Since the cloning of the first inwardly rectifying K+ channel in 1993, a family of related clones has been isolated, with many members being expressed in the heart. Exogenous expression of different clones has demonstrated that between them they encode channels with the essential functional properties of classic inward rectifier channels, ATP-sensitive K+ channels, and muscarinic receptor-activated inward rectifier channels. High-level expression of cloned channels has led to the discovery that classic strong inward, or anomalous, rectification is caused by very steeply voltage-dependent block of the channel by polyamines, with an additional contribution by Mg2+ ions. Knowledge of the primary structures of inward rectifying channels and the ability to mutate them have led to the determination of many of the structural requirements of inward rectification. The implications of these advances for basic understanding and pharmacological manipulation of cardiac excitability may be significant. For example, cellular concentrations of polyamines are altered under different conditions and can be manipulated pharmacologically. Simulations predict that changes in polyamine concentrations or changes in the relative proportions of each polyamine species could have profound effects on cardiac excitability.

A low stringency polymerase chain reaction (PCR) homology screening procedure was used to probe a mouse liver cDNA library to identify novel inward rectifier K+ channel genes. A single gene (mLV1) was identified that exhibited extensive sequence homology with previously cloned inward rectifier K+ channel genes. The mLV1 gene showed greatest sequence identity with genes belonging to the Kir4 subfamily. The amino acid sequence of mLV1 was 96 % identical to a Kir channel cloned from human kidney (hKir4.2), and ≈60 % identical to the Kir4.1 channel cloned from human and rat, so that mLV1 was classified as mKir4.2. Xenopus oocytes injected with cRNA encoding mKir4.2 displayed a large inwardly rectifying K+ current, while control oocytes injected with H2O displayed no similar K+ current. The current was blocked by Ba2+ and Cs+ in a voltage‐dependent fashion and displayed inward rectification that was intermediate between that of the strong inward rectifier Kir2.1 and the weak inward rectifier Kir1.1. The current was weakly blocked by TEA in a voltage‐independent fashion. mKir4.2 current was subject to modulation by several distinct mechanisms. Intracellular acidification decreased mKir4.2 current in a reversible fashion, while activation of protein kinase C decreased mKir4.2 current in a manner that was not rapidly reversible. Incubation of oocytes in elevated [K+] produced a slowly developing enhancement of current. Oocytes co‐injected with cRNA for mKir4.2 and Kir5.1, a protein that does not form functional homomeric channels, displayed membrane currents with properties distinct from those expressing mKir4.2 alone. Co‐injected oocytes displayed larger currents than mKir4.2, with novel kinetic properties and an increased sensitivity to Ba2+ block at negative potentials, suggesting that mKir4.2 forms functional heteromultimeric channels with Kir5.1, as has been shown for Kir4.1 These results demonstrate for the first time that a Kir4.2 channel gene product forms functional channels in Xenopus oocytes, that these Kir channels display novel properties, and that Kir4.2 subunits may be responsible for physiological modulation of functional Kir channels.

1. The effect of protein kinase activators on cloned inward rectifier channels expressed in Xenopus oocytes was examined using a two‐electrode voltage clamp. PKA activators caused no change in KIR1.1, KIR2.1, or KIR2.3 current. The PKC activators phorbol 12‐myristate 14‐acetate (PMA) and phorbol 12, 13‐dibutyrate (PDBu) inhibited KIR2.3 currents, but not KIR2.1 or KIR1.1 current. This inhibition was blocked by staurosporine. An inactive phorbol ester, 4 alpha‐phorbol 12, 13‐didecanoate (4 alpha‐PDD), had no effect on KIR2.3. 2. Upon changing solution from 2 to 98 microM K+, KIR2.3 but not KIR1.1 or KIR2.1 currents typically ‘ran down’ over 5 min to 60‐80% of maximum amplitude. Rundown occurred even if PMA was applied before changing to high [K+] solution, indicating that rundown was independent of PKC activity. Rundown was evoked by substituting NMG+ for Na+, showing that it results from low [Na+] and not from high [K+]. 3. These results suggest that KIR2.3, but not KIR1.1 or KIR2.1, is subject to regulation, both by PKC activation and as a consequence of low [Na+]o. The difference in secondary regulation may account for specific responses to PKC stimulation of tissues expressing otherwise nearly identical KIR channels.

