The central role of T-tubule and sarcoplasmic reticulum (SR) diadic junctions in excitation-contraction (EC) coupling in adult (AD) ventricular myocytes suggests that their absence in newborn (NB) cells may manifest as an altered EC coupling phenotype. We used confocal microscopy to compare fluo-3 [Ca2+]i transients in the subsarcolemmal space and cell center of field-stimulated NB and AD rabbit ventricular myocytes. Peak systolic [Ca2+]i occurred sooner and was higher in the subsarcolemmal space compared with the cell center in NB myocytes. In AD myocytes, [Ca2+]i rose and declined with similar profiles at the cell center and subsarcolemmal space. Disabling the SR (10 micromol/L thapsigargin) slowed the rate of rise and decline of Ca2+ in AD myocytes but did not alter Ca2+ transient kinetics in NB myocytes. In contrast to adults, localized SR Ca2+ release events ("Ca2+ sparks") occurred predominantly at the cell periphery of NB myocytes. Immunolabeling experiments demonstrated overlapping distributions of the Na(+)-Ca2+ exchanger and ryanodine receptors (RyR2) in AD myocytes. In contrast, RyR2s were spatially separated from the sarcolemma in NB myocytes. Confocal sarcolemmal imaging of di-8-ANEPPS-treated myocytes confirmed an extensive T-tubule network in AD cells, and that T-tubules are absent in NB myocytes. A mathematical model of subcellular Ca2+ dynamics predicts that Ca2+ flux via the Na(+)-Ca2+ exchanger during an action potential can account for the subsarcolemmal Ca2+ gradients in NB myocytes. Spatial separation of sarcolemmal Ca2+ entry from SR Ca2+ release channels may minimize the role of SR Ca2+ release during normal EC coupling in NB ventricular myocytes.

Coetzee. Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits.

The liver-type (GLUT2) and brain-type (GLUT3) human facilitative glucose transporters exhibit distinct kinetics (K(m) values for deoxyglucose transport of 11.2 +/- 1.1 and 1.4 +/- 0.06 mM, respectively) and patterns of substrate transport (GLUT2 is capable of D-fructose transport, GLUT3 is not) [Gould, G. W., Thomas, H. M., Jess, T. J., & Bell, G. I. (1991) Biochemistry 30, 5139-5145]. We have generated a range of chimeric glucose transporters composed of regions of GLUT2 and GLUT3 with a view to identifying the regions of the transporter which are involved in substrate recognition and binding. The functional characteristics of these chimeras were determined by expression in Xenopus oocytes after microinjection of cRNA. Replacement of the region from the start of putative transmembrane helix 7 to the C-terminus of GLUT3 with the corresponding region from GLUT2 results in a chimera with the ability to transport fructose and exhibits a K(m) for 2-deoxyglucose transport of close to that observed for wild-type GLUT2 (8.3 +/- 0.3 mM compared to 11.2 +/- 1.1 mM). Replacement of the region in GLUT3 from the end of helix 7 to the C-terminus with the corresponding region from GLUT2 resulted in a species which was unable to transport fructose and whose K(m) for 2-deoxyglucose was indistinguishable from wild-type GLUT3. We have determined the affinity for 2-deoxyglucose, D-fructose, and D-galactose of these and other chimeras. In addition, the Ki for maltose, a competitive inhibitor of 2-deoxyglucose transport, which binds to the exofacial sugar binding site was determined for these chimeras. The results obtained support a model in which the seventh putative transmembrane-spanning helix is intimately involved in the selection of transported substrate and in which this region plays an important role in determining the K(m) for 2-deoxyglucose. Additional data is presented which suggests that a region between the end of putative transmembrane helix 7 and the end of helix 10, together with sequences in the N-terminal half of the protein may also participate in substrate recognition and transport catalysis.

No abstract

The brain-type glucose transporter (GLUT3) is a high-affinity transporter for D-glucose and D-galactose and is a member of a family of mammalian sugar transporters, each of which are proposed to adopt a secondary structure containing 12 transmembrane helices. In an effort to understand structure-function relationships within such transporters, we have employed alanine-scanning mutagenesis to examine the functional importance of each residue within putative transmembrane helix VIII of the human GLUT3 isoform. Each residue in this helix was replaced individually with alanine, and the functional properties of the mutants were examined by microinjection of in vitro transcribed mRNA into Xenopus oocytes. We show that substitution of residues 305, 306, 308-314, and 316-325 with alanine had minimal effect on the functional activity of the transporter, as determined by measurement of the Km for deoxyglucose transport and the Ki for maltose. In contrast, Asn-315 > Ala-315 exhibited a significant increase in the Km for deoxyglucose independently of any effect on the Ki for maltose. This data suggests that, despite the strong sequence conservation in this helix among the GLUT family, no individual residue is absolutely required for transport catalysis by this isoform. We have also examined the role of proline residues in transport catalysis mediated by GLUT3. Substitution of Pro-203 (helix VI), Pro-206, Pro-209 (cytoplasmic loop between helices VI and VII), Pro-381, Pro-383 and Pro-385 (helix X), Pro-399 (intracellular loop between helices X and XI), or Pro-451 (in the carboxy terminus, close to the end of helix XII) with alanine did not change the Km for deoxyglucose transport for any mutant. However, both Pro-381 and Pro-385 when mutated to alanine exhibited a reduction in the Ki for cytochalasin B. In addition, the Ki for maltose inhibition of deoxyglucose transport was increased for mutants Pro206Ala, Pro381Ala, Pro383Ala, and Pro451Ala. These results will be discussed in terms of proposed structural models for the transporters.

Amiodarone rapidly inhibited K(ATP) channel activity by both promoting channel closure and increasing ATP sensitivity. These actions may contribute to the antiarrhythmic properties of amiodarone.

No abstract

105 Structure-function studies of the brain-type glucose transporter, GLUT3 alanine-scanning mutagenesis of putative transmembrane helix 8.