However, in the present experiments, it is clear that cysteine residues lying on the cytosolic surface of the BKCa channel protein(s) are the sites of NEM action. Although positively charged, MTSEA has been reported to cross cell membranes and lipid bilayers (Karlin TH287 & Akabas, 1998). concentration-dependently increased by the concentration of Ca2+ bathing the cytosolic surface of the patch. Each data point represents the average of six to seven experiments. (D) Normalizing individual experiments to the maximum conductance reveals that NEM caused a significant increase in (11.81.5 mV in control 15 nM Ca2+ PSS and 16.91.5 mV after NEM, respectively; were 59.5 and 17 mV, 50.4 and 16.4 mV, 42.3 and 18.8 mV, and 7.4 and 18.7 mV in 15, 150, 300 nM and 1.5 properties of BKCa channels in excised patches of the guinea-pig taenia caeca. BKCa current amplitudes were measured either a 0 mV (ai, plot between C40 TH287 and +80 mV (bii). bii Inset illustrates the current amplitudes in MSET expressed as a fraction of the control channel amplitudes (Rel. Amp.) plotted against plots (plot in the presence of MTSET is markedly reduced such that the slope conductance (at 0 mV) of these BKCa channels was 155 pS in control PSS and 95 pS in MTSET-containing PSS. In addition, the relative amplitude of these BKCa channel currents in MTSET, expressed as a fraction of TH287 their respective control amplitudes (Figure 8bii, inset), decreased at more positive potentials suggesting a voltage-dependent blockade of current flow at positive potentials. Open in a separate window Figure 5 Positively charged MTS reagent MTSET increased BKCa channel activity (at 0 mV) in a manner reversed upon washout. MTSET did not prevent the excitatory actions of NOCys. Application of MTSET (2.5 mM for 5 min) significantly increased subunit and the regulatory subunit. The primary sequence of the subunit obtained from different tissues is almost identical being encoded by a single gene, KCNMA1, previously termed (Butler subunits are encoded by four genes (KCNMB1-4) (Tanaka subunit. In addition to containing the six membrane-spanning domains (S1CS6), the S4 voltage sensor and the pore domain between S5 and S6, which identifies this channel as a members of the S4 superfamily of voltage-gated K+ channels (subunit also contains a Smad7 seventh transmembrane domain (S0) that locates the amino terminus in the extracellular domain. Additional hydrophobic cytoplasmic-located segments (S7CS10) are located within TH287 the long carboxy-terminal domain. A tetramerization domain (BK-T1) has been located in the hydrophilic region between S6 and S7, while Ca2+ binding has been located to a region (calcium bowl’) between S9 and S10, which is highly conserved in all cloned BKCa channels (Orio subunit changes the kinetics and Ca2+ sensitivity of expressed subunit channels. However, larger changes in channel kinetics are generally created when subunits are co-expressed with subunits (Orio subunits (B1CB4) consist of two transmembrane segments connected by an extracellular loop’ so that both termini are in the cytoplasmic domain (Knaus subunit increases the sensitivity of the subunit to Ca2+, particularly at high [Ca]i (McCobb subunit selectivity for K+ ions (McManus subunit is also necessary for the extracellular binding and activation of BKCa channels by 17-estradiol (Valverde ((Figure 3C, D). However, exposure to NEM did not affect the sensitivity of the BKCa channel activation to raising the cytosolic concentration of Ca2+ (Figure 3). These results are consistent with the NEM-evoked decrease in BKCa channel activity in excised patches from the rabbit aorta (Bolotina (channel activity evoked by the NO donor, channel activity may well be arising from differences in the amino-acid sequence and therefore the microenvironment of the native BKCa and the channels under study. In particular, there may well be a number of different spliced variants of the subunit of the BKCa channel being expressed in the smooth muscle cells of the taenia ceca. These differing spliced variants could be combining in varying proportions with a number of differing spliced variants of the subunit to form a variety of BKCa channels, which could explain the increases and decreases in channel gating observed in the presence of oxidizing agents and NEM, respectively. However, in the present experiments, it is clear that cysteine residues lying on the cytosolic surface of the BKCa channel protein(s) are the sites of NEM action. Although positively charged, MTSEA has been reported to cross cell membranes and lipid bilayers (Karlin & Akabas, 1998). Thus, we have assumed that MTSEA will have the greatest access to the cysteine residues of the subunit both within the hydrophobic regions and hydrophilic regions. In our experiments, MTSEA evoked a significant decrease in BKCa channel opening that remained upon washout, suggesting an irreversible binding.