Inward rectification induced by mono- and diaminoalkane application to inside-out membrane patches was studied in Kir2.1 (IRK1) channels expressed in Xenopus oocytes. Both monoamines and diamines block Kir2.1 channels, with potency increasing as the alkyl chain length increases (from 2 to 12 methylene groups), indicating a strong hydrophobic interaction with the blocking site. For diamines, but not monoamines, increasing the alkyl chain also increases the steepness of the voltage dependence, at any concentration, from a limiting minimal value of ∼1.5 (n = 2 methylene groups) to ∼4 (n = 10 methylene groups). These observations lead us to hypothesize that monoamines and diamines block inward rectifier K+ channels by entering deeply into a long, narrow pore, displacing K+ ions to the outside of the membrane, with this displacement of K+ ions contributing to “extra” charge movement. All monoamines are proposed to lie with the “head” amine at a fixed position in the pore, determined by electrostatic interaction, so that zδ is independent of monoamine alkyl chain length. The head amine of diamines is proposed to lie progressively further into the pore as alkyl chain length increases, thus displacing more K+ ions to the outside, resulting in charge movement (zδ) increasing with the increase in alkyl chain length.

Consistent left-right asymmetry requires specific ion currents. We characterize a novel laterality determinant in Xenopus laevis: the ATP-sensitive K+-channel (KATP). Expression of specific dominant-negative mutants of the Xenopus Kir6.1 pore subunit of the KATP channel induced randomization of asymmetric organ positioning. Spatio-temporally controlled loss-of-function experiments revealed that the KATP channel functions asymmetrically in LR patterning during very early cleavage stages, and also symmetrically during the early blastula stages, a period when heretofore largely unknown events transmit LR patterning cues. Blocking KATP channel activity randomizes the expression of the left-sided transcription of Nodal. Immunofluorescence analysis revealed that XKir6.1 is localized to basal membranes on the blastocoel roof and cell-cell junctions. A tight junction integrity assay showed that KATP channels are required for proper tight junction function in early Xenopus embryos. We also present evidence that this function may be conserved to the chick, as inhibition of KATP in the primitive streak of chick embryos randomizes the expression of the left-sided gene Sonic hedgehog. We propose a model by which KATP channels control LR patterning via regulation of tight junctions.

Although Kir4.1 channels are the major inwardly rectifying channels in glial cells and are widely accepted to support K+- and glutamate-uptake in the nervous system, the properties of Kir4.1 channels during vital changes of K+ and polyamines remain poorly understood. Therefore, the present study examined the voltage-dependence of K+ conductance with varying physiological and pathophysiological external [K+] and intrapipette spermine ([SP]) concentrations in Müller glial cells and in tsA201 cells expressing recombinant Kir4.1 channels. Two different types of [SP] block were characterized: "fast" and "slow." Fast block was steeply voltage-dependent, with only a low sensitivity to spermine and strong dependence on extracellular potassium concentration, [K+]o. Slow block had a strong voltage sensitivity that begins closer to resting membrane potential and was essentially [K+]o-independent, but with a higher spermine- and [K+]i-sensitivity. Using a modified Woodhull model and fitting i/V curves from whole cell recordings, we have calculated free [SP](in) in Müller glial cells as 0.81 +/- 0.24 mM. This is much higher than has been estimated previously in neurons. Biphasic block properties underlie a significantly varying extent of rectification with [K+] and [SP]. While confirming similar properties of glial Kir and recombinant Kir4.1, the results also suggest mechanisms underlying K+ buffering in glial cells: When [K+]o is rapidly increased, as would occur during neuronal excitation, "fast block" would be relieved, promoting potassium influx to glial cells. Increase in [K+]in would then lead to relief of "slow block," further promoting K+-influx.

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A mutation in the human FXYD2 polypeptide (Na-K-ATPase gamma subunit) that changes a conserved transmembrane glycine to arginine is linked to dominant renal hypomagnesemia. Xenopus laevis oocytes injected with wild-type FXYD2 or the mutant G41R cRNAs expressed large nonselective ion currents. However, in contrast to the wild-type FXYD2 currents, inward rectifying cation currents were induced by hyperpolarization pulses in oocytes expressing the G41R mutant. Injection of EDTA into the oocyte removed inward rectification in the oocytes expressing the mutant, but did not alter the nonlinear current-voltage relationship of the wild-type FXYD2 pseudo-steady-state currents. Extracellular divalent ions, Ca2+ and Ba2+, and trivalent cations, La3+, blocked both the wild-type and mutant FXYD2 currents. Site-directed mutagenesis of G41 demonstrated that a positive charge at this site is required for the inward rectification. When the wild-type FXYD2 was expressed in Madin-Darby canine kidney cells, the cells in the presence of a large apical-to-basolateral Mg2+ gradient and at negative potentials had an increase in transepithelial current compared with cells expressing the G41R mutant or control transfected cells. Moreover, this current was inhibited by extracellular Ba2+ at the basolateral surface. These results suggest that FXYD2 can mediate basolateral extrusion of magnesium from cultured renal epithelial cells and provide new insights into the understanding of the possible physiological roles of FXYD2 wild-type and mutant proteins.