Intracellular regions of potassium channels: Kv2.1 and heag

Intracellular regions of voltage-gated potassium channels often comprise the largest part of the channel protein, and yet the functional role of these regions is not fully understood. For the Kv2.1 channel, although there are differences in activation kinetics between rat and human channels, there are, for instance, no differences in movement of the S4 region between the two channels, and indeed our mutagenesis studies have identified interacting residues in both the N- and C -terminal intracellular regions that are responsible for these functional effects. Furthermore, using FRET with fluorescent-tagged Kv2.1 channels, we have shown movement of the C-termini relative to the N-termini during activation. Such interactions and movements of the intracellular regions of the channel appear to form part of the channel gating machinery. Heag1 and heag2 channels also display differing activation properties, despite their considerable homology. By a chimeric approach, we have shown that these differences in activation kinetics are determined by multiple interacting regions in the N-terminus and membrane-spanning regions. Furthermore, alanine mutations of many residues in the C-terminal cyclic nucleotide binding domain affect activation kinetics. The data again suggest interacting regions between N- and C- termini that participate in the conformational changes during channel activation. Using a mass-spectrometry approach, we have identified α-tubulin and a heat shock protein as binding to the C-terminus of the heag2 channel, and α-tubulin itself has functional effects on channel activation kinetics. Clearly, the intracellular regions of these ion channels (and most likely many other ion channels too) are important regions in determining channel function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

Roles of surface residues of intracellular domains of heag potassium channels

Ether-a-go-go potassium channels have large intracellular regions containing ‘Per-Ant-Sim’ (PAS) and cyclic nucleotide binding (cNBD) domains at the N- and C-termini, respectively. In heag1 and heag2 channels, recent studies have suggested that the N- and C-terminal domains interact, and affect activation properties. Here, we have studied the effect of mutations of residues on the surfaces of PAS and cNBD domains. For this, we introduced alanine and lysine mutations in heag1 channels, and recorded currents by two-electrode voltage clamp. In both the PAS domain and the cNBD domain, contiguous areas of conserved residues on the surfaces of these domains were found which affected the activation kinetics of the channel. Next, we investigated possible effects of mutations on domain interactions of PAS and cNBD proteins in heag2 by co-expressing these domain proteins followed by analysis with native gels and western blotting. We found oligomeric association between these domains. Mutations F30A and A609K (on the surfaces of the PAS and cNBD domains, respectively) affected oligomeric compositions of these domains when proteins for PAS and cNBD domains were expressed together. Taken together, the data suggest that the PAS and cNBD domains form interacting oligomers that have roles in channel function.     

The roles of intracellular regions in the activation of voltage-dependent potassium channels

The involvement of the transmembrane regions S2, S3 and S4 in the activation of potassium channels by depolarization has been well clarified. However, a role of the intracellular regions in channel function is emerging. Here we review recent evidence for the roles of intracellular regions in the functioning of members of two families of channels. The Kv2.1 potassium channel, a member of the voltage activated Kv family, has long intracellular regions. By mutagenesis studies and expression in oocytes, we identify residues in both the N- and C-terminal regions that contribute to determining activation kinetics of this channel. It seems that the C-terminus wraps around the N-terminus and interacts with it functionally. The voltage-activated ether-a-go-go (eag) channels also have long intracellular regions. Despite considerable homology, eag1 and eag2 channels display different activation kinetics. By making chimeras between these channels and again expressing in oocytes, we show that residues in both the N-terminal region and the membrane-spanning region are involved in determining these differences in activation kinetics. The intracellular N- and C-terminal regions are likely to continue to prove fertile regions in future investigations into the functioning of ion channels.Presented at the Biophysical Society Meeting on Ion channels—from structure to disease held in May 2003, Rennes, France     

Tubulin as a Binding Partner of the Heag2 Voltage-Gated Potassium Channel

The aim of this work was to investigate interactions of the human ether-a-go-go channel heag2 with human brain proteins. For this, we used heag2-GST fusion proteins in pull-down assays with brain proteins and mass spectrometry, as well as coimmunoprecipitation. We identified tubulin and heat shock 70 proteins as binding to intracellular C-terminal regions of the channel. To study functional effects, heag2 channels were expressed in Xenopus laevis oocytes for two-electrode voltage clamping. Coexpression of alpha-tubulin or the application of colchicine significantly prolonged channel activation times. Application at different times of colchicine gave similar results. The data suggest that colchicine application and tubulin expression do not affect heag2 trafficking and that tubulin may associate with the channel to cause functional effects. Coexpression of heat shock 70 proteins had no functional effect on the channel. The role of tubulin in the cell cytoskeleton suggests a link for the heag2 channel in tubulin-dependent physiological functions, such as cellular proliferation.     

