The ATP-sensitive K(+)-channel (K(ATP)) controls early left-right patterning in Xenopus and chick embryos.

Consistent left-right asymmetry requires specific ion currents. We characterize a novel laterality determinant in Xenopus laevis: the ATP-sensitive K(+)-channel (K(ATP)). Expression of specific dominant-negative mutants of the Xenopus Kir6.1 pore subunit of the K(ATP) channel induced randomization of asymmetric organ positioning. Spatio-temporally controlled loss-of-function experiments revealed that the K(ATP) 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 K(ATP) 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 K(ATP) 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 K(ATP) in the primitive streak of chick embryos randomizes the expression of the left-sided gene Sonic hedgehog. We propose a model by which K(ATP) channels control LR patterning via regulation of tight junctions.

Human FXYD2 G41R mutation responsible for renal hypomagnesemia behaves as an inward-rectifying cation channel.

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.

Complex rectification of Müller cell Kir currents.

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.

Polyamine permeation and rectification of Kir4.1 channels.

Inward rectifier K(+) (Kir) channels are expressed in multiple neuronal and glial cells. Recent studies have equated certain properties of exogenously expressed Kir4.1 channels with those of native K(+) currents in brain cells, as well as demonstrating the expression of Kir4.1 subunits in these tissues. There are nagging problems however with assigning native currents to Kir4.1 channels. One major concern is that in many native tissues, the putatively correlated currents show much weaker rectification than typically reported for cloned Kir4.1 channels. We have now examined the polyamine-dependence of Kir4.1 channels expressed at high density in Cosm6 cells, using inside-out membrane patches. The experiments reveal a complex and variable rectification that can help explain the variability reported for candidate Kir4.1 currents in native cells. Most importantly, rectification seems to be incomplete, even at high polyamine concentrations. In excised membrane patches, with high levels of expression, and high concentrations of spermine, there is approximately 15% residual conductance that is insensitive to spermine. From a biophysical perspective, this is a striking finding, and indicates either that a bound spermine fails to completely block permeation or that significant spermine permeation (i.e. 'punchthrough') is occurring. To examine this further, we have examined block by philanthotoxin (PhTx, essentially spermine with a bulky tail). PhTx block, while less potent, is more complete than spermine block. This leads us to propose that spermine 'punchthrough' may be significant in Kir4 channels, and that this may be a major contributor to the weak rectification observed under physiological conditions.

C-terminal determinants of Kir4.2 channel expression.

Inward rectifier potassium (Kir) channels serve important functional and modulatory roles in a wide variety of cells. While the activity of several members of this channel family are tightly regulated by intracellular messengers such as adenosine triphosphate, G proteins, protein kinases and pH, other members are tonically active and activity is controlled only by the expression level of the protein. In a number of Kir channels, sequence motifs have been identified which determine how effectively the channel is trafficked to and from the plasma membrane. In this report, we identify a number of trafficking determinants in the Kir4.2 channel. Using mutational analysis, we found that truncation of the C terminus of the protein increased current density in Xenopus oocytes, although multiple mutations of the C terminus had no effect on current density. Instead, mutation of a unique region of the channel significantly increased current density. Selective mutation of a putative tyrosine phosphorylation site within this region mimicked the increase in current, suggesting that tyrosine phosphorylation of the protein increases channel retrieval from the membrane (or prevents trafficking to the membrane). Mutation of a previously identified trafficking determinant, K110N, also caused an increase in current density, and combining these mutations caused a multiplicative increase in current, suggesting that these two mutations increase current by independent mechanisms. These data demonstrate novel determinants of Kir4.2 channel expression.