ABSTRACTcAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significantly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression ofCFTR protein and an endogenous chloride channel in native cardiac myocytes.In heart cells, elevation of cAMP activates chloride (Cl-) selective channels that contribute to autonomic regulation of action potential duration and resting membrane potential (1-5). Due to similarities in unitary conductance as well as pharmacological and biophysical properties, it has been suggested (6, 7) that cAMP-dependent Cl-channels in heart resemble those of cystic fibrosis transmembrane conductance regulator (CFIR) Cl-channels in epithelial cells, whose regulation and expression is defective in patients with cystic fibrosis. Although recent molecular studies have revealed the presence of CFTR transcript fragments in mammalian myocardium (8, 9), the existing evidence linking the cardiac Clchannel to the CFTR gene product is indirect and circumstantial. Yet recent studies of cardiac cAMP-dependent Cl-channels have proposed a novel model for regulation of CFTR Clchannel gating by phosphorylation and ATP hydrolysis (10, 11). Here, we report the complete sequence of a cDNA isolated from rabbit ventricle that encodes the cardiac cAMPdependent Cl-channel. The sequence indicates that an exon 5 splice variant (exon 5-) of CFTR is expressed in rabbit heart and has >90% identity to human CFTR cDNA present in epithelial cells. cRNA-injected Xenopus oocytes expressed cAMP-dependent Cl-channels with properties similar to those recorded in native cardiac myocytes, and cultured cardiac myocytes exposed to oligodeoxynucleotides complementary (antisense) to the human CFTR transcript exhibited a significantly lower cAMP-dependent Cl-current density compared to cells exposed to control oligodeoxynucleotides homologous (sense) to CFTR. These data provide direct evidence that cAMP-dependent Cl-channels in heart are, in fact, encoded by an alternatively spliced isoform of the epithelial CFTR gene product and argue against earlier suggestions that CFTR exon 5-variants are unlikely to play a physiological role due to defective intracellular processing (12). MATERIALS AND METHODSPoly(A)+ RNA was prepared using the FAST TRACK kit (Invitrogen). First-strand cDNA synthesis was carried out on 0.5 ,tg of poly(A)+...

These data provide evidence that high titers of neutralizing antibodies abolish the in vivo response to interferon beta.

These results clearly demonstrate the molecular expression of CFTR Cl- channels and provide electrophysiological evidence consistent with functional expression of these channels in human atrial and simian ventricular myocardium.

Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT1) receptor-mediated inhibition of total outward K+ current that contributes to an increase in neuronal firing rate. However, the exact K+ conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K+ current revealed a component of K+ current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC50 values of 21.7 micromol/L, 1.49 mmol/L, and 890 micromol/L, respectively, and insensitive to alpha-dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 micromol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K+ current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT2 receptor blocker PD123,319, elicited an inhibition of neuronal K+ current that was abolished by quinine (50 micromol/L). Reverse transcriptase-polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT1 receptor in Xenopus oocytes demonstrated an Ang II-induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT1 receptor-mediated reduction of neuronal K+ current and subsequently to the modulation of cardiovascular function.