Intracellular Polyamines Underlie Physiological Inward Rectification

Although cloning of a voltage-gated potassium channel (Shaker) was first reported in 1988, it was not until 1993 that the first inwardly rectifying K+ channels were successfully cloned (10,11). This advance allowed high-level expression and characterization of channels in heterologous expression systems and led rapidly to the demonstration that polyamines are the cause of inward rectification. In studies of both heterologously expressed and native strong inwardly rectifying K+ channels, it was observed that inward rectification gradually disappeared after excision of a membrane patch from the cell (4,12). A further critical observation was that placing excised membrane patches close to the surface of an oocyte or other cells could restore rectification (4). These data suggested that, rather that being an intrinsic gating mechanism (as is the case for voltage dependence of outward rectification of Kv channels), inward rectification was caused by one or more soluble factors.

Preliminary biochemical purification indicated that these factors were small organic amines, and application of naturally occurring polyamines (spermine, putrescine, cadaverine) to the intracellular face of excised "inside-out" membrane patches containing Kir2 channels was demonstrated to be sufficient to restore all the essential features of inward rectification (4-6,13). Steeply voltage-dependent rectification of Kir2 channels can be induced by application of micromolar spermine and spermidine to the cyto-plasmic face of excised membrane patches. Although less effective and less steeply voltage-dependent, putrescine and cadaverine also cause inward rectification (4,14). Both the voltage dependence and time course of spermine and spermidine unblock match the relaxation time constants of inward rectifiers observed in intact cells, providing further evidence that polyamine block causes inward rectification in vivo (4,5). In contrast to the classical strong inward rectifiers, weakly rectifying channels, such as Kirl.1 (ROMK1) and KATp channels, all exhibit very weak affinity for spermine and other polyamines, and a much shallower voltage dependence of polyamine block. Polyamines have also been demonstrated to cause inward rectification in AMPA/kainate receptors and cyclic nucleotide-gated channels, although block of these channels is not as potent as the block of Kir channels (15,16).

Experimental manipulation of polyamine levels in cells provides further evidence that endogenous polyamines underlie inward rectification. For example, inhibition of the polyamine synthetic enzyme S-adenosylmethionine decarboxylase leads to an increase in cellular putrescine and decrease in spermidine and spermine levels, and consistent with polyamines being a critical element for inward rectification, Bianchi et al. demonstrated that this treatment resulted in relief of inward rectification of endogenous currents in RBL-1 cells (17). Shyng et al. reported similar findings, using a Chinese hamster ovary cell line that is deficient in ornithine decarboxylase activity (18). This cell line requires putrescine in the medium for normal cell growth; removal of putrescine leads to a gradual decline in intracellular levels of putrescine, then sper-midine, and finally spermine. Experimental depletion of polyamines correlated with alterations in Kir2.3 kinetics predicted from excised-patch experiments (18). Finally, manipulation of polyamine levels by genetic approaches in mouse models has provided the most recent evidence of a critical role for polyamines in the regulation of inward rectification in intact tissues. As an example, the Gyro mutant mouse strain lacks the spermine synthase gene, produces no endogenous spermine, and exhibits a significant reduction in the voltage dependence of inward rectification (19).

Relaxation Audio Sounds Babbling Brook

Relaxation Audio Sounds Babbling Brook

This is an audio all about guiding you to relaxation. This is a Relaxation Audio Sounds with sounds from the Babbling Brooks.

Get My Free MP3 Audio

Post a comment