No abstract

It has been known for some years that skeletal muscle develops a high potassium permeability in conditions that produce rigor, where ATP concentrations are low and intracellular Ca2+ is high. It has seemed natural to attribute this high permeability to K channels that are opened by internal Ca2+, especially as the presence of such channels has been demonstrated in myotubes and in the transverse tubular membrane system of adult skeletal muscle. However, as we show here, the surface membrane of frog muscle contains potassium channels that open at low internal concentrations of ATP (less than 2 mM). ATP induces closing of these channels without being split, apparently holding the channels in one of a number of closed states. The channels have at least two open states whose dwell times are voltage-dependent. Surprisingly, we find that these may be the most common K channels of the surface membrane of skeletal muscle.

To enter cells, viruses must fuse their envelope with a host cell membrane. Fusion is mediated by specific, membrane-spanning fusion proteins, of which the influenza virus haemagglutinins (HA) are the best characterized. Several HAs have been sequenced, and the crystal structure of the major part of one HA is known. The conditions for fusion and some of the rearrangements in the HA that accompany fusion are well understood, but it remains unclear how HA causes bilayers to fuse. We have observed, in real time, unitary cell-fusion events caused by HA. Fibroblasts expressing HA were induced to fuse with red blood cells by a rapid drop in pH. Fusion was monitored by fluorescence microscopy, and by measuring the membrane conductance and capacitance of the fibroblast. The earliest event observed was the sudden opening of an aqueous pore connecting the cytoplasms of the fusing cells. Initially, the pore conductance often fluctuated between zero and approximately 600 pS, as if the pore were opening and closing repeatedly. Later, it increased over tens of seconds, as if the pore dilated. We suggest that, as in exocytosis, HA-mediated membrane fusion begins with the formation of a narrow pore. Based on the conductance, we estimate the initial diameter of the pore to be no more than twice that of a gap junction channel.

SUMMARY1. Patch-clamp techniques were used to study adenosine-5'-triphosphate (ATP)-dependent K+ channels in sarcolemmal vesicles from frog skeletal muscle. In addition to its ATP dependence, opening of these channels was voltage dependent, the open-state probability (P1pen) increasing with depolarization.2. The reversal potential of unitary currents changed with external K+ concentration, [K+]o, as expected if the Na-K permeability ratio (PNa/PK) equals 0-015.Unitary conductance increased with increasing [K+]. from 14-8 +±0O5 pS (n = 5) in 2-5 mM-K+ to 42-3 ±1 0 pS (n = 8) in 60 mM-K+. This increase was less than that expected from independence.3. Replacement of 60 mM-external K+ by 60 mM-external Rb+ shifted the reversal potential of unitary currents by -6-7 mV, suggesting that Rb+ enters channels nearly as easily as does K+ (Rb-K permeability ratio, PRb/PK = 0 76). Unitary currents were much smaller in Rb+, consistent with Rb+ binding within the channel.4. The ATP-regulated K+ channel was blocked by both internal and external tetraethylammonium ions (TEA+). 2 mM-TEA+, applied to the cytoplasmic face of membrane patches, interrupted channel openings. Higher concentrations reduced unitary current amplitude, suggesting an increase in the rapidity of TEA+ block.5. The reduction in Popen by ATP was consistent with 1:1 binding and a dissociation constant of 0-135 mm. ATP appeared not to be hydrolysed to close channels. Adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) were less effective than ATP, but retained channel closing properties. Substitution of adenine with other purines or with pyrimidine bases substantially reduced activity, as did substitution of ribose by 2'-deoxyribose or by ribose 2',3'-dialdehyde. 6. Sarcoplasmic Ca2+ did not influence Popen* 7. Myotubes, grown from thigh muscles of new-born rats, appeared to lack ATP-dependent K+ channels. Adult frog muscle appeared to lack high-conductance Cal+-dependent K+ channels, at least in the surface membrane. Such channels were found in myotube membranes.