Molecular identification and characterisation of the human eag2 potassium channel

We report the molecular cloning from foetal brain of the human potassium channel heag2. The cDNA encodes a protein of 988 amino acids, 73% identical to heag1. Heag2 is expressed in the brain, but is also found in a range of tissues including skeletal muscle. In oocytes, the channel is a non-inactivating outward rectifier, with dependence of activation rate on holding potential. Compared with heag1, the conductance-voltage curve for heag2 was shifted to the left, the voltage sensitivity was less, activation kinetics were different, and the sensitivity to terfenadine was lower. The heag2 channel may have important physiological roles.     

The Roles of N- and C-terminal determinants in the activation of the Kv2.1 potassium channel

The human and rat forms of the Kv2.1 channel have identical amino acids over the membrane-spanning regions and differ only in the N- and C-terminal intracellular regions. Rat Kv2.1 activates much faster than human Kv2.1. Here we have studied the role of the N- and C-terminal residues that determine this difference in activation kinetics between the two channels. For this, we constructed mutants and chimeras between the two channels, expressed them in oocytes, and recorded currents by two-electrode voltage clamping. In the N-terminal region, mutation Q67E in the rat channel displayed a slowing of activation relative to rat wild type, whereas mutation D75E in the human channel showed faster activation than human wild type. In the C-terminal region, we found that some residues within the region of amino acids 740-853 ("CTA" domain) were also involved in determining activation kinetics. The electrophysiological data also suggested interactions between the N and C termini. Such an interaction was confirmed directly by using a glutathione S-transferase (GST) fusion protein with the N terminus of Kv2.1, which we showed to bind to the C terminus of Kv2.1. Taken together, these data suggest that exposed residues in the T1 domain of the N terminus, as well as the CTA domain in the C terminus, are important in determining channel activation kinetics and that these N- and C-terminal regions interact.     

Role of the S2 and S3 Segment in Determining the Activation Kinetics in Kv2.1 Channels

We constructed chimeras between the rapidly activating Kv1.2 channel and the slowly activating Kv2.1 channel in order to study to what extent sequence differences within the S1–S4 region contribute to the difference in activation kinetics. The channels were expressed in Xenopus oocytes and the currents were measured with a two-microelectrode voltage-clamp technique. Substitution of the S1–S4 region of Kv2.1 subunits by the ones of Kv1.2 resulted in chimeric channels which activated more rapidly than Kv2.1. Furthermore, activation kinetics were nearly voltage-independent in contrast to the pronounced voltage-dependent activation kinetics of both parent channels. Systematic screening of the S1–S4 region by the replacement of smaller protein parts resolved that the main functional changes generated by the S1–S4 substitution were generated by the S2 and the S3 segment. However, the effects of these segments were different: The S3 substitution reduced the effective gating charge and accelerated both a voltage-dependent and a voltage-independent component of the activation time course. In contrast, the S2 substitution accelerated predominantly the voltage-dependent component of the activation time course thereby leaving the effective gating charge unchanged. It is concluded that the S2 and the S3 segment determine the activation kinetics in a specific manner. Received: 13 November 2000/Revised: 5 April 2001     

Integrated allosteric model of voltage gating of HCN channels

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.     

