Faculty Bibliography

The carboxyl terminal cytoplasmic domain of distinct gap junction proteins may play an important role in assembly of functional channels as well as differential responsiveness to pH, voltage, and intracellular second messengers. Oligonucleotide-directed site-specific mutagenesis in a paired Xenopus laevis oocyte expression system was used to examine the expression of mRNAs encoding wild-type and carboxyl terminal mutant connexin43 (Cx43) proteins. Oocytes were stripped, injected with mRNA or distilled water (dH2O), preincubated for 16-20 hours, and then paired for 5-10 hours; this process was followed by electrophysiological recording using the dual voltage-clamp technique. Initial experiments compared the relative junctional conductances (Gjs) in oocyte pairs expressing Cx43 (382 amino acid residues) and two truncated mutants lacking most or a portion of the cytoplasmic carboxyl terminal. The shortest mutant (M241) contained 240 amino acid residues and was devoid of all phosphorylatable serine residues in the cytoplasmic tail; its length approximated the length of liver connexin26. The longest mutant (M257) tested contained 256 amino acid residues, including two serine residues. Oocyte pairs expressing M241 yielded a Gj similar to that of oocytes injected with dH2O, whereas M257 yielded a Gj similar to that of oocytes injected with Cx43. Immunoprecipitation studies showed that Cx43, M257, and M241 proteins were readily detectable in oocytes injected with their respective mRNAs, indicating that the lack of Gj observed with the M241 mRNA was not due to reduced translation. Immunocytochemical studies revealed that wild-type and both truncated mutants were localized to the area of cell-to-cell contact between the paired oocytes, indicating that protein targeting to the membrane was not inhibited in oocytes injected with M241 mRNA. Oocyte pairs expressing mutants in which serine residues were replaced with nonphosphorylatable amino acids (serine codon No. 255 AGC was converted to GCC, alanine, designated as M255S----A, and serine codon No. 244 AGC was converted to GGC, glycine, designated as M244S----G) showed Gjs similar to M257, indicating that these serine residues and, by inference, their phosphorylation state are not critical for expression of functional channels. The importance of the length of the carboxyl terminus was assessed by comparing the Gjs in a series of mutants that were intermediate in length between M257 and M241. Gradual shortening of the carboxyl terminus produced a gradual reduction of Gj relative to M257. However, simple deletion of amino acid residues 241-257 from the wild-type Cx43 did not affect Gj relative to M257.(ABSTRACT TRUNCATED AT 400 WORDS)

The potassium selective, inward rectifier current (IK1) is known to be responsible for maintaining the resting membrane potential of quiescent ventricular myocytes. However, the contribution of this current to the different phases of the cardiac action potential has not been adequately established. In the present study, we have used the action potential clamp (APC) technique to characterize the dynamic changes of a cesium-sensitive (i.e., Ik1) current which occur during the action potential. Our results show that (a) Ik1 is present during depolarization, as well as in the final phase of repolarization of the cardiac action potential. (b) The current reaches the zone of inward-going rectification before the regenerative action potential ensues. (c) The maximal outward current amplitude during repolarization is significantly lower than during depolarization, which supports the hypothesis that in adult guinea pig ventricular myocytes, Ik1 rectification is accentuated during the action potential plateau. Our results stress the importance of Ik1 in the modulation of cell excitability in the ventricular myocyte