Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Abstract

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg2+. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K+-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K+-flux is convergent with the gating of PIP2 because, at a saturating concentration, PIP2 greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K+-flux gating.

Unconventional voltage sensing in an inwardly rectifying potassium channel

“…Despite these measures, along with extensive perfusion of patches, rectifying features of Kir4.1/Kir5.1 seem to clearly persist. These findings strongly suggest that the previously mentioned activation process involves an unanticipated intrinsic gating mechanism in these channels, perhaps operating alongside their modulation by polyamines ( Marmolejo-Murillo et al, 2021 ).…”

“…While polyamine block typically causes a steep cutoff of current over a narrow voltage range (reflecting a large effective valence associated with blockade), the residual voltage-dependence of Kir4.1/Kir5.1 is weaker and leads to a more gradual inhibition of current at depolarized voltages. With experiments reported thus far, the voltage dependence is difficult to accurately quantify but is certainly smaller than is typically seen for polyamine block; it is apparent in experimental records that even the most negative test voltages (approximately −140 mV in most cases) do not fully activate channels, suggesting a graded channel activity over a very broad voltage range ( Marmolejo-Murillo et al, 2021 ). Interestingly, homomeric Kir4.1 channels do not appear to exhibit any prominent intrinsic voltage dependence, and so it seems likely that the intrinsic gating mechanism must arise from the Kir5.1 subunit.…”

“…With this background in mind, the results presented by Marmolejo-Murillo et al (2021) are interesting and surprising, as they demonstrate that prominent inward rectification persists in Kir4.1/Kir5.1 heteromeric channels, even in the absence of polyamines. These channels are indeed polyamine-sensitive, although not to the same extent as the Kir2.1 channel mentioned above.…”

“…However, other channel subtypes have evolved alternative mechanisms to respond to voltage despite lacking a canonical voltage-sensing domain. In a recent study in the Journal of General Physiology , Marmolejo-Murillo and colleagues report unexpected and previously unrecognized intrinsic voltage dependence of inwardly rectifying potassium (Kir) channels comprised of Kir4.1 and Kir5.1 ( Marmolejo-Murillo et al, 2021 ). These findings test previously held assumptions related to mechanisms of inward rectification—which have been primarily studied in a few model systems, with findings frequently generalized to other members of the Kir channel family ( Hibino et al, 2010 ; Guo and Lu, 2003 ; Kurata et al, 2010b ).…”

“…Over time, optimization of conditions to study Kir channels have generated several tried and true approaches to minimize block by residual polyamines, as employed in this study by Marmolejo-Murillo et al (2021) . This includes replacing organic buffers such as HEPES with phosphate buffers, and including chelating agents such as EDTA (incidentally, most of my electrophysiologist colleagues of course add divalent ions to their recording solutions and believe it is futile to try to record in EDTA, but it works well in my experience).…”