Molecular regions underlying the activation of low- and high-voltage activating calcium channels

We have studied two aspects of calcium channel activation. First, we investigated the molecular regions that are important in determining differences in activation between low- and high-voltage activated channels. For this, we made chimeras between the low-voltage activating Ca_V3.1 channel and the high-voltage activating Ca_V1.2 channel. Chimeras were expressed in oocytes, and calcium channel currents recorded by voltage clamp. For domain I, we found that the molecular region that is important in determining the voltage dependence of activation comprises the pore regions S5-P as well as P-S6, but surprisingly not the voltage sensor S1–S4 region, which might have been expected to play a major part. By contrast, the smaller, but still significant, modulating effects of domain II on activation properties were due to effects involving both S1–S4 and S5–S6 but not the I/II linker. Second, during channel activation we studied movement of the S4 segment in domain I of one of the chimeras, using cysteine-scanning mutagenesis. The reagent parachloromercuribenzensulfonate inhibited currents for mutants V263, A265, L266 and A268, but not for F269 and V271, and voltage dependence of inhibition for residue V263 indicated S4 movement, which occurred before channel opening. The data indicate movement outwards upon depolarisation so as to expose amino acids up to residue 268 in S4.Junying Li and Louisa Stevens contributed equally to this work.     

Store-operated Ca-permeable non-selective cation channels in smooth muscle cells

Over twenty years ago it was shown that depletion of the intracellular Ca²⁺ store in smooth muscle triggered a Ca²⁺ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca²⁺ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca²⁺ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca²⁺ ions. Two activation mechanisms have been identified which involve Ca²⁺ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of α-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.     

Inactivation of cloned Na channels expressed in Xenopus oocytes

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.     

Rundown of the hyperpolarization-activated KAT1 channel involves slowing of the opening transitions regulated by phosphorylation.

Disappearance of the functional activity or rundown of ion channels upon patch excision in many cells involves a decrease in the number of channels available to open. A variety of cellular and biophysical mechanisms have been shown to be involved in the rundown of different ion channels. We examined the rundown process of the plant hyperpolarization-activated KAT1 K+ channel expressed in Xenopus oocytes. The decrease in the KAT1 channel activity on patch excision was accompanied by progressive slowing of the activation time course, and it was caused by a shift in the voltage dependence of the channel without any change in the single-channel amplitude. The single-channel analysis showed that patch excision alters only the transitions leading up to the burst states of the channel. Patch cramming or concurrent application of protein kinase A (PKA) and ATP restored the channel activity. In contrast, nonspecific alkaline phosphatase (ALP) accelerated the rundown time course. Low internal pH, which inhibits ALP activity, slowed the KAT1 rundown time course. The results show that the opening transitions of the KAT1 channel are enhanced not only by hyperpolarization but also by PKA-mediated phosphorylation.     

Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca(2+) ions. Rod CNG channels are directly inhibited by Ca(2+)-calmodulin (Ca(2+)/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca(2+)/CaM-dependent modulation. The molecular basis for Ca(2+)/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca(2+)/CaM to a site in the NH(2)-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH(2)-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca(2+)/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca(2+)/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH(2)- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca(2+)/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH(2)- and COOH-terminal regions underlies Ca(2+)/CaM-dependent inhibition.     

Second transmembrane domain modulates epithelial sodium channel gating in response to shear stress

Na(+) absorption and K(+) secretion in the distal segments of the nephron are modulated by the tubular flow rate. Epithelial Na(+) channels (ENaC), composed of α-, β-, and γ-subunits respond to laminar shear stress (LSS) with an increase in open probability. Higher vertebrates express a δ-ENaC subunit that is functionally related to the α-subunit, while sharing only 35% of sequence identity. We investigated the response of δβγ channels to LSS. Both the time course and magnitude of activation of δβγ channels by LSS were remarkably different from those of αβγ channels. ENaC subunits have similar topology, with an extracellular region connected by two transmembrane domains with intracellular N and C termini. To identify the specific domains that are responsible for the differences in the response to flow of αβγ and δβγ channels, we generated a series of α-δ chimeras and site-specific α-subunit mutants and examined parameters of activation by LSS. We found that specific sites in the region encompassing and just preceding the second transmembrane domain were responsible for the differences in the magnitude and time course of channel activation by LSS.     

