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ABSTRACT: GRP94 is a member of the heat shock protein family normally confined to the endoplasmic reticulum that sometimes escapes the KDEL-mediated retention system. It is overexpressed in some gastric and other gastrointestinal carcinomas, but little is known about the physiological role of GRP94 in gastric mucosa. We investigated the membrane presence of GRP94 in parietal cells, which secrete acid into the gastric lumen, using subcellular fractionation, selective solubilization of membrane proteins, Western blotting, and radio-ligand binding and provided evidence of functional GRP94 expression at the surface of gastric mucosa parietal cells anchored to the basolateral domain. Our results show that GRP94 is not an integral membrane protein since 50 mM Na2CO3 treatment dissociates part of it from the membrane. However, 100 mM Na2CO3 treatment did not extract all GRP94 from the membrane, which indicates that it is strongly associated with it. The presence of GRP94 in isolated plasma membrane was demonstrated by Western blotting and its functionality by radio-ligand binding experiments. Both the KD value obtained in saturation experiments with N-ethylcarboxamido-[3H]adenosine at 4°C, at the nanomolar range, and the inhibition constant of its binding by radicicol, the most specific GRP94 inhibitor, indicate that active receptor regions are exposed at the membrane surface. Western blotting of plasma membrane subfractions showed that GRP94 is mainly expressed in the basolateral membrane of gastric parietal cells, while its presence in the apical domain is negligible, thereby inferring a role for GRP94 in processes operating in this membrane domain.
Biochemistry (Moscow) 01/2014; 79(1):8-15. · 1.15 Impact Factor
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ABSTRACT: To identify the causes for the inhomogeneity of ventricular repolarization and increased QT dispersion in hypothyroid mice.
We studied the effects of 5-propyl-2-thiouracil-induced hypothyroidism on the ECG, action potential (AP) and current density of the repolarizing potassium currents I(to,fast), I(to,slow), I(K,slow) and I(ss) in enzymatically isolated myocytes from three different regions of mouse heart: right ventricle (RV), epicardium of the left ventricle (Epi-LV) and interventricular septum. K(+) currents were recorded with the patch-clamp technique. Membranes from isolated ventricular myocytes were extracted by centrifugation. Kv4.2, Kv4.3, KChIP and Na/Ca exchanger proteins were visualized by Western blot.
The frequency or conduction velocity was not changed by hypothyroidism, but QTc was prolonged. Neither resting membrane potential nor AP amplitude was modified. The action potential duration (APD)(90) increased in the RV and Epi-LV, but not in the septum. Hypothyroid status has no effect either on I(to,slow), I(k,slow) or I(ss) in any of the regions analysed. However, I(to,fast) was significantly reduced in the Epi-LV and in the RV, whereas it was not altered in cells from the septum. Western blot analysis reveals a reduction in Kv4.2 and Kv4.3 protein levels in both the Epi-LV and the RV and an increase in Na/Ca exchanger.
From these results we suggest that the regional differences in APD lengthening, and thus in repolarization inhomogeneity, induced by experimental hypothyroidism are at least partially explained by the uneven decrease in I(to,fast) and the differences in the relative contribution of the depolarization-activated outward currents to the repolarization process.
Acta Physiologica 09/2011; 204(4):502-12. · 4.38 Impact Factor
Biophysical Journal 01/2010; 98(3). · 3.67 Impact Factor