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Functional large-conductance [Ca.sup.2+]--and voltage-activated [K.sup.+] (BK) channels can be assembled from four [alpha] subunits (Slo1) alone, or together with four auxiliary [beta]1 subunits to greatly increase the apparent [Ca.sup.2+] sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the ot subunit, which includes the RCK2 (regulator of [K.sup.+] conductance) domain and [Ca.sup.2+] bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a [Ca.sup.2+] bowl and high affinity [Ca.sup.2+] sensitivity. In the second, the [Ca.sup.2+] bowl was disrupted by mutations that greatly reduce the apparent [Ca.sup.2+] sensitivity. We found that the [31 subunit increased the apparent [Ca.sup.2+] sensitivity of Slol channels, independently of whether the [alpha] subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the [Ca.sup.2+] bowl was mutated. In contrast, [31 subunits no longer increased [Ca.sup.2+] sensitivity when Slo1 tails were replaced by Slo3 tails. The 131 subunits were still functionally coupled to channels with S1o3 mils, as DHS-I and 17 [beta]-estradiol activated these channels in the presence of [beta]1 subunits, but not in their absence. These findings indicate that the increase in apparent [Ca.sup.2+] sensitivity induced by the 131 subunit does not require either the [Ca.sup.2+] bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slol tails. The [beta] subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 mils. In contrast, the [beta] subunit-induced increase in apparent [Ca.sup.2+] sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the [31 subunit may be different. KEY WORDS: [Ca.sup.2+]-activated [K.sup.+] channel * RCK domain * DHS-I * estrogen * Slo3

Presents information on a study which examined the role of the tail domain in modulating the gating and conductance properties of large conductance calcium activated potassium channels. Methodology; Results of the study; Discussion.

Examines the structural features involved in the modulation of calcium sensitivity of functional large-conductance calcium- and voltage-activated (BK) channels. Assembly of BK channels from four alpha sub-units; Reduction of voltage sensitivity; Effect of the beta1 sub-unit on mean burst duration.

Functional large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels can be assembled from four alpha subunits (Slo1) alone, or together with four auxiliary beta1 subunits to greatly increase the apparent Ca(2+) sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the alpha subunit, which includes the RCK2 (regulator of K(+) conductance) domain and Ca(2+) bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca(2+) bowl and high affinity Ca(2+) sensitivity. In the second, the Ca(2+) bowl was disrupted by mutations that greatly reduce the apparent Ca(2+) sensitivity. We found that the beta1 subunit increased the apparent Ca(2+) sensitivity of Slo1 channels, independently of whether the alpha subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca(2+) bowl was mutated. In contrast, beta1 subunits no longer increased Ca(2+) sensitivity when Slo1 tails were replaced by Slo3 tails. The beta1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 beta-estradiol activated these channels in the presence of beta1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca(2+) sensitivity induced by the beta1 subunit does not require either the Ca(2+) bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The beta1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the beta1 subunit-induced increase in apparent Ca(2+) sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the beta1 subunit may be different.

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Electron microscopic examination of the paired coxal glands of the horseshoe crab Limulus polyphemus, focusing on urinary and vascular channels, shows six morphologically distinct regions. Each of four nephridial lobes consists of two cortical layers surrounding a medulla. The outer and inner cortexes contain blood vessels separated by a basement membrane from the urinary space lined by podocytes. Podocyte foot processes are applied to the basement membrane, interdigitate with those from other podocytes, and have a filtration slit diaphragm between them. Cortical morphology demonstrates ultrafiltration of blood, a previously undescribed function of the gland, as well as possible endocytic reabsorption of materials by the podocytes. The medulla drains into the stolon connecting the four lobes. These two areas have urinary tubules of cuboidal epithelium featuring microvillous-like apical projections, cytoplasmic vesicles and vacuoles, elaborate lateral interdigitations with septate junctions, and basal invaginations containing numerous mitochondria. These tubules are closely surrounded by blood channels, lined by a basement membrane containing embedded support cells. The medulla and stolon morphology are suggestive of both ion transport and water movement, in keeping with the gland's role in osmoregulation. The stolon empties into the end sac in the base of the most posterior lobe. It is lined by tall epithelium exhibiting apical overlap, blunt projections into the lumen, apparent endocytic vesicles, and basal plasma membrane infoldings with mitochondria. The end sac drains into the conducting nephric duct, the proximal end of which is lined by a cuticle. J. Morphol. 234:233-252, 1997. © 1997 Wiley-Liss, Inc., (Copyright © 1997 Wiley-Liss, Inc.)