Role of Multiple Calcium and Calcium-Dependent Conductances in Regulation of Hippocampal Dentate Granule Cell Excitability

We have constructed a detailed model of a hippocampal dentate granule (DG) cell that includes nine different channel types. Channel densities and distributions were chosen to reproduce reported physiological responses observed in normal solution and when blockers were applied. The model was used to explore the contribution of each channel type to spiking behavior with particular emphasis on the mechanisms underlying postspike events. T-type calcium current in more distal dendrites contributed prominently to the appearance of the depolarizing after-potential, and its effect was controlled by activation of BK-type calcium-dependent potassium channels. Co-activation and interaction of N-, and/or L-type calcium and AHP currents present in somatic and proximal dendritic regions contributed to the adaptive properties of the model DG cell in response to long-lasting current injection. The model was used to predict changes in channel densities that could lead to epileptogenic burst discharges and to predict the effect of altered buffering capacity on firing behavior. We conclude that the clustered spatial distributions of calcium related channels, the presence of slow delayed rectifier potassium currents in dendrites, and calcium buffering properties, together, might explain the resistance of DG cells to the development of epileptogenic burst discharges.     

Activation of K+ and Cl− channels in MDCK cells during volume regulation in hypotonic media

Single-channel patch-clamp experiments were performed on MDCK cells in order to characterize the ionic channels participating in regulatory volume decrease (RVD). Subconfluent layers of cultured cells were exposed to a hypotonic medium (150 mOsm), and the membrane currents at the single-channel level were measured in cell-attached experiments. The results indicate that MDCK cells respond to a hypotonic swelling by activating several different ionic conductances. In particular, a potassium and a chloride channel appeared in the recordings more frequently than other channels, and this allowed a more detailed study of their properties in the inside-out configuration of the patch-clamp technique. The potassium channel had a linear I/V curve with a unitary conductance of 24 +/- 4 pS in symmetrical K+ concentrations (145 mM). It was highly selective for K+ ions vs. Na+ ions: PNa/PK less than 0.04. The time course of its open probability (P0) showed that the cells responded to the hypotonic shock with a rapid activation of this channel. This state of high activity was maintained during the first minute of hypotonicity. The chloride channel participating in RVD was an outward-rectifying channel: outward slope conductance of 63.3 +/- 4.7 pS and inward slope conductance of 26.1 +/- 4.9 pS. It was permeable to both Cl- and NO3- and its maximal activation after the hypotonic shock was reached after several seconds (between 30 and 100 sec). The activity of this anionic channel did not depend on cytoplasmic calcium concentration. Quinine acted as a rapid blocker of both channels when applied to the cytoplasmic side of the membrane. In both cases, 1 mM quinine reversibly reduced single-channel current amplitudes by 20 to 30%. These results indicate that MDCK cells responded to a hypotonic swelling by an early activation of highly selective potassium conductances and a delayed activation of anionic conductances. These data are in good agreement with the changes of membrane potential measured during RVD.     

A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels,an arachidonate-regulated store-independent Orai channel

The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.     

A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels,an arachidonate-regulated store-independent Orai channel

The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.     

Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End?

Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an approximately 68-residue segment translocated across the membrane. Various experimental interventions could cause a longer or shorter segment within the C domain to be translocated, making us wonder why translocation normally stops where it does, near the amino-terminal end of the C domain (approximately residue 450). We hypothesized that regions upstream from the C domain prevent its amino-terminal end from moving into and across the membrane. To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand. The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane. Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane. All of the colicin Ia carboxy-terminal fragments that we examined form channels that pass from a state of relatively normal conductance to a low-conductance state; we interpret this passage as a transition from a channel with four membrane-spanning segments to one with only three.     

Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region

Shaker potassium channels inactivate and recover from inactivation with multiple exponential components, suggesting the presence of multiple inactivation processes. We describe two different types of inactivation in Shaker potassium channels. N-type inactivation can occur as rapidly as a few milliseconds and has been shown to involve an intracellular region at the amino-terminal acting as a blocker of the pore. C-type inactivation is independent of voltage over a range of -25 to +50 mV. It does not require intact N-type inactivation, but is partially coupled to it. The kinetics of C-type inactivation are quite different for channels with different alternatively spliced carboxy-terminal regions. We have localized the differences in C-type inactivation between the ShB and ShA variants to a single amino acid in the sixth membrane-spanning region. N- and C-type inactivation occur by distinct molecular mechanisms.