of these cells, as shown by single-cell electrophysiological characterization, is a more depolarized resting membrane potential (RMP) compared to native CM. Most reports attribute this to a lower expression of the Kir2.1 potassium channel that generates the I K1 current. However, most RMP recordings are obtained from isolated hSC/hiPSC-CMs whereas in a more native setting these cells are interconnected with neighboring cells by connexin-based gap junctions, forming a syncytium. Hereby, these cells are electrically connected and the total pool of I K1 increases. Therefore, the input resistance (Ri) of interconnected cells is lower than that of isolated cells. During patch clamp experiments pipettes need to be well attached or sealed to the cell, which is reflected in the seal resistance (Rs), because a nonspecific ionic current can leak through this pipette-cell contact or seal and balance out small currents within the cell such as I K1 . By recording the action potential of isolated hSC-CMs and that of hSC-CMs cultured in small monolayers, we show that the RMP of hSC-CMs in monolayer is approximately −20 mV more hyperpolarized compared to isolated cells. Accordingly, adding carbenoxolone, a connexin channel blocker, isolates the cell that is patch clamped from its neighboring cells of the monolayer and depolarizes the RMP. The presented data show that the recorded RMP of hSC-CMs in a syncytium is more negative than that determined from isolated hSC/hiPSC-CMs, most likely because the active pool of Kir2.1 channels increased.

The cardiac Nav1.5 mediated sodium current (I Na) generates the upstroke of the action potential in atrial and ventricular myocytes. Drugs that modulate this current can therefore be antiarrhythmic or proarrhythmic, which requires preclinical evaluation of their potential drug-induced inhibition or modulation of Nav1.5. Since Nav1.5 assembles with, and is modulated by, the auxiliary β1-subunit, this subunit can also affect the channel's pharmacological response. To investigate this, the effect of known Nav1.5 inhibitors was compared between COS-7 cells expressing Nav1.5 or Nav1.5+β1 using whole-cell voltage clamp experiments. For the open state class Ia blockers ajmaline and quinidine, and class Ic drug flecainide, the affinity did not differ between both models. For class Ib drugs phenytoin and lidocaine, which are inactivated state blockers, the affinity decreased more than a twofold when β1 was present. Thus, β1 did not influence the affinity for the class Ia and Ic compounds but it did so for the class Ib drugs. Human stem cell-derived cardiomyocytes (hSC-CMs) are a promising translational cell source for in vitro models that express a representative repertoire of channels and auxiliary proteins, including β1. Therefore, we subsequently evaluated the same drugs for their response on the I Na in hSC-CMs. Consequently, it was expected and confirmed that the drug response of I Na in hSC-CMs compares best to I Na expressed by Nav1.5+β1.

In the Na v channel family the lipophilic drugs/toxins binding sites and the presence of fenestrations in the channel pore wall are well defined and categorized. No such classification exists in the much larger K v channel family, although certain lipophilic compounds seem to deviate from binding to well-known hydrophilic binding sites. By mapping different compound binding sites onto 3D structures of Kv channels, there appear to be three distinct lipid-exposed binding sites preserved in K v channels: the front and back side of the pore domain, and S2-S3/S3-S4 clefts. One or a combination of these sites is most likely the orthologous equivalent of neurotoxin site 5 in Na v channels. This review describes the different lipophilic binding sites and location of pore wall fenestrations within the K v channel family and compares it to the knowledge of Na v channels.

Voltage-gated K+ (Kv) channels mediate the flow of K+ across the cell membrane by regulating the conductive state of their activation gate (AG). Several Kv channels display slow C-type inactivation, a process whereby their selectivity filter (SF) becomes less or nonconductive. It has been proposed that, in the fast inactivation-removed Shaker-IR channel, the W434F mutation epitomizes the C-type inactivated state because it functionally accelerates this process. By introducing another pore mutation that prevents AG closure, P475D, we found a way to record ionic currents of the Shaker-IR-W434F-P475D mutant at hyperpolarized membrane potentials as the W434F-mutant SF recovers from its inactivated state. This W434F conductive state lost its high K+ over Na+ selectivity, and even NMDG+ can permeate, features not observed in a wild-type SF. This indicates that, at least during recovery from inactivation, the W434F-mutant SF transitions to a widened and noncationic specific conformation.

Substituting the pore residue W434 in the Shaker Kv channel by a phenylalanine (W434F) accelerates its C-type inactivation, which involves the collapse of the selectivity filter (SF). In Shaker-W434F this collapse precedes S6-gate opening upon activation and no ionic currents are recorded. However, a valine mutation for the residue T449 (T449V) slows inactivation and introducing this T449V substitution in the Shaker-W434F rescues ionic conduction. The human Shaker-type Kv1.2 channel has a valine at the homologous T449 position (V381). Interestingly, the Kv1.2-W366F mutant yielded ionic current (similar to the Shaker-W434F-T449V combination). Accordingly, substituting V381 in Kv1.2-W366F by a threonine resulted in a similar (non-conducting) phenotype as Shaker-W434F. Thus the rate of SF collapse can be controlled by specific combinations of pore residue mutations. Patch-clamp analysis of the ionic currents of Kv1.2-W366F and Shaker-W434F-T449V showed that the channels remained K þ selective. Thus, the SF of Shaker-W434F-T449V adopts a K þ-selective conformation when the S6-gate opens. To study the SF conformation of Shaker-W434F when the S6-gate closes, we combined Shaker-W434F with the mutation P475D, that by itself prevents complete closing of the S6-gate. We expected to record ionic currents of the double Shaker-W434F-P475D mutant at hyperpolarized potentials when the S6-gate closes and the SF recovers from its collapsed inactivated state. Indeed, Shaker-W434F-P475D resulted in functional voltagedependent channels that were conducting at hyperpolarized potentials and ceased conducting at depolarized potentials. Interestingly, this conductive state of Shaker-W434F-P475D was both Na þ and K þ permeable, i.e. the high K þ selectivity was lost. The mutant remained, however, sensitive to external TEA block. Thus, preventing full closure of the S6-gate appears to affect the recovery process of the SF such that it is trapped in a conformation conducting both Na þ and K þ .