PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR (XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was approximately 90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (approximately 30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in Po. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR.
Gap-junction channels (GJCs) are aqueous channels that communicate adjacent cells. They are formed by head-to-head association of two hemichannels (HCs), one from each of the adjacent cells. Functional HCs are connexin hexamers composed of one or more connexin isoforms. Deafness is the most frequent sensineural disorder, and mutations of Cx26 are the most common cause of genetic deafness. Cx43 is the most ubiquitous connexin, expressed in many organs, tissues and cell types, including heart, brain and kidney. Alterations in its expression and function play important roles in the pathophysiology of very frequent medical problems such as those related to cardiac and brain ischemia. There is extensive information on the relationship between phosphorylation and Cx43 targeting, location and function from experiments in cells and organs in normal and pathological conditions. However, the molecular mechanisms of Cx43 regulation by phosphorylation are hard to tackle in complex systems. Here, we present the use of purified HCs as a model for functional and structural studies. Cx26 and Cx43 are the only isoforms that have been purified, reconstituted, and subjected to functional and structural analysis. Purified Cx26 and Cx43 HCs have properties compatible with those demonstrated in cells, and present methodologies for the functional analysis of purified HCs reconstituted in liposomes. We show that phosphorylation of serine 368 by PKC produces a partial closure of the Cx43 HCs, changing solute selectivity. We also present evidence that the effect of phosphorylation is highly cooperative, requiring modification of several connexin subunits, and that phosphorylation of serine 368 elicits conformational changes in the purified HCs. The use of purified HCs is starting to provide critical data to understand the regulation of HCs at the molecular level.
Gap junction channels are formed by two hemichannels in series (one from each neighboring cell), which are in turn connexin hexamers. Under normal conditions, hemichannels at the plasma membrane are mostly closed but can be opened by changes in membrane voltage, extracellular divalent ion concentration, phosphorylation, pH, and redox potential. Recently, interactions between channels have been found to modulate the activity of several ion channels, including gap junction channels. Here, we studied whether connexin46 (Cx46) hemichannels display such behavior. We studied the response of the Cx46 hemichannels expressed in Xenopus laevis oocytes to consecutive depolarization pulses. Hemichannels formed by wild-type Cx46 and a COOH-terminal domain truncation mutant (Cx46DeltaCT) were activated by voltage pulses. When the hemichannels were depolarized repeatedly from -60 mV to +80 mV, the amplitude of the outward and tail currents increased progressively with successive pulses. This phenomenon ("current facilitation") depended on the amplitude of the depolarization, reaching a maximum at approximately +60 mV in oocytes expressing Cx46, and on the interval between pulses, disappearing with intervals longer than about 20 s. The current facilitation was also present in oocytes expressing Cx46DeltaCT, ruling out a primary role of this domain in the facilitation. Nominal removal of divalent cations from the extracellular side caused maximal current activation of Cx46 and Cx46DeltaCT hemichannels and prevented facilitation. The results suggest that Cx46 hemichannels show a cooperative activation independent of their COOH-terminal domain.
The electrical resistances of the transcellular and paracellular pathways across the toad urinary bladder epithelium (a typical "tight" sodium-transporting epithelium) were determined by two independent sets of electrophysiological measurements: (a) the measurement of the total transepithelial resistance, the ratio of resistance of the apical to the basal cell membrane, and cable analysis of the voltage spread into the epithelium; (b) the measurement of the total transepithelial resistance and the ratio of resistances of both cell membranes before and after replacing all mucosal sodium with potassium (thus, increasing selectively the resistance of the apical membrane). The results obtained with both methods indicate the presence of a finite transepithelial shunt pathway, whose resistance is about 1.8 times the resistance of the transcellular pathway. Appropriate calculations show that the resistance of the shunt pathway is almost exclusively determined by the zonula occludens section of the limiting junctions. The mean resistance of the apical cell membrane is 1.7 times that of the basal cell membrane. The use of nonconducting materials on the mucosal side allowed us to demonstrate that apparently all epithelial cells are electrically coupled, with a mean space constant of 460 microm, and a voltage spread consistent with a thin sheet model.
Continuous intracellular recording of membrane potential with microelectrodes in BSC-1 epithelial cells has been used to study the early ionic effects of the interaction of mitogens with cell surface receptors. Initial results show that (i) there is no significant difference in membrane potential between growing and quiescent cells, (ii) addition of epidermal growth factor or serum to quiescent BSC-1 cells induces a brief and transient depolarization, (iii) the mitogenic response of BSC-1 cells to epidermal growth factor and the transient depolarization show similar concentration dependences, and (iv) serum addition to quiescent BSC-1 cells induces a sustained increase in Na+ influx that is electroneutral and amiloride sensitive. Intracellular pH changes may be a primary event triggering the response of quiescent cells to mitogenic polypeptides.
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