3 Discussion

By selective disruption of one or more subunits, we have studied subunit stoichiometry for the Kir1.1 channel gating by intracellular protons. Although the K80M mutation severely disrupts the pH-dependent channel gating of homomeric Kir1.1 channels, it does not have a dominant-negative effect on the heteromeric channels. Instead, the heteromeric dimers and tetramers show graded reduction in their pH sensitivity with increasing number of the K80M-mutant subunits. Although the full pH sensitivity requires all four functional subunits, one wt subunit is sufficient to enable significant pH sensitivity. Among these subunits a coordination of two subunits in either

trans or cis pair appears important for the pH-dependent gating of Kir1.1 channel.

Kir1 channels including ROMK1 and ROMK2 are expressed in the ascending

limb of Henle and the cortical collecting duct, and play an important role in K+

homeostasis (Wang, 1999; Hebert, 2003). Although they are modulated by several

intracellular messengers such as PIP2, PKA, PKC and WNK4 (McNicholas et al., 1994;

Kahle et al., 2003; Huang, 2001), proton is the major regulator (Tsai et al., 1995; Fakler et al., 1996; Choe et al., 1997; Schulte et al., 1999; Xu et al., 2000a; Xu et al., 2000c; Chanchevalap et al., 2000; McNicholas et al., 1998). While the Kir1 channels are widely open at physiological pH levels, intracellular acidosis causes strong inhibition of these channels (Fakler et al., 1996; Choe et al., 1997; Schulte et al., 1999; Xu et al., 2000a; Xu et al., 2000c; Chanchevalap et al., 2000; McNicholas et al., 1998). Genetic mutations of the Kir1 channels which were found in patients with Bartter syndrome affect the channel

be a critical player in the pH sensitivity, as mutation of this residue to a methionine greatly reduces the pH sensitivity of Kir1 channels (Fakler et al., 1996; McNicholas et al., 1998). Creation of this residue makes the Kir2.1 channel pH-sensitive (Fakler et al., 1996). Such a lysine residue is also found in Kir1.2, Kir4.1 and Kir4.2 channels, in which similar disruption of the pH sensitivity has been observed following site-specific mutation (Choe et al., 1997; McNicholas et al., 1998; Yang et al., 2000; Pessia et al., 2001). In addition to the Lys80, several other residues are involved in the pH sensitivity of the Kir1 channels. Four of six histidine residues located in the C terminus (His225, Hir274, His342 and His354) have been shown to play a part in the channel sensitivity to

pHi. Mutation of any of them reduced the pH sensitivity by 0.2-0.3 pH units

(Chanchevalap et al., 2000). Arg41, Thr51 (in Kir1.2), Val66, Asn171 and Arg311 all contribute to the pH sensitivity of Kir1 channels (Choe et al., 1997; Schulte et al., 1999; Xu et al., 2000a; Xu et al., 2000c). These residues are either non-titratable or titrated at high pH levels, and are believed to shift the pKa of Lys80 to physiological pH levels (Choe et al., 1997; Schulte et al., 1999). Although a number of other residues and protein domains are also involved, the Lys80 is a determinant player in the pH sensitivity of homomeric Kir1.1 channel.

The subunit functional stoichiometry has been studied previously in several Kir channels. Mutation on the signature sequence of the selectivity filter GYG to AAA

results in none conductive K+ channel (Kubo et al., 1993; Kuzhikandathil et al., 2000).

This has been widely used in transgenic mice to suppress certain Kir currents because of its dominant negative effect (McLerie et al., 2003). Some other naturally occurring

mutations in Kir channels that cause diseases also show dominant-negative effect (Flagg et al., 1999; Kunzelmann et al., 2000). However, systematic studies reveal more complicated situations with regard to subunit contributions. The closure of the ATP-

sensitive K+ channels has been shown to be produced by occupation of one of the SUR

subunits (Dorschner et al., 1999). The tetrameric Kir2.1 channel largely retains its

sensitivity to Mg2+ and polyamine when there are three subunits carrying mutations that

abolish Mg2+ and polyamine blockade (Yang et al. 1995). Activation of the GIRK

channels by Gβγ results from graded effects, requiring at least three Gβγ to bind to the

channel protein to achieve full channel activation (Sadja et al., 2002).

