Tolerance for ATP-Insensitive K _ATP Channels in Transgenic Mice

Abstract

Abstract-To examine the role of sarcolemmal K ATP channels in cardiac function, we generated transgenic mice expressing GFP-tagged Kir6.2 subunits with reduced ATP sensitivity under control of the cardiac ␣-myosin heavy chain promoter. Four founder mice were isolated, and both founders and progeny were all apparently normal and fertile. Electrocardiograms from conscious animals also appeared normal, although mean 24-hour heart rate was approximately 10% lower in transgenic animals compared with littermate controls. In excised membrane patches, K ATP channels were very insensitive to inhibitory ATP: mean K 1/2 ([ATP] causing half-maximal inhibition) was 2.7 mmol/L in high-expressing line 4 myocytes, compared with 51 mol/L in littermate control myocytes. Counterintuitively, K ATP channel density was Ϸ4-fold lower in transgenic membrane patches than in control. This reduction of total K ATP conductance was confirmed in whole-cell voltage-clamp conditions, in which K ATP was activated by metabolic inhibition. K ATP conductance was not obvious after break-in of either control or transgenic myocytes, and there was no action potential shortening in transgenic myocytes. In marked contrast to the effects of expression of similar transgenes in pancreatic ␤-cells, these experiments demonstrate a profound tolerance for reduced ATP sensitivity of cardiac K ATP channels and highlight differential effects of channel activity in the electrical activity of the 2 tissues. Key Words:channels are octameric complexes of 2 distinct subunits, a sulfonylurea receptor (SUR) and a pore-forming Kir6.2. 1-7 SUR confers highaffinity block by sulfonylureas and stimulation by K ϩ channel openers (PCOs) and MgADP. 2,8 -13 ATP inhibits channel activity through an interaction with the Kir6.2 subunit. 3,14 -16 Reconstitution experiments indicate that essential properties of the cardiac K ATP channel are reiterated by coexpression of Kir6.2ϩSUR2A channels, 2,17 and K ATP channels are completely absent in myocyte membranes of Kir6.2 knockout animals, 18,19 although antisense oligonucleotide experiments suggest that SUR1 might be a constituent of neonatal ventricular K ATP channels. 20 Complete metabolic inhibition, or anoxia, leads to opening of K ATP channels in the sarcolemmal membrane, 21 and channel density is so high 21,22 that maximal activation of K ATP can block cell-to-cell conduction and completely suppress action potential generation. 23,24 Both experimental data [25][26][27][28] and modeling 25,29 indicate that activation of only 1% of the available K ATP conductance will be sufficient to shorten the action potential Ϸ50%. For normal cardiac function, therefore, it is critically important that K ATP channels remain substantially closed. Consistent with this notion, action potentials are unaltered in myocytes that completely lack K ATP channels. 18 The major mechanism keeping the channels closed under normal conditions is expected to be the high ATP sensitivity: K 1/2 (cytoplasmic [ATP] causing half-maximal inhibition) is normally 25 t...

Consequences of cardiac myocyte-specific ablation of KATPchannels in transgenic mice expressing dominant negative Kir6 subunits

“…The most reasonable explanation is that these mutant subunits competed with native Kir6.2 subunits for the available SUR2A pool, thus preventing the latter from trafficking normally and forming functional K ATP channels. This explanation is consistent with the recent observations that transgenic overexpression of SUR1 and Kir6.2 constructs also demonstrate marked dominant-negative phenotypes (6,13). Regardless of the mechanism, transgenic expression of mutant Kir6.x subunits resulted in hearts lacking K ATP channels, as assessed with patch-clamp methods.…”

Molecular basis of Kir6.2 mutations associated with neonatal diabetes or neonatal diabetes plus neurological features

“…The observed muscle weakness could be of neurological or muscle origin, because Kir6.2 is expressed in both skeletal muscle and peripheral nerve. Interestingly, although Kir6.2 is expressed in the heart, no marked effects were observed in the ECG (4), which agrees with studies on transgenic mice expressing Kir6.2 with reduced ATP sensitivity, which also have a normal ECG (29).…”

ATP-sensitive potassium channelopathies: focus on insulin secretion

“…The muscle weakness could be of neural or muscle origin, as both skeletal muscle (53) and nerve terminals (89) express Kir6.2. Interestingly, the electrocardiogram of patients with PNDM mutations is superficially normal (7,28), as observed for transgenic mice overexpressing a cardiac-targeted gain-of-function mutation in Kir6.2 (90).…”