Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume

Abstract

Large-conductance Ca2+- and voltage-activated K+ (BK) channels have the largest conductance (250–300 pS) of all K+-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA− increased currents and MTSET+ decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high [K+]i. Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance ∼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger.

Structural basis for gating the high-conductance Ca2+-activated K+ channel

“…The S6-lined region of the pore is not constricted sufficiently in the EDTA structure to prevent ions from reaching the selectivity filter from the cytoplasm. This observation is consistent with the demonstrated accessibility of large organic molecules to the central cavity (below the selectivity filter) in the closed conformation of Slo1 channels 31,32,33,34 . To explain closure it has been proposed that S6 helix displacements are coupled to changes in the selectivity filter to prevent ion conduction 31,32,33 .…”

“…This observation is consistent with the demonstrated accessibility of large organic molecules to the central cavity (below the selectivity filter) in the closed conformation of Slo1 channels 31,32,33,34 . To explain closure it has been proposed that S6 helix displacements are coupled to changes in the selectivity filter to prevent ion conduction 31,32,33 . The EDTA structure is consistent with such a mechanism at least in terms of the S6 helices being too open to preclude ion passage (the selectivity filter is unchanged from the open structure).…”

The voltage-dependent gate in MthK potassium channels is located at the selectivity filter

“…This is an important finding because it provides experimental evidence that the fractional voltage drop across the cavity is very small for MthK, consistent with evidence for CNG channels and calculations using continuum electrostatics 50,51 . Other channels with similarly large or larger intracellular cavities, such as BK 29,52 , also likely focus the membrane electric field entirely across the selectivity filter.…”

Lack of negative slope in I-V plots for BK channels at positive potentials in the absence of intracellular blockers

“…Depending on the tissue, functional BK channels can be comprised of four pore-forming α subunits alone, or four α subunits plus accessory β subunits, with the β1 subunit increasing the apparent Ca 2+ sensitivity of BK channels in smooth muscle (Knaus et al, 1995; McManus et al, 1995; Nimigean and Magleby, 1999; Lu et al, 2006). The conductance properties of BK channels have been of considerable interest because BK channels have the highest single-channel conductance of K + -selective channels, of 200–300 pS in symmetrical 150-mM K + solutions (Pallotta et al, 1981; Eisenman et al, 1986; Cox et al, 1997b; Hille, 2001; Brelidze et al, 2003; Nimigean et al, 2003; Geng et al, 2011). Our study was undertaken to address contradictory observations concerning the shape of I-V curves for BK channels.…”