The subunit stoichiometry is well demonstrated in several other intracellular- ligand-gated ion channels. Ruiz and Karpen (1997) have found that the opening of CNG channels requires at least two cyclic nucleotide molecules, while the binding of each successive cyclic nucleotide can further enhance the channel opening. Similar observations were made by Rosenmund et al. (1998) in AMPA type glutamate receptor. In contrast, Liu et al. (1998) and Ulens et al. (Ulens and Siegelbaum, 2003) have shown that the binding of a single cyclic nucleotide molecule is enough to increase the channel opening of the CNG and the hyperpolarization- and cyclic nucleotide-activated HCN channels. They have also found that the successive binding of more ligands provides a

none linear enhancement of the channel activation, and two ligands bound in trans

configuration produce a greater effect than in cis configuration (Liu et al., 1998; Ulens

CNG and HCN channels promote the formation of dimer of dimers and activate the gating mechanisms.

Our stoichiometric studies of the Kir1.1 channel reveal that the K80M mutation

does not have a dominant-negative effect on the heteromeric channels.Indeed, our results

suggest that each individual subunit contributes to pH dependent gating of the Kir1.1 channel. The pH sensitivity of the heteromeric channels drops with a decrease in the number of wt subunits. A reduction of 1 pH units is found when all Lys80 are mutated in a tandem-tetrameric channel. These results thus suggest that one functional subunit is sufficient to act on the pH-dependent gating of the Kir1.1 channel, although the full pH sensitivity requires contributions of all four subunits. This result is in agreement with previous observations on the CNG and HCN channels (Liu et al., 1998; Ulens and Siegelbaum, 2003; Labarca et al., 1995; Schonherr et al., 1999).

These subunit stoichiometric studies allow us to appreciate an interesting finding: most of shifts in pKa and h values results from the introduction of the first single wt subunit, whereas additions of an extra subunit have smaller effects. This result does not support positive cooperativity as suggested by the steep titration curve with high h value in Kir1 channels (Tsai et al., 1995; Fakler et al., 1996; Xu et al., 2000c; McNicholas et al., 1998). Although this finding by itself may not be adequate to indicate negative cooperativity that has been observed previously in several other ion channels and receptors (Gentet and Clements, 2002; MacGregor et al., 2002; Gunderson et al., 1994), it demonstrates a feasibility for further studies. The function attributed to the negative

cooperativity is unclear. It may be involved in subunit coordination in a tetrameric channel.

The pH-dependent gating of Kir1.1 channel relies on specific subunit coordination. The wt Kir1.1 shows a single substate conductance at ~40% of the full conductance level. Disruption of all four subunits results in two new substates of conductance, likely to result from the relieved independence of individual subunits. It is worth noting that the newly appeared substates of conductance cannot be explained with flickering activity, since no second full conductance is seen in these recordings, since a single-substate conductance should not produce two or three substates with amplitudes distinct from the original one. Both these new substates disappear when there are two or more wt subunits in a tetrameric channel, suggesting that the four subunits are coordinated in pairs. Supporting this idea are our data indicating that channels with less than two wt subunits show flickering activity (Fig. I-I-6E,F), new opening events (Fig. I- I-7K,L), and a loss of the long-lasting closures (Fig. I-I-8K,L) during intracellular acidification. Thus, it is possible that four subunits are coordinated in functional dimers as suggested previously in CNG and HCN channels (Liu et al., 1998; Ulens and Siegelbaum, 2003). Although each subunit has significant contribution to the gating process, a bigger step is achieved when the potential functional dimer is recruited. Interestingly, the subunit coordination in the Kir1.1 channel does not require specific

conformation of the functional dimers. Both cis and trans conformations have identical

effects, which makes a clear contrast to that found in the CNG and HCN channels (Liu et al., 1998; Ulens and Siegelbaum, 2003). Since the Lys80 is located behind the TM2

bundle of crossing, the interaction site for the dimers does not seem to occur at this location, perhaps in the C terminal instead. With the functional dimers, the wild types of channel closures, substate conductance and pH sensitivity may be largely maintained when one or two of the subunits are disrupted.

In conclusion, the Kir1.1 gating by intracellular protons requires special subunit stoichiometry. While all four subunits are needed for full strength of channel inhibition, a single subunit is sufficient to activate the pH-dependent gating mechanism. These subunits appear to act as two dynamic functional dimers in the pH-dependent gating of

Kir1.1 channel, and the coordination between two subunits can occur in either trans or cis