We used molecular biological and patch-clamp techniques to identify the Ca(2+)-activated K(+) channel genes in mouse parotid acinar cells. Two types of K(+) channels were activated by intracellular Ca(2+) with single-channel conductance values of 22 and 140 pS (in 135 mM external K(+)), consistent with the intermediate and maxi-K classes of Ca(2+)-activated K(+) channels, typified by the mIK1 (Kcnn4) and mSlo (Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization. The electrophysiological and pharmacological properties of heterologously expressed mIK1 channels matched those of the native current; thus the native, smaller conductance channel is likely derived from the mIK1 gene. We found that parotid acinar cells express a single, uncommon splice variant of the mSlo gene and that heterologously expressed channels of this Slo variant had a single-channel conductance indistinguishable from that of the native, large-conductance channel. However, the sensitivity of this expressed Slo variant to the scorpion toxin iberiotoxin was considerably different from that of the native current. RT-PCR analysis revealed the presence of two mSlo beta-subunits (Kcnmb1 and Kcnmb4) in parotid tissue. Comparison of the iberiotoxin sensitivity of the native current with that of parotid mSlo expressed with each beta-subunit in isolation and measurements of the iberiotoxin sensitivity of currents in cells from beta(1) knockout mice suggest that parotid acinar cells contain approximately equal numbers of homotetrameric channel proteins from the parotid variant of the Slo gene and heteromeric proteins composed of the parotid Slo variant in combination with the beta(4)-subunit.

Mechanisms and residues responsible for slow activation and Ba 2؉ block of the cardiac muscarinic K ؉ channel, Kir3.1/Kir3.4, were investigated using site-directed mutagenesis. Mutagenesis of negatively charged residues located throughout the pore of the channel (in H5, M2, and proximal C terminus) reduced or abolished slow activation. The strongest effects resulted from mutagenesis of residues in H5 close to the selectivity filter; mutagenesis of residues in M2 and proximal C terminus equivalent to those identified as important determinants of the activation kinetics of Kir2.1 was less effective. In giant patches, slow activation was present in cell-attached patches, lost on excision of the patch, and restored on perfusion with polyamine. Mutagenesis of residues in H5 and M2 close to the selectivity filter also decreased Ba 2؉ block of the channel. A critical residue for Ba 2؉ block was identified in Kir3.4. Mutagenesis of the equivalent residue in Kir3.1 failed to have as pronounced an effect on Ba 2؉ block, suggesting an asymmetry of the channel pore. It is concluded that slow activation is principally the result of unbinding of polyamines from negatively charged residues close to the selectivity filter of the channel and not an intrinsic gating mechanism. Ba 2؉ block involves an interaction with the same residues.

summaryThe effects of Ba¥, Mg¥, Ca¥ and Na¤ as blocking ions were investigated in 90 and 10 mÒ extracellular K¤ solutions on the cloned inward rectifying K¤ channel Kir2.1 expressed in Xenopus oocytes. Some data were also obtained using another inward rectifying K¤ channel Kir3.1ÏKir3.4. The addition of Ba¥ caused a concentration-, voltage-and time-dependent block of both channels. Decreasing the extracellular K¤ concentration augmented the block. The data suggest that Ba¥ blocks the channels by binding to a site within the channel pore and that the electrical binding distance, ä, of the site is significantly different for Kir2.1 and Kir3.1ÏKir3.4 (•0·38 and •0·22, respectively). Mg¥ and Ca¥ caused an instantaneous concentration-and voltage-dependent block of both channels. With Kir2.1, decreasing the K¤ concentration augmented the block. The voltage dependence of the block was less than that of Ba¥ (ä, •0·1), indicating a more superficial binding site for these ions within the channel pore. The affinity of the channels for Mg¥ and Ca¥ was •1000-fold lower than that for Ba¥. Addition of Na¤ resulted in a concentration-, voltage-and time-dependent block of Kir2

Block of K+ channels can be influenced by the ability of charged residues on the protein surface to accumulate cationic blocking ions to concentrations greater than those in bulk solution. We examined the ionic strength dependence of extracellular block of Shaker K+ channels by tetraethylammonium ions (TEA+) and by a trivalent quaternary ammonium ion, gallamine3+. Wild-type and mutant channels were expressed in Xenopus oocytes and currents recorded with the cut-open oocyte technique. Channel block by both compounds was substantially increased when the bathing electrolyte ionic strength was lowered, but with a much larger effect for trivalent gallamine. These data were quantitatively well described by a simple electrostatic model, accounting for accumulation of blocking ions near the pore of the channel by surface charges. The surface charge density of the wild-type channel consistent with the results was -0.1 e nm-2. Shaker channels with T449Y mutations have an increased affinity for both TEA and gallamine but the ionic strength dependence of block was described with the same surface charge density as wild-type channels. Much of the increased sensitivity of Shaker K+ channels to gallamine may be due to a larger local accumulation of the trivalent ion. The negative charge at position 431 contributes to the sensitivity of channels to TEA (MacKinnon & Yellen, 1990). A charge reversal mutation at this location had little effect on the ionic strength dependence of quaternary ammonium ion block, suggesting that the charge on this amino acid may directly affect binding affinity but not local ion accumulation.

The effects of Ba2+, Mg2+, Ca2+ and Na+ as blocking ions were investigated in 90 and 10 mM extracellular K+ solutions on the cloned inward rectifying K+ channel Kir2.1 expressed in Xenopus oocytes. Some data were also obtained using another inward rectifying K+ channel Kir3.1/Kir3.4. The addition of Ba2+ caused a concentration-, voltage- and time-dependent block of both channels. Decreasing the extracellular K+ concentration augmented the block. The data suggest that Ba2+ blocks the channels by binding to a site within the channel pore and that the electrical binding distance, delta, of the site is significantly different for Kir2.1 and Kir3. 1/Kir3.4 (0.38 and 0.22, respectively). Mg2+ and Ca2+ caused an instantaneous concentration- and voltage-dependent block of both channels. With Kir2.1, decreasing the K+ concentration augmented the block. The voltage dependence of the block was less than that of Ba2+ ([delta], 0.1), indicating a more superficial binding site for these ions within the channel pore. The affinity of the channels for Mg2+ and Ca2+ was 1000-fold lower than that for Ba2+. Addition of Na+ resulted in a concentration-, voltage- and time-dependent block of Kir2.1, similar to that observed with Ba2+. The competition between the blocking cations (for Kir2.1: Ba2+, Mg2+, Ca2+; for Kir3. 1/Kir3.4: Ba2+) and extracellular K+ suggests that the binding sites for the blocking cations may be sites to which K+ binds as part of the normal passage of K+ through the channels. It is possible that under normal physiological conditions naturally occurring extracellular cations may partly block the two inward rectifying K+ channels.