Intracellular pH regulates the strength of the intrinsic inward rectification of Kir4.1/Kir5.1 channels.

Kir4.1/Kir5.1 channels play a crucial role in important physiological functions, notably in the kidneys and brain. A hallmark of these channels is the coexistence of two mechanisms of inward rectification: the classical "extrinsic" inward rectification induced by polyamines and Mg²⁺ blocking the pore, and a novel "intrinsic" voltage-dependent mechanism driven by K⁺ flux. Previous studies have shown that Kir4.1/Kir5.1 channels are modulated by the intracellular pH in the physiological range. Here, we investigated the influence of the intracellular pH on the extent of the intrinsic inward rectification of Kir4.1/Kir5.1 channels expressed in HEK-293 cells and recorded using the inside-out configuration of the patch-clamp technique. We found that mutations that are known to modulate the pH sensitivity of Kir4.1/Kir5.1 channels attenuated inward rectification. The combination of these mutations in the triple mutant channel Kir4.1(K67M)/Kir5.1(N161E-R230E) virtually abolished inward rectification at pH 7.4; however, this property was re-established at acidic pH values. Consistently, the strong inward rectification of wild-type Kir4.1/Kir5.1 channels was reduced by intracellular alkalinization and further enhanced by acidification. Altogether, these experiments indicate that the intracellular pH strongly regulates the strength of the intrinsic inward rectification. Furthermore, triple mutant channels retained the extrinsic mechanism of inward rectification at pH 7.4, as can be blocked by spermine, but lost the ability to respond to elevated levels of PIP_2, unlike wild-type channels. Interestingly, whole-cell recordings of wild-type and triple mutant channels imply that the mechanism of intrinsic inward rectification is an important contributor to the overall rectification of Kir4.1/Kir5.1 channels in basal conditions.